Mastocitos en placa de Peyer de ratón / Mast cells in mouse's Peyer patches

Los mastocitos son células que participan en actividades de hipersensibilidad inmediata y tardía, inmunorregulación e inflamación. Recientemente se han detectado dos grupos de ellos: a) los que se localizan en tejido conjuntivo y b) los de la mucosa intestinal. Entre ellos hay diferencias morfológicas y funcionales. Los de mucosa intestinal son T dependientes y proliferan durante las parasitosis así como en los procesos de hipersensibilidad intestinal a diversos antígenos. Las placas de Peyer (PP) son los sitios principales donde se captan antígenos e inician las respuestas inmunitarias en el intestino, por lo que en este trabajo investigamos la relación morfológica entre las células de las PP y los mastocitos. Las PP de segmentos proximal, medial y distal del intestino delgado de ratones Balb/c se procesaron histológicamente, tiñeron con azul de toluidina y cuantificaron los mastocitos en las diferentes capas del intestino. Los datos se analizaron mediante una prueba de doble T pareada para diferencia de medios. Se observó mayor cantidad de mastocitos en la zona marginal de la PP en comparación con las capas: muscular resistente, submucosa y corion. La abundancia de mastocitos en relación con la PP sugiere que probablemente tenga influencia moduladora sobre la unción de las células linfoides de la PP

Pigment resembling atmospheric dust in Peyer's patches.

Terminal ileal pigmentation was observed during colonoscopy, in surgically resected specimens, and autopsy cases. Microscopically, black pigment was seen within macrophages in the lamina propria and submucosa, closely related to the Peyer's patches. Three ilia from autopsies with no macroscopic pigmentation showed deposits following digestion and X-ray microanalysis. X-ray microanalysis of tissue sections and digestates revealed a heterogenous population of particles. Approximately one third of the particles contained calcium and phosphorus and were considered endogenous. The rest of the particles were predominantly aluminum and magnesium-rich silicates, which were considered exogenous. Analysis of particulate extracted from lungs and ilea of four autopsy cases demonstrated remarkable similarities in composition. These findings suggest that the ileal deposits are derived from atmospheric dust. This pigment is believed to migrate into the Peyer's patches through the M cells of the follicle associated epithelium, although other mechanisms for pigment deposition cannot be ruled out.

Neutralization of cholera toxin by rat IgA secretory antibodies induced by a free synthetic peptide.

Secretory immunoglobulin A (sIgA) is the major immunoglobulin in the bile of several species. They contribute to local immune defences of the gut. The protection against cholera toxin (CT) is due to the presence of specific sIgA in the bile and in the gut. We have already reported that oral administration of the peptide corresponding to the sequence 50-75 of cholera toxin B subunit elicits serum antibodies neutralizing CT activity, and that IgA and local protection are observed in the intestine of P50-75 orally immunized mice. In this study, we demonstrate the potential of this synthetic peptide as immunogen without carrier or adjuvant, not only in a strain known to be sensitive to CT, but also in an outbred one. Furthermore, this peptide stimulates the mucosal immunity, since we show that P50-75 induced-sIgA purified from rats bile and serum, are capable of neutralizing CT activity in the in vivo intestinal ligated loop test.

Human lamina propria lymphocytes bear homing receptors and bind selectively to mucosal lymphoid high endothelium.

It has been hypothesized that the selective recognition of tissue-specific endothelial cell molecules helps determine the in vivo distribution of lymphoid effector cells by controlling the extravasation of their circulating precursors. Here we report (a) immunofluorescence studies of the cell surface phenotype of human lamina propria lymphocytes (LPL), including staining with monoclonal antibody Hermes-1, which defines a 90-kDa lymphocyte surface glycoprotein involved in recognition of high endothelial venules (HEV); and (b) functional analyses of the ability of LPL to bind to HEV in frozen sections of mucosal lymphoid tissues (appendix or Peyer's patch) vs. peripheral lymph nodes. Essentially all LPL bear the Hermes-1 antigen, over 90% at levels comparable to those of circulating PBL. As a population, LPL display a quantitative preference for adherence to mucosal HEV, binding 0.8-1.5 times as well as PBL to mucosal HEV, but only 0.1-0.5 times as well to HEV in peripheral lymph nodes. Of particular interest was the behavior of the lymphoblast fraction, which typically constituted 3-7% of LPL. These cells, defined by size, consisted of a mixture of T cells and surface IgA+ blasts. One hundred percent were Hermes-1 bright, and they bound 4-8 times more efficiently to mucosal HEV than PBL while failing to bind detectably to lymph node HEV. LPL binding to mucosal HEV involves the gp90 Hermes, since the monoclonal anti-gp90 antibody, Hermes-3, and a polyclonal anti-gp90 antiserum inhibit the binding of small LPL and of LP blasts. The remarkable efficiency and specificity of binding by LP blasts may reflect retention of homing properties of the blood-borne precursors of these blasts and is discussed in relation to the capacity of immunoblasts in mesenteric nodes and in thoracic duct lymph to traffic selectively to mucosal lymphoid and extralymphoid sites. The demonstration of organ-specific endothelial cell recognition by LP lymphoblasts provides considerable support for the concept that selective interactions with endothelium play an important role in directing the distribution of activated lymphocyte subsets in vivo.

