Identification of M-cells in the rabbit tonsil by vimentin immunohistochemistry and in vivo protein transport

The rabbit palatine tonsil was studied by electron microscopy to determine whether M-cells similar to those of the Peyer's patches exist in the tonsil epithelium. A subpopulation of crypt epithelial cells was found with long, irregularly shaped microvilli, small cytoplasmic vesicles and engulfing intraepithelial lymphocytes and macrophages. Using ultrastructural immunohistochemistry, vimentin, the rabbit marker for intestinal and bronchial M-cells, was detected in the cytoplasm of these cells, whereas no vimentin immunoreactivity was found in the remaining epithelial cells. The vimentin filaments surrounded the nucleus of the presumed M-cells and lay beneath the plasma membrane that surrounded intraepithelial lymphocytes. In vivo experiments using horseradish peroxidase as tracer revealed that this protein was endocytosed by the presumed M-cells and transported to the intercellular spaces between epithelial cells and lymphocytes. The results indicate that specialized epithelial cells exist in the tonsil which have morphological characteristics similar to those of intestinal M-cells, are in close contact to cells of the immune system, are positive for the rabbit M-cell marker vimentin, and are capable of antigen uptake and transcytosis. It is therefore concluded that M-cells are present in the tonsil and probably play a role in the initiation of immune responses to orally delivered antigens.     

Vimentin-positive cells in the epithelium of rabbit ileal villi represent cup cells but not M-cells.

Membranous (M)-cells are specialized epithelial cells of the Peyer's patch domes that transport antigens from the intestinal lumen to the lymphoid tissue. Vimentin is a reliable marker for M-cells in rabbits. Using immunohistochemistry (IHC), a subpopulation of epithelial cells has recently been identified in ordinary rabbit ileal villi, which are vimentin-positive and share morphological characteristics with the M-cells of the domes. To test the hypothesis that these cells represent M-cells outside the organized lymphoid tissue, lectin labeling and tracer uptake experiments were performed. Lectins specific for N-acetyl-glucosamine oligomers selectively bound to the vimentin-positive villous cells but not to M-cells in the domes. Microbeads instilled into the ileal lumen were taken up by M-cells within 45 min but not by the vimentin-positive cells in the villi. Lectin-gold labeling on ultrathin sections revealed that the lectin binding sites were located in the brush border and in vesicles in the apical cytoplasm. The vimentin/lectin-positive cells shared ultrastructural characteristics with the so-called "cup cells." We conclude (a) that the vimentin-positive cells in ordinary villi represent cup cells but not M-cells, (b) that they are readily detectable by (GlucNAc)(N)-specific lectins, and (c) that they do not transcytose experimental tracers. Although the specific function of cup cells is still obscure, they most probably represent a cell type distinct from M-cells of the domes with respect to both function and expression of the two new markers.     

Formation of spontaneous and immune rosettes by the cells of the thymus and other formations of the rabbit lymphoid system]

Only T1--RFC (rosette-forming cells) are revealed in the thymus of nonimmunized rabbits. Their number is 2--2.5 times less than in the palatine tonsils, submaxillary lymph nodes and the spleen. T2--RFC are present in these lymphoid formations. There is an increase in the T1--RFC in the thymus after the intravenous immunization of rabbits with sheep erythrocytes. In other lymphoid formations the correlation of the population of cells of the thymus origin altered as a result of increase in the number of T2--RFC. B--RFC accumulated in considerable amounts. Dynamics of T2 and B--RFC accumulation in the lymphoid formations corresponded to the highest antibody titres in the rabbit blood. In the formation of primary immune response the amount of the T1 and T2-RFC in the formations of rabbit lymphoid system depended on the dose of the antigen.     

Tonsil-derived mesenchymal progenitor cells acquire a follicular dendritic cell phenotype under cytokine stimulation

Tonsils comprise part of the mucosal immune system and contain lymphocytes, macrophages, and follicular dendritic cells (FDCs). FDCs are located in the B cell area of the follicles of secondary lymphoid organs, such as the spleen, tonsils, or lymph nodes, and they trap and retain immune complexes on their surfaces to regulate B cell activation and maturation. Stromal cells from the palatine tonsils are often used for FDC in vitro studies, and it has been reported that human palatine tonsils may be a good source of multipotent mesenchymal cells. Therefore, we assessed whether tonsil-derived mesenchymal stromal cells could differentiate into a FDC-like phenotype. We discovered that stromal cells isolated from human tonsils not only had the potential to differentiate into various cell types of mesenchymal origin, but they also could differentiate into FDC-like cells under cytokine stimulation in vitro.     

