Epithelial M cells in the rabbit caecal lymphoid patch display distinctive surface characteristics

The follicle-associated epithelium (FAE) in the rabbit caecal lymphoid patch is characterised by the presence of membranous (M) cells, which are believed to be functionally equivalent to those present at other sites of gut-associated lymphoid tissue (GALT). Caecal patch M cells display distinctive features compared with those of other GALT sites, despite similar general morphology and expression of the M cell marker vimentin, suggesting marked heterogeneity in the apical surface of M cells at discrete GALT sites. Electron microscopy reveals that rabbit caecal patch M cells differ from those in the small intestinal Peyer's patch FAE: the former have a prominent aspect within the epithelium and possess microvilli which are longer than those of adjacent enterocytes. Many of the M cells in peripheral regions of the caecal patch FAE are not associated with leucocytes and may thus represent an immature M cell population. The M cells are also histochemically distinct from adjacent enterocytes and from Peyer's patch M cells, showing greater expression of brush-border alkaline phosphatase activity and affinity for certain lectins (peanut and wheat germ agglutinins, Bandeiraea simplicifolia agglutinin II). The differences in the brush-border morphology and glycocalyx structure between M cells at different GALT sites may affect their function at these sites by influencing the interaction of luminal antigens and microorganisms with the M cell surface. The present data also support the hypothesis that M cells arise directly from differentiation of crypt stem cells and not from the transformation of existing fully differentiated enterocytes.     

Enterocytes in the follicle-associated epithelia of rabbit small intestine display distinctive lectin-binding properties

Glycoconjugate expression in follicle-associated epithelia has been examined by application of a panal of lectins to fixed preparations of rabbit small intestine, including Peyer's patches. Each of the lectins examined (wheat germ agglutinin, peanut agglutinin,Ulex europaeus agglutinin I andBandeiraea simplicifolia agglutinin II) exhibited a lower affinity for the apical surface of the specialised M cells than to columnar enterocytes within the Peyer's patch follicle-associated epithelium. Peanut agglutinin differed from the other lectins examined in that it displayed a markedly higher affinity for enterocytes within the follicle-associated epithelium than the neighbouring villi. This observation reveals that the specialised development of the follicle-associated epithelium involves expression of distinctive surface properties within the enterocyte population in addition to the more widely documented heterogeneous development of enterocytes and the specialised M cells.     

Identification of M cells and their distribution in rabbit intestinal Peyer's patches and appendix

The distribution of intestinal membranous (M) cells has been studied within the follicle-associated epithelium of rabbit Peyer's patches and appendix. Vimentin expression has been assessed as a primary criterion to identify rabbit M cells in tissue sections and in whole tissue preparations. This criterion has been compared to the use of the absence of alkaline phosphatase which, due to its heterogeneous distribution within the enterocyte population, is less reliable than vimentin expression as a marker for rabbit M cells. The pattern of vimentin immunostaining revealed that the majority of M cells are located in the periphery of the follicle-associated epithelium, the dome apex being largely free of M cells. This distribution was confirmed by scanning electron microscopy. Vimentin is also expressed by follicle-associated epithelial cells in the vicinity of crypts which lack the typical lymphocyte-containing pocket of M cells. Cytoplasmic peanut agglutinin binding coincides with vimentin-expression throughout the follicle-associated epithelium but is absent from vimentin-negative enterocytes. The co-localisation of these two phenotypic markers in both M cells and epithelial cells adjacent to crypts, which lack the typical morphology of fully developed rabbit M cells, suggests that they correspond to immature M cells which by their location appear to derive directly from undifferentiated crypt stem cells and not from mature columnar enterocytes.     

