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.     

The viral sigma1 protein and glycoconjugates containing alpha2-3-linked sialic acid are involved in type 1 reovirus adherence to M cell apical surfaces

Type 1 reoviruses invade the intestinal mucosa of mice by adhering selectively to M cells in the follicle-associated epithelium and then exploiting M cell transport activity. The purpose of this study was to identify the apical cell membrane component and viral protein that mediate the M cell adherence of these viruses. Virions and infectious subviral particles of reovirus type 1 Lang (T1L) adhered to rabbit M cells in Peyer's patch mucosal explants and to tissue sections in an overlay assay. Viral adherence was abolished by pretreatment of sections with periodate and in the presence of excess sialic acid or lectins MAL-I and MAL-II (which recognize complex oligosaccharides containing sialic acid linked alpha2-3 to galactose). The binding of T1L particles to polarized human intestinal (Caco-2(BBe)) cell monolayers was correlated with the presence of MAL-I and MAL-II binding sites, blocked by excess MAL-I and -II, and abolished by neuraminidase treatment. Other type 1 reovirus isolates exhibited MAL-II-sensitive binding to rabbit M cells and polarized Caco-2(BBe) cells, but type 2 or type 3 isolates including type 3 Dearing (T3D) did not. In assays using T1L-T3D reassortants and recoated viral cores containing T1L, T3D, or no sigma1 protein, MAL-II-sensitive binding to rabbit M cells and polarized Caco-2(BBe) cells was consistently associated with the T1L sigma1. MAL-II-recognized oligosaccharide epitopes are not restricted to M cells in vivo, but MAL-II immobilized on virus-sized microparticles bound only to the follicle-associated epithelium and M cells. The results suggest that selective binding of type 1 reoviruses to M cells in vivo involves interaction of the type 1 sigma1 protein with glycoconjugates containing alpha2-3-linked sialic acid that are accessible to viral particles only on M cell apical surfaces.     

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.     

Differential surface characteristics of M cells from mouse intestinal Peyer''s and caecal patches

Summary The mouse caecal patch is located near the blind end of the caecum, and consists of a group of lymphoid follicles. In common with the Peyer's patches, the follicle-associated epithelium overlying these follicles is largely composed of enterocytes, goblet cells and membranous epithelial (M) cells. Each of these types of cell was readily identified by electron microscopy, although caecal patch enterocytes and M cells were morphologically distinct from those of the Peyer's patches. Staining for alkaline phosphatase activity demonstrated that the majority of caecal follicle-associated epithelial cells were alkaline phosphatase-negative, positive cells consisting of a mixture of enterocytes and M cells. In contrast, it has previously been found that Peyer's patch enterocytes are positive for alkaline phosphatase while the M cells are relatively lacking in alkaline phosphatase activity. Lectin histochemistry revealed that surface glycoconjugate expression differs between the caecal and Peyer's patch follicle-associated epithelial cells; in particular, the characteristic staining of Peyer's patch M cells by Ulex europaeus agglutinin 1 was absent on the caecal patch follicle-associated epithelium. These altered surface characteristics indicate that the development of the caecal patch follicle-associated epithelial cells is influenced by the local environment, and these altered properties may be indicative of modified functional roles for the cells at this site.     

Interactions with M cells and macrophages as key steps in the pathogenesis of enterohemorrhagic Escherichia coli infections

Enterohemorrhagic Escherichia coli (EHEC) are food-borne pathogens that can cause serious infections ranging from diarrhea to hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS). Translocation of Shiga-toxins (Stx) from the gut lumen to underlying tissues is a decisive step in the development of the infection, but the mechanisms involved remain unclear. Many bacterial pathogens target the follicle-associated epithelium, which overlies Peyer's patches (PPs), cross the intestinal barrier through M cells and are captured by mucosal macrophages. Here, translocation across M cells, as well as survival and proliferation of EHEC strains within THP-1 macrophages were investigated using EHEC O157:H7 reference strains, isogenic mutants, and 15 EHEC strains isolated from HC/HUS patients. We showed for the first time that E. coli O157:H7 strains are able to interact in vivo with murine PPs, to translocate ex vivo through murine ileal mucosa with PPs and across an in vitro human M cell model. EHEC strains are also able to survive and to produce Stx in macrophages, which induce cell apoptosis and Stx release. In conclusion, our results suggest that the uptake of EHEC by M cells and underlying macrophages in the PP may be a critical step in Stx translocation and release in vivo. A new model for EHEC infection in humans is proposed that could help in a fuller understanding of EHEC-associated diseases.     

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.     

Selective binding and transcytosis of Ulex europaeus 1 lectin by mouse Peyer's patch M-cells in vivo

The in vivo interaction of the lectin Ulex europaeus agglutinin 1 with mouse Peyer's patch follicle-associated epithelial cells was studied in the mouse Peyer's patch gut loop model by immunofluorescence and electron microscopy. The lectin targets to mouse Peyer's patch M-cells and is rapidly endocytosed and transcytosed. These processes are accompanied by morphological changes in the M-cell microvilli and by redistribution of polymerised actin. The demonstration of selective binding and uptake of a lectin by intestinal M-cells in vivo suggests that M-cell-specific surface glycoconjugates might act as receptors for the selective adhesion/uptake of microorganisms.This work was supported under the LINK programme in Selective Drug Delivery and Targeting, funded by SERC/MRC/DTI and industry (SERC grant GR/F 09747). M.A.J. was also supported by a Wellcome Postdoctoral Research Followship (039684/Z/93/Z). Additional support was provided by the Royal Society for equipment.     