Differentiation of the palatine tonsillar tissues of the human fetus]

Differentiation of epithelial and lymphoid tissues of the palatine tonsils was studied in human embryos at the age of 8-34 weeks of development by means of histochemical, immunomorphological and morphometric methods. The anlage of the palatine tonsils appears at the age of 9 weeks of fetal development. At the age of 13-14 weeks of fetal development the tonsil suspension contains 2 subpopulations of lymphocytes possessing properties of T-cells differing in the ability of their superficial receptors to interact with sheep erythrocyte antigens forming rosettes (RFC--rosette forming cells) and with antigens of their own erithrocytes (autoRFC). The number both increases sharply by the 16th week of gestation. Simultaneously, essential alterations are noted in epithelial and lymphoid tissues. In epithelium of crypts cornified cells appear; the amount of lymphoid tissue increases sharply, primary follicles without reactive centers appear, lymphocytic infiltration of epithelium occurs. The amount of RFC does not change considerably, and the amount of autoRFC has a tendency towards some increase. From the data obtained, it is possible to suggest that human palatine tonsils already at embryonic period participate in functioning of immunogenic organs and in maintaining of immunologic homeostasis of the fetal organism.     

Morphofunctional Organization of Lymphoepithelial Organs of the Human Pharynx

We present the current concepts of morphofunctional organization of the lymphoepithelial organs in the human pharynx based on the published data and authors" results. Functional compartmentation of palatine and pharyngeal tonsils is considered, which reflects cooperative cell interactions in the immune response; B- and T-areas have been structurally isolated and functionally substantiated. A special attention is paid to the fine structure of cryptal epithelium and its interactions with lymphoid cells infiltrating the epithelial sheet: lymphoepithelial symbiosis. Attention is also paid to structural homology of the lymphoepithelial compartment of palatine tonsils and thymus. The problem concerns the place of lymphoepithelial organs in hierarchy of the immune system as secondary organs with their own immunoregulatory area and having the functions of a regional center controlling the mucosal immunity.     

Differentiated S100A7 expression in infected tonsils and tonsils from allergic individuals

Palatine tonsils are continuously exposed to microorganisms and antigens and secrete antimicrobial peptides as a first line of defense. S100A7 is a protein with antimicrobial and chemotactic properties. Our aim was to investigate how the expression of S100A7 in human palatine tonsils is affected by inflammatory processes. Tonsils obtained from 109 patients undergoing tonsillectomy were divided into groups of infected and noninfected as well as allergic and nonallergic, based on the results from tonsillar core culture tests and Phadiatop analysis, respectively. Western blot and immunohistochemistry were used to assess protein expression and real-time PCR was used to quantify mRNA levels. To explore the induction of S100A7, tonsils were stimulated with lipopolysaccharide in vitro. The immunohistochemical staining for S100A7 was most intense in the tonsillar epithelium, but the protein was also detected in B- and T-cell regions, which was confirmed with Western blot on isolated B and T cells. The S100A7 expression appeared to be the highest in CD8+ T cells. Reduced mRNA levels of S100A7 were detected in infected tonsils as well as in tonsils from allergic individuals. In vitro stimulation of tonsils with lipopolysaccharide did not have any effect on the expression. The results suggest a role for S100A7 in recurrent tonsillitis and allergic disease.     

M-cells: origin, morphology and role in mucosal immunity and microbial pathogenesis

M-cells are specialized cells found in the follicle-associated epithelium of intestinal Peyer's patches of gut-associated lymphoid tissue and in isolated lymphoid follicles, appendix and in mucosal-associated lymphoid tissue sites outside the gastrointestinal tract. In the gastrointestinal tract, M-cells play an important role in transport of antigen from the lumen of the small intestine to mucosal lymphoid tissues, where processing and initiation of immune responses occur. Thus, M-cells act as gateways to the mucosal immune system and this function has been exploited by many invading pathogens. Understanding the mechanism by which M-cells sample antigen will inform the design of oral vaccines with improved efficacy in priming mucosal and systemic immune responses. In this review, the origin and morphology of M-cells, and their role in mucosal immunity and pathogenesis of infections are discussed.     