Automated histochemical analysis of cell populations in the intact follicle-associated epithelium of the mouse Peyer's patch

Summary A new technique of quantitative histochemistry has been developed to study the cellular composition of the follicle-associated epithelium of the mouse Peyer's patch. This technique involves applying naphthol AS-BI phosphate to the surface of intact tissue where it is hydrolysed by alkaline phosphatase present in the luminal membrane of the epithelial cells. Naphthol AS-BI produced by this reaction is then coupled to Fast Red TR diazonium salt at the site of hydrolysis. M cells present in the epithelium contain little alkaline phosphatase activity and, therefore, remain white. Treatment with Alcian Blue is finally used to label goblet cells. Subsequent quantitative analysis of alkaline phosphatase-rich cells is carried out by scanning microdensitometry. Using this technique it is possible to detect two populations of alkaline phosphatase-containing cells in mice reared in a normal animal house environment.These results are discussed in relation to possible interactions taking place between enteric antigens and the gut-associated lymphoid tissue which could reduce the ability of follicle-associated enterocytes to express alkaline phosphatase.     

Vimentin-positive cells in the villus epithelium of the rabbit small intestine

In rabbit intestinal epithelium, vimentin intermediate filaments are selectively expressed in the M cells of follicle-associated epithelium (FAE). To find intestinal epithelial cells belonging to the M cell lineage, vimentin was detected immunohistochemically in the rabbit small and large intestines. Vimentin-positive columnar cells were scattered throughout the villus epithelium of the small intestine. In their cytoplasm, vimentin was located from the perinuclear region to the cell membrane touching intraepithelial lymphocytes. These cells had microvilli shorter than those of absorptive cells, and the alkaline phosphatase activity of the microvilli was markedly weaker than that of absorptive cell microvilli. Glycoconjugates on the surface of the microvilli were alcian blue positive and periodic acid-Schiff negative. The morphological and histochemical features of these vimentin-positive villus epithelial cells differed from those of adjacent absorptive cells and closely resembled those of the M cells in FAE covering Peyer's patches and solitary lymphatic nodules. These results suggest that the vimentin-positive cells in the villus epithelium belong to the M cell lineage.     

Immune cells associated with M cells in the follicle-associated epithelium of Peyer's patches in the rat

Summary Follicle-associated epithelium of Peyer's patches can be differentiated from nearby villous epithelium by the presence of M cells which are antigen-sampling epithelial cells, and by an increase in intraepithelial lymphocytes that are in close contact with M cells. The phenotype of the immune cells close to the M cells of the follicle-associated epithelium of rat Peyer's patches was determined by immunohistochemistry and compared with that of the intra-epithelial lymphocytes of the villous epithelium. Lymphoid T cells, predominantly of the cytotoxic/suppressor phenotype, were observed both in follicle-associated epithelium and in villous epithelium. Lymphoid B cells, mainly immunoblasts and plasma cells containing intracytoplasmic IgM, were present only in the follicle-associated epithelium, near M cells. Macrophages were also present, in contact with M cells, in follicle-associated epithelium, but not in villous epithelium. In addition, M cells bore Ia molecules on their apical membranes. These findings reinforce the concept of immune specialization of the follicle-associated epithelium, by demonstrating that this epithelium contains all the effector cells of immune responses.     

Lectin binding reveals divergent carbohydrate expression in human and mouse Peyer's patches

The nature of cell-associated carbohydrates in the human intestine that may mediate transepithelial transport of bacterial and dietary lectins and their processing by the lymphoid cells of Peyer's patches is not known. Because the cell surface carbohydrate receptors for lectins may vary in different species, the glycoconjugates of human and mouse follicle-associated epithelium and gut-associated lymphoid tissue were compared. A panel of 27, mainly recently isolated, lectins were used to identify glycoconjugate expression in M-cells, enterocytes, goblet cells, lymphocytes and macrophages in mouse and human intestine. Mouse M-cells were exclusively labelled by fucose-specific lectins but in human follicle-associated epithelium no distinct M-cell staining pattern was observed. In the human Peyer's patches,Bryonia dioica lectin bound selectively to paracortical T-lymphocytes andChelidonium majus lectin to germinal centre B-cells. Certain mannose-specific lectins (Galanthus nivalis, Hippeastrum hybrid) stained the tingible body macrophages in the germinal centre of human Peyer's patches but labelled the macrophages in the paracortical T-cell region of the mouse. The results indicate distinct differences in glycosylation between mouse and human Peyer's patches and their associated lymphoid cells. When considering cell surface glycoconjugates as target molecules for the gut immune system, care has to be taken to choose the appropriate lectin for each species.     

