Scanning electron microscopy of swine lymphoid organs

The aim of this investigation was to study by scanning electron microscopy the structure of several swine lymphoid organs (lymph nodes, Peyer's patches, and tonsil). Two groups of animals were used: six-month-old pigs and six- to nine-day-old piglets. Samples were jet-washed to eliminate most free cells in order to observe the reticular framework of these organs more clearly. Peyer's patches in piglets showed two types of villi. In one of them the cellular types were absorptive cells and goblet cells. The second type of villi were shorter and wider, with M cells characterized by presenting long, thick microvilli over their surfaces. Peyer's patches of pigs did not show this second type of villi but were usually covered by absorptive villi. The soft palate tonsil was similar in both groups of animals with its surface epithelial cells full of microfolds, partially and frequently obscured by microorganisms. The appearance of the surface epithelium in the same crypt was different depending on the area. There was a large number of holes through which cells apparently passed towards the crypt lumen. The medulla in the lymph nodes was at the periphery and showed a dense reticular framework. Cortex-like lymphoid tissue was formed by lymphoid follides and diffuse lymphoid tissue with high endothelid venules and lymphatic sinuses. The serosal surface of lymphoid organs was formed either by a typical mesothelial cell layer (small intestine) or by loosely arranged connective fibers (lymph nodes).     

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.     

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.     

Apoptotic process of porcine intestinal M cells

Membranous (M) cells of the follicle-associated epithelium (FAE) are believed to sample antigens from the gut lumen. However, the origin, differentiation mechanism, and cell death of M cells are still a matter of controversy. Therefore, we investigated the process of M cell differentiation and determined their fate in the intestine of three-way crossbred female pigs. We used anti-cytokeratin 18 and anti-PCNA antibodies to distinguish M cells and proliferative cells and performed immunohistochemistry, enzyme histochemistry, and scanning electron microscopy on fresh ileal Peyer’s patches. Cell migration and apoptotic cells were detected by BrdU labeling and the TUNEL method, respectively. The turnover of the FAE was similar to that of the villi. M cells were mostly observed from the FAE crypt to the FAE periphery, but not in the FAE apex. As proliferative M cells (cytokeratin 18⁺/PCNA⁺ cells) have previously been detected in the FAE crypt, porcine M cells may be directly derived from intestinal epithelial stem cells and committed as a distinct cell lineage in the crypts. M cells from the FAE periphery were unstained or only weakly stained for alkaline phosphatase, whereas cytokeratin 18⁺/alkaline phosphatase⁺ cells lying near to the FAE apex showed a columnar shape similar to that of adjacent enterocytes. These data suggest that the committed M cells differentiate to mature M cells by contact with lymphocytes at the FAE periphery, and that they trans-differentiate to enterocytes and are finally excluded near the FAE apex. This investigation was supported by a Grant-in-Aid for Scientific Research (16658107) from the Ministry of Education, Culture, Sports, Science and Technology, by two grants (Prion Project and Secure and Healthy Livestock Farming Project) from the Ministry of Agriculture, Forestry and Fisheries, and by a grant from the Naito Foundation.     

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.     

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.     

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.     

Psg18 is specifically expressed in follicle-associated epithelium

Pregnancy-specific glycoproteins (Psgs) secreted by the placenta regulate the immune system to ensure the survival of the fetal allograft by inducing IL-10, an anti-inflammatory cytokine. However, it is unknown whether Psgs are involved in more general aspects of immune response other than maternal immunity. Here, we report that Psg18 is highly expressed in the follicle-associated epithelium (FAE) overlaying Peyer's patches (PPs). Bioinformatics analysis with Reference Database for Immune Cells (RefDIC) as well as RT-PCR data demonstrated that Psg18 is exclusively expressed in FAE in adult mice, in contrast to other Psg family members that are either not expressed or only slightly expressed in FAE. Psg18 expression was observed in FAE of germ-free-conditioned mice, and was slightly upregulated after bacterial inoculation. In situ hybridization analysis revealed that Psg18 is widely expressed throughout FAE. Furthermore, Psg18 protein is deposited on the extracellular matrix in the subepithelial dome beneath FAE, where antigen-presenting cells accumulate. These results suggest that Psg18 is an FAE-specific marker protein that could promote interplay between FAE and immune cells in mucosa-associated lymphoid tissues.     