Clusterin in human gut-associated lymphoid tissue, tonsils, and adenoids: localization to M cells and follicular dendritic cells

The follicle-associated epithelium (FAE) overlying the follicles of mucosa-associated lymphoid tissue is a key player in the initiation of mucosal immune responses. We recently reported strong clusterin expression in the FAE of murine Peyer’s patches. In this study, we examined the expression of clusterin in the human gut-associated lymphoid tissue (GALT) and Waldeyer’s ring. Immunohistochemistry for clusterin in human Peyer’s patches, appendix and colon lymphoid follicles revealed expression in M cells and in follicular dendritic cells (FDCs). Using cryo-immunogold electron microscopy in Peyer’s patches, we observed cytosolic immunoreactivity in M cells and labeling in the ER/Golgi biosynthetic pathway in FDCs. In palatine tonsils and adenoids, we demonstrated clusterin expression in germinal centers and in the lymphoepithelium in the crypts where M cells are localized. In conclusion, clusterin is expressed in M cells and follicular dendritic cells at inductive sites of human mucosa-associated lymphoid tissue suggesting a role for this protein in innate immune responses. Moreover, the use of clusterin as a human M cell marker could prove to be a valuable tool in future M cell research.     

Cathepsin E in follicle associated epithelium of intestine and tonsils: localization to M cells and possible role in antigen processing

A specific rabbit anti-human serum was used selectively to localize the aspartic proteinase cathepsin E to follicle associated epithelium (FAE) of human and rat intestine, including jejunum, ileum, appendix, colon and rectum, as well as of human palatine, pharyngeal and lingual tonsils. Coexpression of class II histocompatibility antigen HLA-DR antigen has been observed in some of the cathepsin E-positive epithelial cells. In addition, cathepsin E has been detected in a few mononuclear cells of intestinal lymphoid structures and tonsils resembling interdigitating reticulum cells of lymph nodes. Another aspartic proteinase, cathepsin D, has been found to be poorly represented in FAE and intensely expressed by macrophages. Electron immunocytochemistry localized cathepsin E to endosomal vesicles and endoplasmic reticulum of M cells in rat and human ileum as well as of M-like cells in human palatine tonsil. The results suggest a possible role of endosomal cathepsin E in the processing of macromolecules and microorganisms transported by M cells and related epithelial cells to mucosal associated lymphoid tissue (MALT).     

Histological studies on the development of porcine tonsils after birth

Tonsils form the topographically first immune barrier of an organism against the invasion of pathogens. We used histology to study the development of tonsils of pigs after birth. At birth, the tonsils consist of diffuse lymphoid tissue without any lymphoid follicle aggregations. At the age of 7 days, lymphoid follicles appeared in the soft palate tonsil. The lymphoid layer of the nasopharyngeal tonsil, soft palate tonsil, and lingual tonsil became thicker, and lymphoid follicles in the lamina propria were clearly visible at the age of 21 days. Secondary lymphoid follicles were present in the nasopharyngeal tonsil at the age of 50 days, and in the soft palate tonsil at the age of 120 days. Dendritic cells (DCs), CD3⁺ T cells and IgA⁺ B cells in the soft palate tonsil, nasopharyngeal tonsil and lingual tonsil increased continuously, especially during the first 21 days. The results suggested that tonsils have an important role in local immune defense against invading antigens after birth and will be beneficial for understanding the mechanisms of immunity in these animals after nasal and oral vaccination.     