Lectin binding defines and differentiates M-cells in mouse small intestine and caecum

M-cell surface glycoconjugate expression was investigated by applying a panel of lectins to whole fixed mouse Peyer's and caecal patches. While the majority of lectins failed to identify mouse M-cells, the lectinEuonymus europaeus differentially stained the surface of M-cells in both mouse Peyer's and caecal patches, and the lectinsUlex europaeus II andBandeiraea simplicifolia I isolectin B₄ identified M-cells in the Peyer's and caecal patch follicle associated epithelium, respectively. These three mouse M-cell markers failed to identify rat and rabbit Peyer's patch M-cells, although bothEuonymus europaeus andUlex europaeus II differentially stained M-cells in the periphery of rabbit caecal patch domes. These site and species related variations in M-cell surface glycoconjugate expression may reflect the local microorganism populations and will have important implications if orally delivered vaccines and drugs are to be targeted to M-cells via their surface glycoconjugates.     

Gene expression profiling of jejunal Peyer’s patches in juvenile and adult pigs

Peyer's patches are organized lymphoid tissues of the small intestine that play a critical role in disease resistance and oral tolerance. Peyer's patches in the jejunum contain lymphocytes, dendritic cells, macrophages, villous epithelium, and specialized follicle-associated epithelium. Little is known about the mechanisms and processes by which cells of the Peyer's patches discriminate food nutrients and commensal microflora from pathogenic microbiota. We hypothesize that the jejunal Peyer's patches express genes that mediate and regulate its essential functions. Expression patterns of approximately 2600 cDNAs from a porcine Peyer's patch subtracted library were examined by microarray profiling. Individual mRNAs of interest were further examined by quantitative RT-PCR. Innate immunity-associated genes, including complement 3 and lysozyme, and the genes for epithelial chloride channel and trappin 1 were highly expressed by jejunal Peyer's patch in both juvenile and adult pigs. The growth- and apoptosis-associated genes CIDE-B, GW112, and PSP/Reg I (pancreatic stone protein or regenerating gene) were differentially expressed in juvenile pig Peyer's patches. Many sequences which were highly expressed in jejunal Peyer's patches have previously been described with functions in epithelial cells. Animal-to-animal variation in basal jejunal Peyer's patch gene expression was considerable and reflects the dynamic physiological environment of the gut in addition to genetic, epigenetic, and microbiological variation in the small intestine.     

Selective expression of brush border hydrolases by mouse Peyer's patch and jejunal villus enterocytes

Developmental profiles describing the expression of lactase, alpha-glucosidase, and alkaline phosphatase activities have been determined quantitatively in mouse jejunal enterocytes during migration over villi and Peyer's patch lymphoid tissue. The predicted maximal lactase and alpha-glucosidase activities expressed by enterocytes migrating over Peyer's patch follicles were about one-quarter and one-half of values found in control villi. Alkaline phosphatase activity was, on the other hand, one third greater in Peyer's patch compared with villus enterocytes. Expression of lactase and alpha-glucosidase activities was initially less in enterocytes migrating along interfollicular compared with control villi. Subsequent increase in hydrolase activities occurred during the later stages of enterocyte migration over interfollicular villi. Lactase activity in athymic mice Peyer's patch enterocytes was identical to that recorded for control mice. The corresponding value for villus lactase was, however, only half that found in control tissue. Factors produced locally in lymphoid follicles are probably responsible for selective effects on enterocyte differentiation.     

