Rabbit Peyer's Patches Research Articles

IMMUNOHISTOCHEMICAL CHARACTERISATION OF LYMPHOID POPULATIONS IN RABBIT JEJUNAL PEYER’S PATCH

Rabbits are an important livestock animal species, which are used for their meat and fur. Nowadays they are also becoming more popular as pets. Furthermore, rabbits are commonly used in research, inter alia in immunological studies and for studying pathogenesis of human and animal diseases. The lymphoid tissue is abundant in the rabbit intestine and a lot of it is concentrated in Peyer's patches, the majority of which is located in the jejunum. Understanding of the rabbit Peyer's patches functions is essential for the prevention and treatment of their diseases. In order to enhance it, accurate knowledge of its lymphocyte population composition is needed. At present, the cellular composition of the rabbit gut-associated lymphoid tissue remains insufficiently studied. Therefore, the aim of our study was to establish the content and localization of lymphoid populations in the domestic rabbit jejunal Peyer’s patches. We performed immunohistochemical studies using monoclonal antibodies CD3, CD10 and CD20 (DAKO, Denmark) to identify the distribution of lymphoid populations in jejunal Peyer's patches of a mature domestic rabbit. The results, obtained in our study, complement the existing data and determine the main lymphoid populations in the rabbit jejunal Peyer's patches. The presented data are fundamental for further studies of the rabbit gut-associated lymphoid tissue. We found that lymphoid nodules of jejunal Peyer's patches contains a predominant CD20+ cell population (B cells). It also contains many CD10+ cells (precursors of T cells and B cells), which are mostly concentrated in the germinal centers of lymphoid nodules. In the diffuse form of the lymphoid tissue the vast majority of cells are CD3+ cells (T cells)

Localization of arabinogalactan-proteins (AGP) in aerial parts of Echinacea purpurea and of AGP binding sites in rabbit Peyer's patches by immunofluorescence

Localization of arabinogalactan-proteins (AGP) in aerial parts of Echinacea purpurea and of AGP binding sites in rabbit Peyer's patches by immunofluorescence

Comparison of Poly(dl-Lactide-co-glycolide) and Polystyrene Microsphere Targeting to Intestinal M Cells

The interaction of poly(dl-lactide-co-glycolide) (PLG) and polystyrene microspheres with the follicle-associated epithelium (FAE) of rabbit Peyer's patches was compared. Binding by PLG microspheres to the FAE was an order of magnitude lower than that of polystyrene microspheres of equivalent size (0.5–0.6 μm diameter). Although PLG microspheres, are not selectively targeted to the M cell surface, as is the case with polystyrene microspheres, a high proportion of those that bind to M cells are transcytosed, resulting in the transepithelial delivery of 1.5×104 PLG microspheres/mm2 FAE. This represents the first direct demonstration of transepithelial delivery of PLG microspheres by M cells, which is crucial to the potential use of such vehicles for the oral delivery of drugs and vaccines. As native PLG microspheres are not optimally targeted to the M cell surface, there is scope for the further improvement of their efficacy by surface modification.

Tacrolimus (FK506) and cyclosporine reduce the uptake and transport of particles into rabbit Peyer's patches.

