Transfer of carbonic anhydrase-positive particles from the follicle-associated epithelium to lymphocytes of Peyer's patches in foetal sheep and lambs

Summary Carbonic anhydrase cytochemistry of the ileal Peyer's patch in foetal and neonatal lambs has indicated secretion from the follicle-associated epithelium to the follicles. Reaction for carbonic anhydrase in the follicle-associated epithelium was found in the luminal plasma membrane, in cytoplasmic vesicles, and in vacuoles containing 50-nm membrane-bounded particles that seemed to be shed to the intercellular space. The lateral plasma membrane was negative for carbonic anhydrase, indicating that formation of carbonic anhydrase-positive particles was restricted to vacuoles. Administration of ferritin to ileal loops of sheep foetuses showed ferritin localized in vesicles and vacuoles of the follicle-associated epithelium followed by exocytosis, together with carbonic anhydrase-positive particles, into the indentations of the lateral cell border. The carbonic anhydrase-positive particles seemed to be transported to the centres of lymphoid follicles where many were attached to the plasma membrane of lymphocytes. Carbonic anhydrase-positive particles were also seen in vesicles and sometimes free in the cytoplasm of the lymphocytes or attached to their nuclear envelope. Light microscopically, carbonic anhydrase reactivity of the follicle-associated epithelium was associated with the early formation of the ileal Peyer's patch at about 100 days gestation. At this time the follicle-associated epithelium showed a strong luminal but at most a week lateral staining. With further foetal development there was a progressive increase in the amount of carbonic anhydrase-positive reaction product in extracellular particles, both along the lateral cell borders of the follicle-associated epithelium and among the lymphocytes of the follicle centres.     

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.     

Differentiation of the follicle-associated epithelium in ileal Peyer’s patch and production of 50-nm particles are maintained in B-cell-depleted fetal sheep

To evaluate the dependence of the differentiation of the follicle-associated epithelium (FAE) on the presence of follicular B-cells, the FAE of ileal Peyers patch follicles was examined in B-cell-depleted fetal lambs. The FAE of these rudimentary follicles, which are devoid of lymphocytes, showed normal differentiation, including carbonic anhydrase reactivity and ultrastructural characteristics of transcytosis, extensive interdigitation of the lateral plasma membrane and the shedding of membrane-bounded particles, approximately 50 nm in size, resembling exosomes. These 50-nm membrane-bounded particles were abundant in the extracellular space of the epithelium and the dome but no particles were found in the rudimentary follicles. This study confirms that the rudimentary follicles consist of clusters of follicular dendritic cells. Our findings suggest that the differentiation of FAE of ileal Peyers patch and the production of the 50-nm particles constitute features that appear to be independent of B-cells. This study was supported in part by grants from the Norwegian Research Council     

Ontogeny of reticular cells in the ileal Peyer's patch of sheep and goats.

The ontogeny of reticular cells in the ileal Peyer's patch of sheep from 70 days gestational age was studied by light and electron microscopy and by enzyme histochemistry. Small to medium-sized lymphocytes were seen in the lamina propria at 97 days, when the stroma was essentially still mesenchymal. By 110 days, the stromal cells in the dome/follicle primordia had differentiated into reticular fibroblasts, whose processes and fibers were seen to surround groups of lymphocytes. With advancing age the number and size of primordia increased, and proliferation was obvious among the lymphocytes. Processes of reticular cells increased in number and penetrated between individual lymphocytes of the groups. Coarser desmosome-like contacts were seen between the reticular cells from 115 days onwards. A central light area in the follicle was apparent from 130 days onwards. The fine structure of the stromal cells in this light follicle center developed towards but never became similar to that of follicular dendritic cells in a typical germinal center. The fine interdigitating end branches of the stromal cells were less numerous, and the dense homogeneous material present in between the end branches was not observed in the ileal Peyer's patch follicle. Instead, small particles and vesicles were seen between the various cell types of the light center and were not restricted to the intercellular spaces between the stromal cells. In the dark peripheral zone of the follicle, the stromal cells retained more immature features. The follicle became bordered by a capsule at an early stage. This capsule was formed by multiple layers of flattened fibroblasts separated by small amounts of intercellular material only. The alkaline phosphatase, Mg(2+)-dependent adenosine triphosphatase and 5' nucleotidase reactivities of the follicular dendritic cells in the ileal Peyer's patch were similar to those of early prenatal primary follicles of sheep lymph nodes. This study indicates that the stromal cells of the ileal Peyer's patch are mesenchymal in nature and different from those of germinal centers and the epithelial stromal cells of bursa Fabricii of birds.     