CCL9 is secreted by the follicle-associated epithelium and recruits dome region Peyer's patch CD11b+ dendritic cells

The follicle-associated epithelium (FAE) secretes chemokines important in the recruitment of various cell types including CCL20 (MIP-3alpha). CCL20 is chemotactic to the CD11b(+) dendritic cells (DCs) distributed in the subepithelial dome regions of the Peyer's patches, and mice deficient in the receptor for CCL20, CCR6, have been reported to be devoid of the CD11b(+) DCs in the dome regions. Here, we describe another chemokine specifically secreted from the FAE of mouse Peyer's patches, CCL9 (MIP-1gamma, CCF18, MRP-2). By in situ hybridization, we demonstrated that CCL9 mRNA was expressed by the FAE but not by the villus epithelium. At the protein level, CCL9 was detected on the FAE and on extracellular matrix structures within the dome regions of the Peyer's patches. By RT-PCR, we demonstrated that one of the putative receptors for CCL9, CCR1, was expressed by the Peyer's patch CD11b(+) DCs and in a chemotaxis assay, CD11b(+) DCs migrated toward CCL9. To compare the abilities of the chemokines CCL20 and CCL9 to recruit CD11b(+) DCs to the dome regions, we examined the in vivo distribution of these cells in CCR6-deficient, CCL9-blocked wild type, or CCL9-blocked CCR6-deficient mice. To our surprise, using a sensitive immunofluorescence analysis, we observed that CD11b(+) DCs were present in the dome regions of the CCR6-deficient mice. In contrast, Ab neutralization of CCL9 in vivo resulted in significant reduction of the CD11b(+) DC number in the subepithelial dome regions of Peyer's patches of both wild type and CCR6 -/- mice. Taken together, these results demonstrate an important role of CCL9 in CD11b(+) DC recruitment to the dome regions of mouse Peyer's patches.     

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.     

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.     

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.     

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.     

Co-expression of transcription factor AP-2beta (TFAP2B) and GATA3 in human mammary epithelial cells with intense,apicobasal immunoreactivity for CK8/18

AP-2β is a new mammary epithelial differentiation marker and its expression is preferentially retained and enhanced in lobular carcinoma in situ and invasive lobular breast cancer. In normal breast epithelium AP-2β is expressed in a scattered subpopulation of luminal cells. So far, these cells have not been further characterized. Co-expression of AP-2β protein and luminal epithelium markers (GATA3, CK8/18), hormone receptors [estrogen receptor (ER), androgen receptor (AR)] and candidate stem cells markers (CK5/14, CD44) were assessed by double-immunofluorescence staining in normal mammary gland epithelium. The subpopulation of AP-2β-positive mammary epithelial cells showed an almost complete, superimposable co-expression with GATA3 and a peculiar intense, ring-like appearing immunoreactivity for CK8/18. Confocal immunofluorescence microscopy revealed an apicobasal staining for CK8/18 in AP-2β-positive cells, which was not seen in in AP-2β-negative cells. Furthermore, AP-2β-positive displayed a partial co-expression with ER and AR, but lacked expression of candidate stem cell markers CK5/14 and CD44. In summary, AP-2β is a new luminal mammary epithelial differentiation marker, which is expressed in the GATA3-positive subpopulation of luminal epithelial cells. These AP-2β-positive/GATA3-positive cells also show a peculiar CK8/18-expression which may indicate a previously unknown functionally specialized mammary epithelial cell population.