Co-expression of vimentin and cytokeratins in M cells of rabbit intestinal lymphoid follicle-associated epithelium

Summary Membranous epithelial (M) cells within the follicle-associated epithelium which overlies gut-associated lymphoid tissue in Peyer's patches and of appendix have been shown by immunocytochemical staining, in rabbit, to contain both vimentin- and cytokeratin-type intermediate filaments. The specificity of vimentin immunostaining has been confirmed by blocking with purified vimentin and by immunoblotting. No evidence was obtained for the expression of vimentin in rat, mouse or human M cells. The possible significance of vimentin-expression in these specialized epithelial cells and the potential use of vimentin as a positive marker for M cells are discussed.     

Differentiation of a murine intestinal epithelial cell line (MIE) toward the M cell lineage

M cells are a kind of intestinal epithelial cell in the follicle-associated epithelium of Peyer's patches. These cells can transport antigens and microorganisms into underlying lymphoid tissues. Despite the important role of M cells in mucosal immune responses, the origin and mechanisms of differentiation as well as cell death of M cells remain unclear. To clarify the mechanism of M cell differentiation, we established a novel murine intestinal epithelial cell line (MIE) from the C57BL/6 mouse. MIE cells grow rapidly and have a cobblestone morphology, which is a typical feature of intestinal epithelial cells. Additionally, they express cytokeratin, villin, cell-cell junctional proteins, and alkaline phosphatase activity and can form microvilli. Their expression of Musashi-1 antigen indicates that they may be close to intestinal stem cells or transit-amplifying cells. MIE cells are able to differentiate into the M cell lineage following coculture with intestinal lymphocytes, but not with Peyer's patch lymphocytes (PPL). However, PPL costimulated with anti-CD3/CD28 MAbs caused MIE cells to display typical features of M cells, such as transcytosis activity, the disorganization of microvilli, and the expression of M cell markers. This transcytosis activity of MIE cells was not induced by T cells isolated from PPL costimulated with the same MAbs and was reduced by the depletion of the T cell population from PPL. A mixture of T cells treated with MAbs and B cells both from PPL led MIE cells to differentiate into M cells. We report here that MIE cells have the potential ability to differentiate into M cells and that this differentiation required activated T cells and B cells.     

Validation and quantitation of an in vitro M-cell model

This study has evaluated an in vitro model of the follicle-associated epithelia that overlie Peyer's patches of the small intestine. The model shares many phenotypic characteristics of M cells in vivo. Co-cultures of the human adenocarcinoma cell line Caco-2 and freshly isolated Peyer's patch cells were established. Fluorescence microscopy and quantitative image analysis were used to validate the model against known markers of M-cell phenotype. Apical expression of alkaline phosphatase was down-regulated in co-cultures and villin was re-distributed from the apical membrane to the cytoplasm. alpha5beta1 integrin was found on the apical surfaces of the monolayers and B and T lymphocytes integrated into the monolayers. Particle transport was temperature-dependent in co-cultures, indicating that a transcytotic route was responsible. This model provides opportunities to study factors that influence M-cell development, assess putative Peyer's patch targeting in oral vaccine technologies, and study intestinal uptake in vitro.     

Reversible differentiation of Caco-2 cells reveals galectin-9 as a surface marker molecule for human follicle-associated epithelia and M cell-like cells

M cells interspersed in the follicle-associated epithelium of Peyer's patches represent the major antigen sampling cells of the intestinal mucosa providing immune surveillance for particulate antigens. Despite their crucial role in immune defense our knowledge about these elusive cells is still only rudimentary. A Caco-2 co-culture model for the induction of M cell-like cells and DNA microarray analysis for differential gene expression profiling were employed to identify (a) putative suitable surface marker(s). Induction of M cell-like cells was demonstrated morphologically by electron microscopy, evaluated by infection with Yersinia enterocolitica and enteropathogenic Escherichia coli strain E2348/69 and further monitored by changes in binding of the lectin UEA-1. The differentiation of Caco-2 cells was found to be reversible, dependent on (a) lymphocyte-derived soluble factor(s) and accompanied by the up-regulation of the glycoprotein lectin galectin-9, which was specifically expressed on these cells as well as on human follicle-associated epithelial (FAE) cells. Galectin-9 represents a novel surface marker which might be employed for molecular targeting to the Peyer's patches thereby opening new opportunities for drug and vaccine development.     