Tacrolimus (FK) and cyclosporine (Cs) are potent immunosuppressants that effectively prevent the rejection of transplanted organs including liver and small intestine. Our study examined the effects of these immunosuppressants on Peyer's patches, which play an important role in mucosal immune response through uptake and transport of enteric microorganisms and macromolecules in the gut-associated lymphoid tissues. After administration of FK and Cs, we assessed changes in lymphoid follicle structure and quantified the uptake and transport of particles in the follicle associated epithelium (FAE) including M cells, using fluorescent latex microspheres in rabbit Peyer's patches. Rabbits, five in each group, received oral administration of FK (3.2 mg/kg), Cs (10 mg/kg), or phosphate-buffered saline daily for 7 days. After 2 days of withdrawal, rabbits were anesthetized, and received injections with 2 ml of the suspension of 0.5-microm fluorescent microspheres (1010/ml) into ligated intestinal segments containing Peyer's patches. After 2 hr of gentle agitation, segments were removed, rinsed, fixed with periodate-lysine-2% paraformaldehyde, frozen, and sections were stained with fluorescent phalloidin to label brush border actin filaments. The size of the lymphoid follicles in each group was measured under a light microscope. The number of microspheres in follicles was assessed in graphically defined areas of follicles from each group. In addition, immunohistochemical analysis of CD43 and MHC-II positive cells in FAE of lymphoid follicles of each group was performed. FK and Cs significantly reduced the height of lymphoid domes and the height and width of follicles, as compared to those of controls. In both FK and Cs groups, the numbers of microspheres that adhered, were taken up and were transported into lymphoid follicles were smaller than in controls, indicating that their movement rates into deep layers were markedly reduced. Furthermore, FK and Cs reduced the mean numbers of CD43 and MHC-II positive cells in FAE per unit area (mm2) as compared with controls. These findings suggest that FK and Cs may produce immunosuppressive effects, at least in part, through reduction of the uptake and transport of particles into Peyer's patches, and by reduction of the number of immunoreactive cells in FAE of Peyer's patches.

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.

Up-regulation of microsphere transport across the follicle-associated epithelium of Peyer's patch by exposure to Streptococcus pneumoniae R36a.

Transport of antigens through the follicle-associated epithelium (FAE) of Peyer's patch (PP) is the critical first step in the induction of mucosal immune responses. We have previously described that short-term exposure to Streptococcus pneumoniae R36a induced dramatic morphological alterations of the FAE in rabbit PP. These results prompted us to investigate whether the pneumococci-induced modifications were accompanied by enhanced ability of the FAE to transport antigens. We addressed this problem by evaluating the ability of the FAE to bind, internalize, and transport fluorescent polystyrene microparticles, highly specific to rabbit M cells, after exposure to S. pneumoniae. Quantitative study revealed a marked increase in the number of microspheres in PP tissues exposed to S. pneumoniae compared to tissues exposed to either phosphate-buffered saline or Escherichia coli DH5alpha as controls. No sign of bacterially induced damage to the epithelial barrier was observed. Further confocal microscopy analysis of the FAE surface showed that a significant increase in the number of cells that showed both morphological and functional features of M cells took place within pneumococci-treated PP tissues. These data provide the first direct evidence that the FAE-specific antigen sampling function may be manipulated to improve antigen and drug delivery to the intestinal immune system.

Identification of a phosphopeptide in bovine alpha s1-casein digest as a factor influencing proliferation and immunoglobulin production in lymphocyte cultures.

Digestion of bovine alpha s1-casein with bovine trypsin produced peptide(s) with an inhibitory effect on concanavalin A-induced proliferation of mouse spleen cells. One of these peptides was isolated from the alpha s1-casein digest following ultrafiltration, hydroxyapatite chromatography and reversed-phase HPLC, and amino acid composition and sequence analyses showed it to be sequence 59-79 from the phosphoserine-rich region of alpha s1-casein. The isolated peptide significantly inhibited the concanavalin A-induced proliferation of mouse spleen cells and rabbit Peyer's patch cells, whereas it enhanced the lipopolysaccharide- and phytohaemagglutinin-induced proliferation of both cells. The peptide displayed mitogenic activity in the cell cultures without the commercial mitogen, and significantly enhanced immunoglobulin production. Moreover, residues 1-25 from the phosphoserine-rich region of bovine beta-casein had a similar effect on the proliferation of mouse spleen cells and rabbit Peyer's patch cells stimulated or not stimulated by the commercial mitogen. These results indicate that caseinophosphopeptides may act as a humoral immunostimulator in cell cultures.

Mucosal and systemic antibody responses to a C4/V3 construct following DNA vaccination of rabbits via the Peyer's patch.