Immunogenicity of Bifidobacterium breve and change in antibody production in Peyer's patches after oral administration.

Immunogenicity in the intestine of Bifidobacterium breve, included in fermented milk, was compared with that of Bacteroides thetaiotaomicron, also predominant in human intestine. In vivo, serum antibody to B. breve was detected first in mice fed the organism for 33 d; antibody decreased in mice fed these for more than 33 d. Serum antibody to Bact. thetaiotaomicron was detected in mice fed the organism for 7 d and was maintained at the same level in mice fed these for more than 7 d. From in vitro tests, the optimal doses of B. breve and Bact. thetaiotaomicron to induce antibody production by Peyer's patch cells, intestinal lymphoid tissue cells, were 5 x 10(8) and 5 x 10(7) bacteria/ml, respectively. Therefore, it was suggested that immunogenicity of B. breve is weaker than that of Bact. thetaiotaomicron. Furthermore, the change of antibody production to the organism by Peyer's patch cells in the mice administered B. breve orally was tested by the Peyer's patch cell culture method. Antibody production against B. breve by Peyer's patch cells in mice given B. breve for 25 and for 33 d increased and decreased, respectively, in comparison with the control. These results suggest that when serum antibody to B. breve increases significantly, anti-B. breve antibody production by Peyer's patch cells is suppressed, and thereafter, serum antibody to B. breve decrease and is not detected. These findings favor the view that serum antibody production to B. breve is regulated in Peyer's patches.

Detection of IL-1 alpha and IL-1 beta gene expression by in situ hybridization. Tissue localization of IL-1 mRNA in the normal C57BL/6 mouse.

IL-1 is a cytokine with a wide variety of effects on cells involved in inflammatory and immune responses, hemopoiesis, and bone formation. Many cell types have been shown to produce IL-1 in vitro; however, very little is known about the source and role of IL-1 in vivo. By using in situ hybridization, we examined the tissue distribution of cells containing IL-1 mRNA in normal C57BL/6 mice. The results show that many organs contain IL-1 mRNA-positive cells, but the highest frequency was found in lymphoid organs. The distribution and localization of these cells suggest that many of the IL-1 mRNA-producing cells are tissue macrophages. Organs exposed to environmental Ag and microbial products (lymph nodes, liver, intestine, lung, and uterus) had high frequencies of IL-1 mRNA-producing cells, suggesting that IL-1 is produced in local inflammatory or immune responses in vivo. The production of IL-1 mRNA in the thymus and in the bone marrow suggests that IL-1 is available to play physiologic roles in T cell differentiation and in hemopoiesis.

Immunohistochemical examination of Peyer's patches in autoimmune mice.

The distribution of T-cells and B-cells in Peyer's patches was examined in three autoimmune model mice, MRL/Mp-lpr/lpr, BXSB, NZBWF1/J mice and normal BALB/c mice, between one and ten months old. A multiple layering technique was used for immunohistochemical detection of lymphocyte surface antigens of T-cells (Thy1.2, Lyt1, Lyt2) and B-cells (surface IgM) and peanut agglutinin receptor for germinal center cells. The T-cell population of female MRL/Mp-lpr/lpr mice increased markedly with age, and the B-cell population of the male BXSB mouse tended to increase. However, little change was observed with age in the NZBWF1/J mice. The immunohistochemical properties of the Peyer's patches in the three autoimmune model mice were different.

Vasoactive intestinal peptide-containing nerves in Peyer's patches.