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.     

Generation and characterization of monoclonal antibodies recognizing follicle epithelial M cells in rabbit gut-associated lymphoid tissues

A panel of mouse B cell hybridomas producing monoclonal antibodies (mAb) directed against rabbit M cell-containing epithelia was developed. By immunohistochemistry, the mAb 5D9, 5B11, 1D9, and 4G2 were found to label approximately 50% of the follicle-associated epithelial (FAE) cell populations overlying lymphoid follicles in Peyer's patches, cecal patch, sacculus rotundus, and appendix. The cell staining was localized to FAE cell basolateral surfaces outlining the M cell pockets which enclosed clusters of mononuclear leukocytes, and extended from the crypts of Peyer's patches and sacculus rotundus, and appendiceal crevices, to the apices of domes. In contrast, the stem cell and proliferative regions facing the lamina propria were devoid of immunologically reactive sites. The mAb 5D9, 1D9, and 4G2 did not recognize antigens associated with non-FAE cells in the intestinal lymphoid tissues examined. Only the mAb 5B11 labeled apical surfaces of Peyer's patch and cecal patch non-FAE. However, this mAb did not label interdomal colonic epithelial cells in sacculus rotundus and appendix. Besides recognizing FAE cells, the mAb 4G2 recognized a cross-reactive antigen displayed by dome and lymphoid follicle lymphocytes. By flow cytometry, the mAb 5D9, 5B11, and 1D9 were shown to stain from 14 to 29% of the cells in M cell-enriched populations prepared from Peyer's patches, sacculus rotundus, and appendix, whereas mAb 4G2 was found to recognize 44-54% of the cells. Two-color flow cytometric analysis showed that the mAb stained a functionally distinct subpopulation of Peyer's patch phagocytic cells and did not recognize spleen macrophages. These findings indicate that the panel of mAb recognized novel antigens expressed by FAE cells overlying intestinal lymphoid aggregates, and that the mAb allow identification of phagocytic M cells in suspensions of FAE cells.     

Histological and ultrastructural studies of the marsupial Didelphis albiventris Peyer's patches

Differing from the studied Eutheria the white belly opossum Peyer's patches do not present a conspicuous dome. M cells are located in the inner layer of bilaminal formed at the bottom of the villi. A great variation in the morphology of M cells was observed. The enterocytes located at the epithelial inner layer may present endocytic vesicles, and the microvilli are shorter than the microvilli of enterocytes lining the small intestine. As these morphological aspects have been described to exist in the enterocytes of the lactent opossum small intestine it was surmised that the opossum Peyer's patches special epithelium could represent the persistence in adult animals of a cellular pattern established before the intestinal maturation had occurred.     

Prion uptake in the gut: identification of the first uptake and replication sites

After oral exposure, prions are thought to enter Peyer's patches via M cells and accumulate first upon follicular dendritic cells (FDCs) before spreading to the nervous system. How prions are actually initially acquired from the gut lumen is not known. Using high-resolution immunofluorescence and cryo-immunogold electron microscopy, we report the trafficking of the prion protein (PrP) toward Peyer's patches of wild-type and PrP-deficient mice. PrP was transiently detectable at 1 day post feeding (dpf) within large multivesicular LAMP1-positive endosomes of enterocytes in the follicle-associated epithelium (FAE) and at much lower levels within M cells. Subsequently, PrP was detected on vesicles in the late endosomal compartments of macrophages in the subepithelial dome. At 7-21 dpf, increased PrP labelling was observed on the plasma membranes of FDCs in germinal centres of Peyer's patches from wild-type mice only, identifying FDCs as the first sites of PrP conversion and replication. Detection of PrP on extracellular vesicles displaying FAE enterocyte-derived A33 protein implied transport towards FDCs in association with FAE-derived vesicles. By 21 dpf, PrP was observed on the plasma membranes of neurons within neighbouring myenteric plexi. Together, these data identify a novel potential M cell-independent mechanism for prion transport, mediated by FAE enterocytes, which acts to initiate conversion and replication upon FDCs and subsequent infection of enteric nerves.     