A plasmid encoding T1-SP10MN(A), a peptide derived from immunodominant regions of human immunodeficiency virus type 1 gp120, was delivered to rabbit Peyer's patches using a helium-driven gene gun. Six weeks thereafter, 2 of 5 animals were given an intradermal booster immunization. Blood, feces, and vaginal washes were collected weekly and assayed by ELISA. High titer T1-SP10MN(A)-specific fecal and vaginal secretory IgA responses were observed, and the response appeared to be augmented following dermal booster immunizations. Specific serum IgG was also detected within 1 week of immunization and remained elevated through week 20 in the 2 animals receiving dermal boosts (titers > or = 6400). This study establishes the Peyer's patch as a promising target tissue for DNA vaccination and demonstrates the efficacy of gene gun-mediated delivery of foreign DNA to a mucosal tissue for the induction of an immune response.

Where do M cells go? identification of their desquamation sites in rabbit peyer's patches and appendix by immunohistochemical labeling of M cell cytoplasmic vimentin

Where do M cells go? identification of their desquamation sites in rabbit peyer's patches and appendix by immunohistochemical labeling of M cell cytoplasmic vimentin

Immunohistochemical and antigen uptake studies in rabbit palatine tonsils and peyer's patches

Immunohistochemical and antigen uptake studies in rabbit palatine tonsils and peyer's patches

Increase of polypeptide transport by lectin conjugation across rabbit Peyer's patches

The transport of bovine serum albumin (BSA) across Peyer's patches (PP) was increased by conjugate synthesis with lectin concanavalin A (Con A). The transport of the intact form was increased by the conjugation and significantly reduced by colchicine. The increase is thought to be due to the endocytosis activity of PP in response to lectin Con A. The transport of the conjugate across the jejunal epithelium (JE) was also increased, and it was concluded that the increase was due to the increase in stability against enzymatic degradation. These results suggest that conjugate synthesis with lectin is an effective method to improve the poor absorption of polypeptides and protein drugs in the intestine.

In vivo apoptosis in Shigella flexneri infections.

Shigella flexneri, an etiological agent of bacillary dysentery, causes apoptosis in vitro. Here we show that it also induces apoptosis in vivo. We were able to quantify the number of apoptotic cells in rabbit Peyer's patches infected with S. flexneri by detecting cells with fragmented DNA. Infection with virulent S. flexneri results in massive numbers of apoptotic cells within the lymphoid follicles. In contrast, neither an avirulent strain nor an avirulent strain capable of colonizing Peyer's patches increases the background level of apoptotic cells. Macrophages, T cells, and B cells are shown to undergo apoptosis in vivo. These results indicate that apoptosis may play a crucial role in the pathogenesis of shigellosis.

Modifications of the follicle-associated epithelium by short-term exposure to a non-intestinal bacterium.

This study was undertaken in order to study the effects of short-term exposure of the follicle-associated epithelium (FAE) of rabbit Peyer's patches to a non-intestinal, Gram-positive bacterium. Isolated ileal loops, each containing one Peyer's patch (PP), were stimulated for short periods of time (30 and 60 min) with Streptococcus pneumoniae R36a, a micro-organism normally not present in the intestinal area. Samples from antigen-stimulated and control Peyer's patches were analysed by light (LM), transmission electron (TEM), and scanning electron microscopy (SEM). Stimulation with living pneumococci induced dramatic changes in FAE architecture and morphology. A massive passage of cells from lymphoid tissue to the FAE was rapidly detectable, accompanied by alterations of the FAE surface, with a marked increase of M-cell area. Furthermore, TEM analysis revealed that M cells were able to internalize living pneumococci. S. pneumoniae R36a is a valid experimental model for the further study of the unique antigen sampling function which characterizes the highly specialized FAE in Peyer's patches.

Role of the glycocalyx in regulating access of microparticles to apical plasma membranes of intestinal epithelial cells: implications for microbial attachment and oral vaccine targeting.