Uptake and transport of copolymer biodegradable microspheres by rabbit Peyer's patch M cells

In this study, we demonstrate the role of M cells in uptake of poly(D-L-lactic-co-glycolic acid) (PLGA) microspheres and transport into rabbit Peyer's patches. Microspheres 1 to 10 m in diameter composed of 50:50 lactic acid:glycolic acid were instilled into in-testinal segments containing jejunal or ileal Peyer's patches, and uptake by M cells was examined by electron microscopy. PLGA microspheres visualized as electron-lucent, spherical particles were taken up by M cells by pseudopod-like extensions of the M cell apical membrane and translocated to the pocket region containing mononuclear leukocytes within 60 min. These results indicate that PLGA microspheres can be directed to M cell apical surfaces for delivery to immunocompetent cells in gut-associated lymphoid tissues.     

IL-6 is a potent cofactor of IL-1 in IgM synthesis and of IL-5 in IgA synthesis

In these studies we determined the capacity of IL-6 to act as a differentiation cofactor for murine Peyer's patch B cells producing different Ig classes and subclasses. In preliminary studies we determined that sufficient endogenous IL-6 was produced in LPS-induced cell systems to obscure responses to exogenous IL-6. We therefore studied IL-6 effects on Peyer's patch B cells (T cell-depleted cell populations) in the absence of LPS, relying on responses of in vivo-activated cells. rIL-1 alpha or purified IL-6 only slightly enhanced synthesis of IgM over minimal baseline levels in Peyer's patch T cell-depleted cell cultures; however, when IL-6 was added to cultures also containing rIL-1, IgM synthesis was very substantially increased. In addition, rIL-5 alone gave rise to a modest increase in IgM synthesis and its effect was not enhanced by either rIL-1 or IL-6. IgG production (mainly IgG3) followed a similar pattern. In contrast, IgA production was only modestly increased above baseline by rIL-1, rIL-5, or IL-6 alone or by rIL-1 and IL-6 in combination, but was greatly increased by rIL-5 and IL-6 in combination. The effect of IL-6 on Ig synthesis in the above studies was not due to an effect on cell proliferation. In summary, these data indicate that B cells differ in respect to the cytokines supporting maximal terminal differentiation and thus the class of Ig produced may depend on the presence of a particular combination of cytokines and lymphokines.     

Selective adherence of IgA to murine Peyer's patch M cells: evidence for a novel IgA receptor

M cells represent the primary route by which mucosal Ags are transported across the intestinal epithelium and delivered to underlying gut-associated lymphoid tissues. In rodents and rabbits, Peyer's patch M cells selectively bind and endocytose secretory IgA (SIgA) Abs. Neither the nature of the M cell IgR nor the domains of SIgA involved in this interaction are known. Using a mouse ligated ileal loop assay, we found that monoclonal IgA Abs with or without secretory component, but not IgG or IgM Abs, bound to the apical surfaces of Peyer's patch M cells, indicating that the receptor is specific for the IgA isotype. Human serum IgA and colostral SIgA also bound to mouse M cells. The asialoglycoprotein receptor or other lectin-like receptors were not detected on the apical surfaces of M cells. We used recombinant human IgA1 and human IgA2 Abs and domain swapped IgA/IgG chimeras to determine that both domains Calpha1 and Calpha2 are required for IgA adherence to mouse Peyer's patch M cells. This distinguishes the M cell IgA receptor from CD89 (FcalphaI), which binds domains Calpha2-Calpha3. Finally, we observed by immunofluorescence microscopy that some M cells in the human ileum are coated with IgA. Together these data suggest that mouse, and possibly human, M cells express an IgA-specific receptor on their apical surfaces that mediates the transepithelial transport of SIgA from the intestinal lumen to underlying gut-associated organized lymphoid tissues.     