Secretory immunoglobulin A antibodies against the sigma1 outer capsid protein of reovirus type 1 Lang prevent infection of mouse Peyer's patches

Reovirus type 1 Lang (T1L) adheres to M cells in the follicle-associated epithelium of mouse intestine and exploits the transport activity of M cells to enter and infect the Peyer's patch mucosa. Adult mice that have previously cleared a reovirus T1L infection have virus-specific immunoglobulin G (IgG) in serum and IgA in secretions and are protected against reinfection. Our aim in this study was to determine whether secretory IgA is sufficient for protection of Peyer's patches against oral reovirus challenge and, if so, against which reovirus antigen(s) the IgA may be directed. Monoclonal antibodies (MAbs) of the IgA isotype, directed against the sigma1 protein of reovirus T1L, the viral adhesin, were produced and tested along with other, existing IgA and IgG MAbs against reovirus T1L outer capsid proteins. Anti-sigma1 IgA and IgG MAbs neutralized reovirus T1L in L cell plaque reduction assays and inhibited T1L adherence to L cells and Caco-2(BBe) intestinal epithelial cells in vitro, but MAbs against other proteins did not. Passive oral administration of anti-sigma1 IgA and IgG MAbs prevented Peyer's patch infection in adult mice, but other MAbs did not. When anti-sigma1 IgA and IgG MAbs were produced in mice from hybridoma backpack tumors, however, the IgA prevented Peyer's patch infection, but the IgG did not. The results provide evidence that neutralizing IgA antibodies specific for the sigma1 protein are protective in vitro and in vivo and that the presence of these antibodies in intestinal secretions is sufficient for protection against entry of reovirus T1L into Peyer's patches.     

Cytokeratin 18 is a specific marker of bovine intestinal M cell

Microfold (M) cells in the follicle-associated epithelium (FAE) of Peyer's patches have an important role in mucosal immune responses. A primary difficulty for investigations of bovine M cells is the lack of a specific molecular marker. To identify such a marker, we investigated the expression of several kinds of intermediate filament proteins using calf Peyer's patches. The expression patterns of cytokeratin (CK) 18 in jejunal and ileal FAE were very similar to the localization pattern of M cells recognized by scanning electron microscopy. Mirror sections revealed that jejunal CK18-positive cells had irregular and sparse microvilli, as well as pocket-like structures containing lymphocytes, typical morphological characteristic of M cells. However, CK18-negative cells had regular and dense microvilli on their surface, typical of the morphology of enterocytes. In contrast, CK20 immunoreactivity was detected in almost all villous epithelial cells and CK18-negative cells in the FAE. CK18-positive proliferating transit-amplifying cells in the crypt exchanged CK18 for CK20 above the mouth of the crypt and after moving to the villi; however, CK18-positive M cells in the crypt continued their expression of CK18 during movement to the FAE region. Terminal deoxynucleotidyl-transferase-mediated deoxyuridine-triphosphate-biotin nick-end labeling-positive apoptotic cells were specifically detected at the apical region of villi and FAE in the jejunum and ileum, and all were also stained for CK20. These data indicate that CK18 may be a molecular marker for bovine M cells in FAE and that M cells may transdifferentiate to CK20-positive enterocytes and die by apoptosis in the apex of the FAE.