Transepithelial transport of antigens and pathogens across the epithelial barrier by M cells may be a prerequisite for induction of mucosal immunity in the intestine. Efficient transport of antigens and pathogens requires adherence to M cell apical surfaces. Coupling of antigen-containing particles to the pentameric binding subunit of cholera toxin (CTB) has been proposed as a means for increasing antigen uptake because the CTB receptor, ganglioside GM1, is a glycolipid present in apical membranes of all intestinal epithelial cells. To test the accessibility of enterocyte and M cell membrane glycolipids to ligands in the size ranges of viruses, bacteria, and particulate mucosal vaccines, we analyzed binding of CTB probes of different sizes to rabbit Peyer's patch epithelium. Soluble CTB-fluorescein isothiocyanate (diameter 6.4 nm) bound to apical membranes of all epithelial cells. CTB coupled to 14 nm colloidal gold (final diameter, 28.8 nm) failed to adhere to enterocytes but did adhere to M cells. CTB-coated, fluorescent microparticles (final diameter, 1.13 microns) failed to adhere to enterocytes or M cells in vivo or to well-differentiated Caco-2 intestinal epithelial cells in vitro. However, these particles bound specifically to GM1 on BALB/c 3T3 fibroblasts in vitro and to undifferentiated Caco-2 cells that lacked brush borders and glycocalyx. Measurements of glycocalyx thickness by electron microscopy suggested that a relatively thin (20 nm) glycocalyx was sufficient to prevent access of 1-micron microparticles to glycolipid receptors. Thus, the barrier function of the intestinal epithelial cell glycocalyx may be important in limiting microbial adherence to membrane glycolipids, and in CTB-mediated targeting of vaccines to M cells and the mucosal immune system.

Infection of rabbit Peyer's patches by Shigella flexneri: effect of adhesive or invasive bacterial phenotypes on follicle-associated epithelium.

In order to invade the colonic mucosa, the bacterial pathogen Shigella flexneri must find a site of entry. Experiments with the rabbit ligated intestinal loop model described here confirm that M cells of the follicle-associated epithelium (FAE) that covers lymphoid structures of the Peyer's patches represent a major site of entry for invasive microorganisms. In addition, in an isogenic Shigella background, expression of an adhesive phenotype, or of an invasive phenotype, is required for bacteria to efficiently colonize the FAE. A nonadhesive, noninvasive mutant barely interacted with FAE. Adhesive and invasive strains induced dramatic but different alterations on FAE. Invasive strain M90T caused major inflammation-mediated tissue destruction after 8 h of infection. Adhesive strain BS15 caused limited inflammation, but major architectural changes, characterized by an increase in the size of M cells that became stretched over large pockets containing an increased number of mononuclear cells, were observed. M cells progressively occupied large surface areas of the FAE at the expense of enterocytes. This contributed to enterocytes losing contact with the lumen. These experiments demonstrate that various remodeling patterns may occur in Peyer's patches in response to bacterial pathogens, depending on the virulence phenotype expressed by the pathogenic strain.

A simplified procedure for studies of intestinal immunity in rabbits

As interest in the development of oral vaccines continues to rise, alternative animal models for studies of mucosal immunity are needed. The present study examines a simplified procedure for delivering antigen to rabbit Peyer's patches via an indwelling cannula. The cannula was placed 3–4 cm proximal to the Peyer's patch, and was used to deliver four weekly doses of the potent mucosal immunogen, cholera toxin (CT). Anti-CT specific fecal secretory IgA (S-IgA), serum IgG and serum IgA were found in essentially equal amounts in rabbits with cannulas and in rabbits fitted with Thiry-Vella (T-V) isolated ileal loops. In contrast to animals with T-V loops, the intestinal flora of animals with cannulas contained less bacterial overgrowth with Pseudomonas sp. Further, the villus architecture remained histologically normal in appearance and there were fewer post-surgical complications associated with this technique than with T-V loops. This simplified technique should allow wider use of rabbits in studies of mucosal immunity.

Three-dimensional (3D-) reconstruction of M cells in rabbit Peyer's patches: definition of the intraepithelial compartment of the follicle-associated epithelium.