Analysis of T cell and B cell function in Peyer's patch and lamina propria of New Zealand Black and DBA/2 mice

We have analyzed gastrointestinal immune function in both DBA/2 and spontaneously autoimmune New Zealand Black (NZB) mice. We have studied both in vitro proliferation and differentiation of Peyer's patch cells and have measured immunoglobulin (Ig) secretion by cultured jejunal segments. Peyer's patch B cells and T cells from both DBA/2 and NZB mice showed similar proliferative responses to Con A and lipopolysaccharide (LPS), respectively. Unlike NZB splenic B cells, isolated Peyer's patch B cells from NZB mice did not spontaneously secrete Ig of any isotype. Seven-day cultures of equal numbers of Peyer's patch T cells and B cells resulted in similar patterns of secretion of IgA, IgG, and IgM in both strains. The addition of Con A to cultures of DBA/2 Peyer's patch cells consistently resulted in a onefold to threefold increase in IgA secretion after 7 days. Con A stimulation of NZB Peyer's patch cells did not produce any increment in IgA secretion. LPS stimulation of Peyer's patch cells from either strain resulted in a similar increase in IgG secretion with little effect on IgA secretion. The in vivo correlate of this finding was seen in the IgA to IgG ratio of Ig secreted by cultured jejunal fragments. In DBA/2 mice the rates of IgA/IgG varied from 2.36 to 4.85, whereas in NZB mice the ratio never exceeded 0.5. These experiments show that defects on the T cell compartment of NZB mice encompass gut-associated lymphoid tissue. The possible relationship of these findings and previously observed defects in oral tolerance is discussed.     

Export of virulence genes and Shiga toxin by membrane vesicles of Escherichia coli O157:H7

Membrane vesicles released by Escherichia coli O157:H7 into culture medium were purified and analyzed for protein and DNA content. Electron micrographs revealed vesicles that are spherical, range in size from 20 to 100 nm, and have a complete bilayer. Analysis of vesicle protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates vesicles that contain many proteins with molecular sizes similar to outer membrane proteins and a number of cellular proteins. Immunoblot (Western) analysis of vesicles suggests the presence of cell antigens. Treatment of vesicles with exogenous DNase hydrolyzed surface-associated DNA; PCR demonstrated that vesicles contain DNA encoding the virulence genes eae, stx1 and stx2, and uidA, which encodes for beta-galactosidase. Immunoblot analysis of intact and lysed, proteinase K-treated vesicles demonstrate that Shiga toxins 1 and 2 are contained within vesicles. These results suggest that vesicles contain toxic material and transfer experiments demonstrate that vesicles can deliver genetic material to other gram-negative organisms.     

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.     

Lacrimal gland-directed B cell responses

Although it is accepted that IgA plasma cells predominate in the lacrimal gland, the factors leading to this prevalence are not known. A series of 4-day LPS-driven co-culture experiments performed with dissociated lacrimal gland and lymphoid cell populations was employed to study the direct effect of lacrimal gland cells on B cell differentiation. Lacrimal gland cells, when co-cultured with spleen or mesenteric lymph node cells, were found to suppress differentiation of cells to IgA, IgG, and IgM production. Furthermore, suppression of IgG and IgM responses occurred after co-culture of lacrimal gland cells with Peyer's patch cells. However, these Peyer's patch co-cultures led to a stimulation of the IgA response, a condition that was abrogated by removal of Peyer's patch T cells before co-culturing. Pretreatment of lacrimal gland cells with mitomycin C eliminated the suppression and stimulation previously observed. These results demonstrate the effects of lacrimal gland, both directly and indirectly through T cells, on B cell differentiation. These findings explain in part the preferential accumulation of IgA-plasma cells within the gland.     

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.     

Export of Virulence Genes and Shiga Toxin by Membrane Vesicles of Escherichia coli O157:H7

Membrane vesicles released by Escherichia coli O157:H7 into culture medium were purified and analyzed for protein and DNA content. Electron micrographs revealed vesicles that are spherical, range in size from 20 to 100 nm, and have a complete bilayer. Analysis of vesicle protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates vesicles that contain many proteins with molecular sizes similar to outer membrane proteins and a number of cellular proteins. Immunoblot (Western) analysis of vesicles suggests the presence of cell antigens. Treatment of vesicles with exogenous DNase hydrolyzed surface-associated DNA; PCR demonstrated that vesicles contain DNA encoding the virulence genes eae, stx1 and stx2, and uidA, which encodes for β-galactosidase. Immunoblot analysis of intact and lysed, proteinase K-treated vesicles demonstrate that Shiga toxins 1 and 2 are contained within vesicles. These results suggest that vesicles contain toxic material and transfer experiments demonstrate that vesicles can deliver genetic material to other gram-negative organisms.     

cDNA genes formed after infection with retroviral vector particles lack the hallmarks of natural processed pseudogenes.