Vasoactive intestinal peptide (VIP) is a neuropeptide that is well represented in the gut tract. Previous work has demonstrated that murine T lymphocytes have high-affinity specific receptors for VIP and has implicated their interaction with VIP in the control of T-cell migration into Peyer's patches in vivo. It was postulated that this effect was mediated by interactions in the vicinity of the specialized endothelium of the postcapillary venules of Peyer's patches. We report the localization of VIP-like immunoreactivity in mouse Peyer's patches. Immunohistochemical staining was performed with heterologous antiserum against VIP using the avidin-biotin-peroxidase complex technique. VIP positivity was present near vessels of various sizes and in close proximity to small caliber vessels lined with specialized polygonal endothelial cells. These findings provide an anatomical basis for the concept that VIP may be available as a local neurophysiological signal during the migration of lymphocytes from the blood into Peyer's patches.

Comparison of immunoglobulin heavy chain isotype expression in Peyer's patch and splenic B-cells.

Past work has shown that B lymphocytes from Peyer's patches (PP) and from spleen differ from each other in the following ways: (1) PP are enriched for IgA-producing precursor cells while splenic B-cells are a rich source of IgM-precursor cells; and (2) PP are a poor source of antibody-secreting cells when compared to the spleen. The reasons for these differences are unclear. In this work B-cells have been examined expressing newly regenerated surface immunoglobulin heavy chains in murine PP and spleens for immunoglobulin isotype expression. Dual color immunofluorescence demonstrated higher levels of B-cells regenerating two surface isotypes in the PP (IgM+ IgG+ = 14.9%; IgM+ IgA+ = 9.4%) than in the spleen (IgM+ IgG+ = 0.4%; IgM+ IgA+ = 0.2%). Poly A+ RNA was purified from these B-cells and compared for immunoglobulin heavy chain isotype expression by DNA-excess slot blot hybridization. Splenic B-cells contained higher amounts (at least two-fold) of Ig heavy chain-specific mRNA per microgram of polysomal RNA than did PP B-cells. Peyer's patches B-cells demonstrated slightly lower mu:alpha ratios than splenic B-cells. In vitro translation of the RNAs suggested higher levels of translatable alpha-specific Poly A+ RNA in PP B-cells than in B-cells from MLN or spleen; furthermore, splenic B-cell RNA contained higher levels of translatable mu-RNA than did the other tissues examined. Northern blot analysis of RNA derived from these tissues identified major mu-, gamma 2b-, and alpha-hybridizing sequences, though PP-derived B-cell preparations were shown to be enriched for the membrane forms of mRNA for gamma 2b and alpha when compared to the spleen-derived B-cell preparations. These results suggest that the level of differentiation of PP B-cells (that are capable of regeneration of surface Ig) may differ significantly from that of splenic B-cells.

Structural features of and cholesterol distribution in M-cell membranes in guinea pig, rat, and mouse Peyer's patches.

Evidence suggests that M cells, which are antigen-sampling epithelial cells that overlie the domes of Peyer's patches, have an apical plasma membrane that differs from that of absorptive cells. We examined the structural features of rat, mouse, and guinea pig M-cell apical membranes and compared them with those of dome and villus absorptive cell apical membranes using electron microscopy of thin sections and freeze-fracture replicas. We also determined the distribution of morphologically detectable cholesterol in M-cell plasma and intracellular membranes in dome epithelium exposed to the polyene antibiotic, filipin. The areal density of P-face intramembrane particles was significantly less on M-cell microvilli than on microvilli of dome absorptive cells and villus absorptive cells. Areal densities of E-face intramembrane particles were similar on M-cell and absorptive-cell microvilli. M-cell apical plasma membranes were rich in cholesterol, displaying numerous filipin-induced membrane lesions in thin sections and freeze-fracture replicas. In contrast, apical membrane endocytic pits and coated vesicles in M cells failed to show filipin-induced membrane lesions. These findings suggest that, compared with the apical membranes of absorptive cells, those of M cells have a low protein to lipid ratio and an abundance of morphologically detectable cholesterol except in domains involved in endocytosis. Additionally, a subpopulation of dome epithelial cells displayed distinctive tight junctions with high strand counts and unusual depth. Although the cell type associated with these modified tight junctions could not be identified with certainty, their interspecies frequency paralleled the interspecies frequency of M cells in dome epithelium. These expanded tight junctions may result from physical tension caused by epithelial shape changes induced by intraepithelial lymphoid trafficking or, alternatively, may help buttress M cells that have attenuated cytoplasmic processes due to the presence of central hollows.

Relative contribution of Peyer's patches to intestinal DNA content and tritiated thymidine content of the mouse small intestine.