One of the major cell components of the rabbit follicle-associated epithelium is represented by the M cells. M cells are able to harbour variable amounts of immunocompetent cells inside peculiar invaginations of their basolateral cytoplasmic membrane, currently referred to as "pockets." This study provides a description of the exact spatial relationships between the M cells and the cells harboured in these so-called "pockets." Pieces of Peyer's patches, taken from the small intestine of an adult male rabbit, were treated as usual for conventional electron microscopy. Consecutive semithin and ultrathin sections were made through the entire thickness of the follicle-associated epithelium along planes parallel to the mucosal surface. Micrographs, taken from the ultrathin sections, were transposed into a software MacDraw Pro to obtain a computerized three-dimensional reconstruction. Three-dimensional reconstruction of the M cells showed that the "pockets" were not formed by mere invaginations of the cytoplasmic membrane, but that they resulted from the branching of the supranuclear portion of the M cell cytoplasm around the M cell-infiltrating lymphocytes. These intrusive cells could be found inside the "pockets" or lined up with one another, in vertical columns, bordering on the basal aspect of the M cells. The particular arrangement of the M cell apical cytoplasm created a labyrinth within the follicle-associated epithelium, which could be assumed as a real intraepithelial compartment expandable virtually throughout all the epithelium. The functional meaning of the intraepithelial compartment delimited by the M cells and its possible role is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

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 lectin Euonymus europaeus differentially stained the surface of M-cells in both mouse Peyer's and caecal patches, and the lectins Ulex europaeus II and Bandeiraea 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 both Euonymus europaeus and Ulex 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.

Inhibition of proliferative responses of mouse spleen lymphocytes and rabbit Peyer's patch cells by bovine milk caseins and their digests

The modulating effect of bovine milk casein components and their digests on the proliferative responses of mouse spleen lymphocytes and rabbit Peyer's patch cells induced or not induced by mitogens has been studied with a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. All the casein components and their digests tested had little mitogenic effect on the proliferative responses of mouse spleen lymphocytes and rabbit Peyer's patch cells. Intact kappa-casein significantly inhibited the proliferative responses of mouse spleen lymphocytes and Peyer's patch cells induced by mitogens such as lipopolysaccharide from Salmonella typhimurium, concanavalin A, phytohaemagglutinin and pokeweed mitogen. In contrast, intact alpha s1-casein and beta-casein had little effect. kappa-Casein had an inhibitory effect after digestion by pancreatin or trypsin, but not after pepsin or chymotrypsin digestion. Both pancreatin and trypsin digests of alpha s1-casein and beta-casein significantly inhibited the proliferative responses of mouse spleen lymphocytes and rabbit Peyer's patch cells induced by mitogens, whereas pepsin and chymotrypsin digests of both caseins were without effect. Moreover, the trypsin digest of each casein component had an inhibitory effect on mouse spleen lymphocyte proliferation in the absence of mitogen. Since trypsin is a major proteinase in pancreatin, the substrate specificity of trypsin seems to be important for the formation of the inhibitory peptides from casein components. These observations suggest that intact kappa-casein and some peptides formed from milk casein components by the action of trypsin may suppress the immune responsiveness of neonates.

Inhibition of mitogen-induced proliferative responses of lymphocytes by bovine kappa-caseinoglycopeptides having different carbohydrate chains.

Bovine kappa-caseinoglycopeptides (i.e. residues 106-169, CGP) were prepared from kappa-casein digested with rennin and a commercial whey protein concentrate. CGP from whey protein concentrate was further divided into seven CGP fractions having different carbohydrate compositions using FPLC. Unfractionated CGP inhibited lipopolysaccharide (LPS)- and phytohaemagglutinin (PHA)-induced proliferative responses of mouse spleen cells and rabbit Peyer's patch cells. The unfractionated CGP also inhibited antibody responses to sheep red blood cells in mouse spleen cell cultures. However, seven CGP fractions having zero to five N-acetylneuraminic acid (NANA) residues had different inhibitory effects on both LPS- and PHA-induced proliferative responses of mouse spleen cells. The inhibitory effect on PHA-induced proliferative responses increased with increasing numbers of NANA residues, whereas that on LPS-induced proliferation was highest with the CGP fraction having two NANA residues. Both inhibitory effects decreased significantly after neuraminidase or chymotrypsin digestion. These findings indicate that both the carbohydrate (particularly the NANA residues) and the polypeptide portions are essential for inhibitory effects on LPS- and PHA-induced proliferative responses of mouse spleen cells.