Retroviral proteins can encapsidate RNAs without retroviral cis-acting sequences. Such RNAs are reverse transcribed and inserted into the genomes of infected target cells to form cDNA genes. Previous investigations by Southern blot analysis of such cDNA genes suggested that they were truncated at the 3' and the 5' ends (R. Dornburg and H. M. Temin, Mol. Cell. Biol. 8:2328-2334, 1988). To analyze such cDNA genes further, we cloned three cDNA genes (derived from a hygromycin B phosphotransferase gene) in lambda vectors and analyzed them by DNA sequencing. We found that they did not correspond to the full-length mRNA: they were truncated at both the 3' and the 5' ends, did not contain a poly(A) tract, and were not flanked by direct repeats. The 3'-end junctions to chromosomal DNA of five more cDNA genes were amplified by polymerase chain reaction, cloned in pUC vectors, and sequenced. All of these cDNA genes had 3'-end truncations, and no poly(A) tracts were found. Further polymerase chain reaction experiments were performed to detect hygromycin B phosphotransferase cDNA genes with a poly(A) tract in DNA extracted from a pool of about 500 colonies of cells containing cDNA genes. No hygromycin B phosphotransferase cDNA gene with a poly(A) tract was found. Investigation of two preintegration sites by Southern analysis revealed that deletions were present in chromosomal DNA at the site of the integration of the cDNA genes. Naturally occurring processed pseudogenes correspond to the full-length mRNA, contain a poly(A) sequence, and are flanked by direct repeats. Our data indicate that cDNA genes formed by infection with retrovirus particles lack the hallmarks or natural processed pseudogenes. Thus, it appears that natural processed pseudogenes were not generated by retrovirus proteins.     

Cytopathogenic effect of Salmonella typhi GIFU 10007 on M cells of murine ileal Peyer's patches in ligated ileal loops: an ultrastructural study

An electron microscopic study revealed that, within 30 min after inoculation into the ligated ileal loop of anesthetized mice, cells of Salmonella typhi GIFU 10007 adhered to the M cell surface of Peyer's patch lymphoid follicle epithelium, and induced almost complete destruction of M cells. The M cell cytoplasms were pinched off and extruded from the epithelial lining into the luminal space together with the lymphoid cells primarily enfolded into the corresponding M cells. When two or more M cells were destroyed, a large defect in the epithelial lining was apparent, and a number of bacteria appeared near the basal lamina of the epithelial lining. These findings suggest, as far as anesthetized murine ileal loops and strain 10007 are concerned, that ileal M cells are the target cell at an early stage of S. typhi infection and the infection may further progress to deeper tissues and to the general circulation.     

Retention of ingested latex particles in Peyer's patches of germfree and conventional mice

Conventional and germfree mice ingested a suspension of 2-micron latex particles in drinking water for a 15-day period. Number and distribution of intestinal Peyer's patches did not differ significantly in the two types of mice. Cleared Peyer's patches were compared with regard to size and particle content. The location of particles within Peyer's patch follicles of germfree mice was similar to that of conventional mice, but the latter had significantly larger follicles and greater accumulations of latex particles. Latex concentration varied with patch location. Proximal patches contained the majority of particles in germfree mice, whereas particles were most abundant in distal patches of conventional mice. The results show that particle uptake into Peyer's patches takes place even in the complete absence of bacteria in the gut.     