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.     

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.     

Rapid in vivo transport of proteins from digested allergen across pre-sensitized gut

Although the route of sensitization to food allergens is still the subject of debate, it is generally accepted the gut immune system plays a pivotal role. However, hitherto the transport of allergens across the normal, pre-sensitized gut epithelium remained largely unknown. Our aim was to identify the route through which protein bodies and soluble proteins from digested peanuts penetrated the pre-sensitized gut epithelium in vivo and the specific cell types involved in the transport. Digestion of peanuts released a large number of protein bodies that are exclusively transported across the epithelium by specialized antigen-sampling M cells and delivered to the lymphoid tissue of Peyer's patch. Intracellular transport of soluble protein also occurred almost exclusively via M cells and it was negligible across absorptive enterocytes. We hypothesize that these conditions which are known to favour strongly the induction of immune responses rather than oral tolerance may play a significant role in the genesis of allergic reactions.     

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.     

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.     

Altered expression of inflammatory cytokine receptors in response to LPS challenge through interaction between intestinal epithelial cells and lymphocytes of Peyer's patch

The intense innate immunological activities occurring at the enteric mucosal surface involve interactions between intestinal epithelial cells and immune cells. Our previous studies have indicated that Peyer's patch lymphocytes may modulate intestinal epithelial barrier and ion transport function in homeostasis and host defense via cell-cell contact as well as cytokine signaling. The present study was undertaken using the established co-culture system of Caco-2 epithelial cells with lymphocytes of Peyer's patch to investigate the expression of IL-8 and IL-6 cytokines and cytokine receptors in the co-culture system after challenge with Shigella F2a-12 lipopolysaccharide (LPS). The human colonic epithelial cell line Caco-2 was co-cultured with freshly isolated lymphocytes from the murine Peyer's patch either in the mixed or separated (isolated but permeable compartments) co-culture configuration, and was challenged with Shigella F2a-12 LPS for 8 h. The level of mRNA expressions of human interleukin-8 (hIL-8), human interleukin-8 receptor (hIL-8R), mouse interleukin-8 receptor (mIL-8R), mouse interleukin-6 (mIL-6), mouse interleukin-6 receptor (mIL-6R) and human interleukin-6 receptor (hIL-6R) was examined by semi-quantitative PCR. In both co-culture groups, hIL-8 expression of Caco-2 cells was decreased, and hIL-8R expression was increased compared to the Caco-2 alone group. Upon LPS challenge, hIL-8 expression from the Caco-2 cells of both co-culture groups was higher than in the Caco-2 control group. The increased hIL-8 expression of Caco-2 cells in the separated co-culture group is correlated with a decreased hIL-8R expression and an increased mIL-8R expression. In the mixed co-culture group, the increased expression of hIL-8 was associated with the upregulated hIL-8R expression on Caco-2 cells and downregulated mIL-8R on murine Peyer's patch lymphocytes (PPL). mIL-6 expression from mouse PPL was also upregulated by LPS in mixed co-culture. However, upon the treatment with LPS, hIL-6R expression of Caco-2 cells was decreased in the mixed co-culture, but increased in separated co-culture. The data suggest that release of hIL-8 from epithelial cells may act on lymphocytes through a paracrine pathway, but it may also act on the epithelial cells themselves via an autocrine pathway. The data also suggest that the release of mIL-6 from Peyer's patch lymphocytes affects epithelial cells in a paracrine fashion.     

Translocation of enteropathogenic Escherichia coli across an in vitro M cell model is regulated by its type III secretion system

Enteropathogenic Escherichia coli (EPEC) is an extracellular pathogen that utilizes a type III secretion system (TTSS) to modulate diverse host cell processes including cytoskeletal dynamics, tight junction permeability and macrophage phagocytosis. Some EPEC strains exhibit selective tropism for the specialized follicle-associated epithelium (FAE) overlying lymphoid follicles in the gut, which is a major site of uptake of inert particulates and pathogens, but do not translocate from the intestinal lumen in significant numbers. We have investigated the interaction of EPEC with FAE using an established in vitro model of the specialized FAE in which polarized enterocyte-like Caco-2 cells cocultured with the Raji B cell line undergo a phenotypic switch to a form that morphologically and functionally resembles the specialized antigen-transporting M cells found within FAE. Having confirmed that coculture with Raji B cells induces brush border reorganization and enhances particle transport across Caco-2 cells, we investigated translocation of bacteria across the M cell model. While Salmonella translocation was markedly upregulated by Raji coculture, transport of wild-type EPEC occurred at similarly low levels across both native Caco-2 and Caco-2/Raji-cocultured layers. Translocation rates were markedly higher for EPEC strains lacking either functional TTSS or the effector protein EspF. These observations resemble previously reported data on the inhibition of macrophage phagocytosis by EPEC, which has also been reported to be dependent on TTSS and EspF. Furthermore, as with macrophage phagocytosis, enhanced translocation of a TTSS mutant was blocked by wortmannin, implicating inhibition of phosphatidyl inositol 3-kinase-mediated signalling in the regulation of M cell translocation by EPEC.     