The quantification of mucosal DNA as a measure of epithelial cellularity has been criticized because it would include the non-epithelial cell populations present in the mucosa. The weight, DNA content and tritiated thymidine uptake of Peyer's patches and the remaining mucosa were measured and compared. Although the Peyer's patches only contribute 2.5% to the weight of the small intestine, they contained 12% of the DNA and 7% of the tritiated thymidine which would be found in a mucosal scrape.

Immunoperoxidase localization of large granular lymphocytes in normal tissues and lesions of athymic nude rats.

The immunocytochemical localization of cells reacting with the OX-8 monoclonal antibody was studied in athymic nude rats by the avidin-biotin-peroxidase complex (ABC) immunoperoxidase technique. By a number of criteria, including morphology and tissue distribution of OX-8-positive cells, it is postulated that the majority of lymphocytes reacting with OX-8 antibody in nude rats are LGL. The cell surface of large lymphocytes reacted with the OX-8 antibody in fixed tissue sections. The tissues with the greatest density and percentages of these reactive cells included the paracortex of lymph nodes in association with interdigitating cell (IDC) hyperplasia, bronchial-associated lymphoid tissue (BALT), the medullary cords and sinuses of lymph nodes, and intestinal epithelium. Ultrastructure of the paracortical areas of IDC hyperplasia revealed granular lymphocytes in close association with IDC. Tissues with the fewest positive cells included bone marrow, B cell areas of lymphoid tissues, and parenchymal epithelial organs. In healthy and cachectic nude rats, various incidental and pathologic lesions contained a significant number of OX-8-positive cells. Large numbers of positive lymphocytes were seen in suppurative pneumonic lesions, enteritis, focal ulcerative epithelial lesions, and papovaviral sialoadenitis. The observation of a large number of OX-8-positive lymphocytes in nonlymphoid organs supports the hypothesis that these cells play a major role in the first line of defense against not only tumors but also infectious agents.

Membrane fluorescent probes for the demonstration of lymphocyte population heterogeneity. I T. and B lymphocytes of mice and rats.

Non-fixed lymphocytes of rats and mice were stained with the membrane fluorescent probe, 3-methoxybenzanthrone. The probe is capable of binding preferentially with lymphocyte membranes and fluoresces in the green spectral region. Microfluorometry of single cells showed that lymphocytes differ in all lymphoid organs and these may be a 3-10-fold variation in fluorescence intensity. Lymphocytes can be divided into two groups according to fluorescence intensity: "bright" and "dim". The proportions of "bright" and "dim" cells were determined in rats and mice for various lymphoid organs in the normal state, after thymectomy and cyclophosphamide treatment, and also after lymphocyte separation on a nylon wool column. In all cases the proportion of bright lymphocytes corresponded to the B-cell content, and the proportion of dim lymphocytes corresponded to the content of T-cells.

Intestinal barrier to large particulates in mice.

Intestinal barrier function in mice was assessed after acute or chronic oral administration of 15.8- and 5.7-micron synthetic spherical particles. The results failed to confirm previous reports that ingested particles rapidly appear in blood. Furthermore, 15.8-micron particles did not accumulate in intestinal Peyer's patches, mesenteric lymph nodes, or other organs of the reticuloendothelial system, even after the maximum dosage of 8 X 10(6) particles per day for 60 d. However, the 5.7-micron particles were demonstrated in Peyer's patches, mesenteric lymph nodes, and lungs after the maximum dosage of 4.5 X 10(8) particles per day for 60 d. At 77 d after the termination of ingestion, 5.7-micron particles were still present in these tissues. The 5.7-micron particles were not found in spleen; retention in liver was equivocal. The site of uptake of particles capable of penetrating the intestinal mucosa appears to be the Peyer's patches. It is suggested that most absorbed particles are sequestered in Peyer's patch macrophages. Particles that escape sequestration are transported by lymph rather than by portal blood. The findings indicate that hazards associated with intestinal uptake of large (> 5 micron) particulates exist, but that the frequency of such penetration is still unclear.

Characteristics of mesenteric lymph node cells homing to gut-associated lymphoid tissue in syngeneic mice.

A subpopulation of cells in murine mesenteric lymph nodes, about 15% of those synthesizing DNA at any given time, homes specifically to the gut and mesenteric nodes of syngeneic recipients within 1 day of i.v. transfer. In contrast, cells from Oeyer's patches or peripheral lymph nodes do not. A large proportion of the B blasts which home to the small intestine has surface Ig, but lacks complement receptors. Thy-1-positive T blasts home to the gut to a lesser extent than B blasts. However, it is probable that equal fractions of B and T blasts home to mesenteric nodes. Homing is not affected by measures calculated to interfere with the combination of cell surface IgA and secretory component.