Isotype-specific immunoregulation; characterization and function of Fc receptors on T-T hybridomas which produce murine IgA-binding factor

Several methods have been used in the present study to characterize Fc receptors (FcR) expressed on T-T hybridomas derived from mouse Peyer's patch T helper (Th) cell clones that preferentially support IgA responses. These T hybridomas (designated Th HA cells) produce IgA-binding factor (IBF alpha) which regulates antigen-dependent IgA responses. The ultrastructure of Th HA cells and the distribution of Fc alpha R on these cell lines were determined by colloidal gold (CG) immunoelectron microscopy (IEM). When Th HA cells were incubated with purified mouse IgA followed by CG-labeled anti-IgA, an even pattern of CG was distributed on the cell membrane. To ensure that binding occurred through Fc alpha R, Th HA cells were mixed with MOPC 315 IgA anti-DNP, followed by staining with CG-labeled TNP-human serum albumin. This resulted in an identical pattern of gold particle distribution, confirming expression of Fc alpha R on Th HA cells. No Fc mu R or Fc gamma 1R were detectable on Th HA cells by IEM. Immunocytoadherence with TNP-conjugated erythrocytes confirmed that Th HA cells were Fc alpha R+; however, no IgM or IgG rosettes were seen. When these cell lines were analyzed by flow cytometry (FACS) using IgA, IgM, or IgG1 and FITC-labeled anti-H chain-specific antibodies, 55 to 65% of cultured Th HA cells expressed Fc alpha R, and 11 to 18% expressed Fc mu R; however, no Fc gamma 1R was detectable on Th HA cells. The use of ELISA with Th HA cells as antigen confirmed the expression of Fc alpha R and the presence of less Fc mu R on these two cell lines. Solubilized membrane fractions derived from Th HA cells were tested for the presence of FcR by ELISA and for biologic function for support of IgA responses in Peyer's patch B cell cultures. Both Fc alpha R and Fc mu R were detected in fractions derived from Th HA cells. Furthermore, these fractions supported in vitro IgA anti-sheep erythrocyte responses, comparable to those obtained with Th HA cell culture supernatants containing IBF alpha. These studies show that Th HA cells express Fc alpha R with less Fc mu R, and the solubilized form of Fc alpha R exhibits IBF alpha-like activity. The significance of FcR expression by Th cell clones and cell lines and the relationship of soluble Fc alpha R and IBF alpha for IgA response regulation are discussed.     

cDNA representational difference analysis of ileal Peyer's patches in lambs after oral inoculation with scrapie

cDNA representational difference analysis (RDA) was used to study gene expression profiles in the ileal Peyer's patch of a lamb 1 week after oral inoculation with the scrapie agent. Twenty-five differentially expressed cDNA fragments were identified and cloned. Sequence analysis indicated seven novel gene sequences. Other clones shared sequence homology with genes encoding ribosomal and mitochondrial proteins, the translation initiation factor EIF4GII and the bovine pancreatic thread protein. Reverse Northern was used to confirm the differential expression in another four lambs inoculated with scrapie and the tissue distribution of the novel genes was examined using Northern blot analysis.     

Response of Peyer's patch lymphocyte subsets to Giardia muris infection in BALB/c mice. I. T-cell subsets

Initial cellular events in the intestinal immune response occur within Peyer's patches and are subject to complex regulation by T cells. The aim of this study was to analyze the response of murine Peyer's patch T, T-helper (Th), and T-suppressor (Ts) cells to Giardia muris infection. Immunocompetent BALB/c mice were infected with G. muris cysts and, at serial times during the infection, Peyer's patch leukocyte suspensions were incubated with fluorescent monoclonal antibodies that identified murine leukocytes, T, Th, or Ts lymphocytes. These suspensions were examined by flow cytometry to quantify each T-cell subset as a percentage of total leukocytes. Total Peyer's patch leukocytes more than doubled in number during the course of G. muris infection and returned to control levels as the infection was cleared. The percentages of Peyer's patch Th and Ts lymphocytes were 34.1 +/- 0.8% (mean +/- SEM) and 6.2 +/- 0.3%, respectively, in the absence of infection, and did not change significantly during Giardia infection. The Th/Ts ratio in Peyer's patches was 5.6 +/- 0.2 in uninfected BALB/c mice and also did not change significantly during clearance of G. muris. We conclude that Peyer's patch leukocytes double in number in response to G. muris infection in immunocompetent mice, G. muris infection does not lead to altered percentages of Peyer's patch T, Th, or Ts lymphocytes, and clearance of G. muris infection is associated with a Peyer's patch Th/Ts ratio of greater than 5.