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.     

Transport of membrane-bound macromolecules by M cells in follicle-associated epithelium of rabbit Peyer's patch

Summary M cells in Peyer's patch epithelium conduct transepithelial transport of luminal antigens to cells of the mucosal immune system. To determine the distribution of specific lectin-binding sites on luminal membranes of living M cells and to follow the transport route of membranebound molecules, lectin-ferritin conjugates and cationized ferritin were applied to rabbit Peyer's patch mucosa in vivo and in vitro. The degree to which binding enhances transport was estimated by comparing quantitatively the transport of an adherent probe, wheat germ agglutinin-ferritin, to that of a nonadherent BSA-colloidal gold probe. When applied to fixed tissue, the lectins tested bound equally well to M cells and columnar absorptive cells. On living mucosa, however, ferritin conjugates of wheat germ agglutinin and Ricinus communis agglutinins I and II bound more avidly to M cells. Absorptive cells conducted little uptake and no detectable transepithelial transport. Lectins on M cell membranes were endocytosed from coated pits, rapidly transported in a complex system of tubulocisternae and vesicles, and remained adherent to M cell basolateral membranes. Cationized ferritin adhered to anionic sites and was similarly transported, but was released as free clusters at M cell basolateral surfaces. When applied simultaneously to Peyer's patch mucosa, wheat germ agglutinin-ferritin was transported about 50 times more efficiently than was bovine serum albumin-colloidal gold.     

Translocation of Yersinia entrocolitica across reconstituted intestinal epithelial monolayers is triggered by Yersinia invasin binding to beta1 integrins apically expressed on M-like cells

Yersinia enterocolitica cross the intestinal epithelium via translocation through M cells, which are located in the follicle-associated epithelium (FAE) of Peyer's patches (PP). To investigate the molecular basis of this process, studies were performed using a recently developed in vitro model, in which the enterocyte-like cell line Caco-2 and PP lymphocytes are co-cultured in order to establish FAE-like structures including M cells. Here, we demonstrate that Y. enterocolitica does not adhere significantly to the apical membrane of differentiated enterocyte-like Caco-2 cells that express binding sites for Ulex europaeus agglutinin (UEA)-1. In contrast, Y. enterocolitica adhered to, and was internalized by, cells that lacked UEA-1 binding sites and displayed a disorganized brush border. These cells were considered to be converted to M-like cells. Further analysis revealed that part of these cells expressed β1 integrins at their apical surface and, as revealed by comparison of wild-type and mutant strains, interacted with invasin of Y. enterocolitica . Consistently, anti-β1 integrin antibodies significantly inhibited internalization of inv -expressing yersiniae. Experiments with Yersinia mutant strains deficient in YadA or Yop secretion revealed that these virulence factors play a minor role in this process. After internalization, yersiniae were transported within LAMP-1-negative vacuoles to, and released at, the basal surface. Internalization and transport of yersiniae was inhibited by cytochalasin D, suggesting that F-actin assembly is required for this process. These results provide direct evidence that expression of β1 integrins at the apical surface of M cells enables interaction with the invasin of Y. enterocolitica , and thereby initiates internalization and translocation of bacteria.     

TLRs regulate the gatekeeping functions of the intestinal follicle-associated epithelium

Initiation of adaptive mucosal immunity occurs in organized mucosal lymphoid tissues such as Peyer's patches of the small intestine. Mucosal lymphoid follicles are covered by a specialized follicle-associated epithelium (FAE) that contains M cells, which mediate uptake and transepithelial transport of luminal Ags. FAE cells also produce chemokines that attract Ag-presenting dendritic cells (DCs). TLRs link innate and adaptive immunity, but their possible role in regulating FAE functions is unknown. We show that TLR2 is expressed in both FAE and villus epithelium, but TLR2 activation by peptidoglycan or Pam(3)Cys injected into the intestinal lumen of mice resulted in receptor redistribution in the FAE only. TLR2 activation enhanced transepithelial transport of microparticles by M cells in a dose-dependent manner. Furthermore, TLR2 activation induced the matrix metalloproteinase-dependent migration of subepithelial DCs into the FAE, but not into villus epithelium of wild-type and TLR4-deficient mice. These responses were not observed in TLR2-deficient mice. Thus, the FAE of Peyer's patches responds to TLR2 ligands in a manner that is distinct from the villus epithelium. Intraluminal LPS, a TLR4 ligand, also enhanced microparticle uptake by the FAE and induced DC migration into the FAE, suggesting that other TLRs may modulate FAE functions. We conclude that TLR-mediated signals regulate the gatekeeping functions of the FAE to promote Ag capture by DCs in organized mucosal lymphoid tissues.     

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.