     

Differential display analysis of gene expression during the induction of mucosal immunity

One approach to understanding the physiologically relevant events during the induction of an immune response is to identify genes that are expressed when the immune system first encounters antigen. Such an investigation requires a naive but fully functional immune system, and the fetal lamb provides these conditions during the last trimester of gestation. 'Intestinal segments,' containing a jejunal Peyer's patch, were surgically prepared in fetal lambs (>120 days gestation) and individual 'intestinal segments' were injected with either culture medium or infectious bovine rotavirus. Peyer's patch tissue was collected 18 h postinfection. Histology and virus culture confirmed that bovine rotavirus had infected the mucosal epithelium. RNA was extracted from jejunal Peyer's patch tissue and mRNA differential display was used to identify genes expressed following rotavirus infection. Ten cDNAs were identified by differential display and these cDNAs were isolated, cloned, and sequenced. One of the cDNAs sequenced, displayed homology to the gene encoding the sperm surface protein Sp17. Differential expression of this gene in antigen-exposed jejunal Peyer's patches was confirmed by Northern blot and RT-PCR. The complete sequence for sheep Sp17 mRNA was obtained from a lambda cDNA library, prepared from the jejunal Peyer's patch of a young lamb. Sp17 expression was detected by RT-PCR in a variety of mucosa-associated lymphoid tissues but not in primary or other secondary lymphoid tissues. Thus, the fetal lamb model may be appropriate for identifying genes relevant to mucosal immunity.     

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.     

Peptidoglycan recognition protein expression in mouse Peyer's Patch follicle associated epithelium suggests functional specialization

Mammalian Peyer's Patches possess specialized epithelium, the follicle associated epithelium (FAE), and specialized cells called M cells which mediate transcytosis of antigens to underlying lymphoid tissue. To identify FAE specific genes, we used TOGA gene expression profiling of microdissected mouse Peyer's Patch tissue. We found expression of laminin beta3 across the FAE, and scattered expression of peptidoglycan recognition protein (PGRP)-S. Using the M cell specific lectin Ulex europaeus agglutinin 1 (UEA-1), PGRP-S expression was nearly exclusively co-localized with UEA-1+ M cells. By contrast, the related gene PGRP-L was expressed among a subset of UEA-1 negative FAE cells. Expression of these proteins in transfected cells demonstrated distinct subcellular localization. PGRP-S showed a vesicular pattern and extracellular secretion, while PGRP-L showed localization to both the cytoplasm and the cell surface. The potential function of these PGRP proteins as pattern recognition receptors and their distinctive cellular distribution suggests a complex coordination among specialized cells of the FAE in triggering mucosal immunity and innate immune responses.     

Cloning and expression analysis of an ovine PAP-like protein cDNA, a gene differentially expressed in scrapie

In a previous study, the mRNA level of a pancreatitis-associated protein (PAP)-like protein was found to be elevated in the ileal Peyer's patch of lambs during the early phase of scrapie infection. Here, we report the isolation of the ovine PAP-like protein cDNA which encodes a putative 178 amino acid protein with a signal peptide and a C-lectin binding domain. Comparisons of REG/PAP proteins between various species showed that the deduced amino acid sequences were conserved. The overall amino acid identity between the ovine PAP-like protein and bovine, human and rat REG/PAP proteins varied from 23% to 85%. In Northern blot analysis the expression of the ovine PAP-like protein mRNA was restricted to the ileal and jejunal Peyer's patches. The cellular expression of the PAP-like protein mRNA in the ovine intestine was further characterized by in situ hybridization. PAP-like protein mRNA was detected in cells of the epithelial lining in most crypts and in some intestinal villi in the ileum and jejunum while in the colon and rectum, the PAP-like protein mRNA expression was only detected in the deep portion of a few crypts. The data provided will offer the possibility to search for a link between this PAP-like protein and early events in the development of scrapie.