Functional analysis of cloned germinal center CD4+ cells with natural killer cell-related features. Divergence from typical T helper cells

Granular lymphocytes co-expressing the Leu-7 (NK-related) and CD4 (T helper cell) markers are selectively localized in the germinal centers of lymphoid tissues. Leu-7+ cells (greater than 98% of which co-expressed CD4) were isolated from inflammatory tonsils and were cloned by the limiting dilution technique. Clones were analyzed for their phenotypic and functional characteristics. CD4+-Leu-7+ cell-derived clones retained their CD3 and CD4 surface antigens, lost the Leu-7 marker, and acquired HLA-DR determinants. In comparison with clones derived from peripheral blood or tonsil CD4+ cells, CD4+-Leu-7+ tonsil cell-derived clones showed similar low frequencies of cytotoxic precursors. In contrast, the frequency of interleukin 2 (IL 2) and B cell growth factor producing clones was much lower for tonsil CD4+-Leu-7+ cells than for CD4+ blood or tonsil progenitors. We conclude that germinal center CD4+-Leu-7+ cells are a subset of T cells unable to produce IL 2 in response to phytohemagglutinin or anti-CD3 stimulation, which is effective on the majority of T helper cells.     

Expression of 5-lipoxygenase in specialized epithelial cells of nasopharyngeal-associated lymphoid tissue

Leukotrienes are lipid mediators that are produced primarily by certain types of leukocytes. The synthesis of the leukotriene LTB₄ is initiated by the enzyme 5-lipoxygenase and completed by LTA₄ hydrolase. Epithelial cells constitutively express LTA₄ hydrolase but normally lack 5-lipoxygenase. In this study, we report that the stratified squamous epithelial cells from inflamed or hyperplastic tissues of palatine and pharyngeal tonsils (nasopharyngeal-associated lymphoid tissue) express 5-lipoxygenase protein. The localization of 5-lipoxygenase was indicated by immunohistochemical staining and presence confirmed by immunoblot. Positive staining for 5-lipoxygenase in infiltrating leukocytes in inflamed tissues served as internal positive controls for immunohistochemical staining. Staining for 5-lipoxygenase in appendix tissue was negative for epithelial cells while positive for polymorphonuclear leukocytes, indicating that 5-lipoxygenase expression is not a general feature of epithelial cells in mucosa-associated lymphoid tissue. In tonsils, 5-lipoxygenase staining was pronounced in broad regions but reduced or absent in others, suggesting regional regulation of expression. Epithelial cells of tonsils were also positive for 5-lipoxygenase activating protein and leukotriene A₄ hydrolase, indicating a capacity to produce LTB₄. Taken together, these results suggest that the specialized epithelial cells of the mucosa-associated lymphoid tissue of human tonsils can synthesize LTB₄. This lipid mediator may serve to modulate the function of cells within the lymphoid tissue as well as promote an inflammatory response.     

Immunologic 'ignorance' of vascularized organ transplants in the absence of secondary lymphoid tissue

Secondary lymphoid organs (the spleen, lymph nodes and mucosal lymphoid tissues) provide the proper environment for antigen-presenting cells to interact with and activate naive T and B lymphocytes. Although it is generally accepted that secondary lymphoid organs are essential for initiating immune responses to microbial antigens and to skin allografts, the prevailing view has been that the immune response to primarily vascularized organ transplants such as hearts and kidneys does not require the presence of secondary lymphoid tissue. The assumption has been that the immune response to such organs is initiated in the graft itself when recipient lymphocytes encounter foreign histocompatibility antigens presented by the graft's endothelial cells. In contrast to this view, we show here that cardiac allografts are accepted indefinitely in recipient mice that lack secondary lymphoid tissue, indicating that the alloimmune response to a vascularized organ transplant cannot be initiated in the graft itself. Moreover, we demonstrate that the permanent acceptance of these grafts is not due to tolerance but is because of immunologic 'ignorance'.     

Innate Response Activator (IRA) B Cells Reside in Human Tonsils and Internalize Bacteria In Vitro

Innate response activator (IRA) B cells have been described in mice as a subset of B-1a B cells that produce granulocyte/macrophage colony-stimulating factor (GM-CSF) and have been found in the spleen upon activation. In humans, identification, tissue localization and functionality of these lymphocytes are poorly understood. We hypothesized that IRA B cells could reside in human palatine tonsils, which are a first line of defense from infection of the upper respiratory tract. In the present work, we used flow cytometry and confocal microscopy to identify and characterize human IRA (hIRA) B cells in tonsils. We show that CD19⁺CD20⁺GM-CSF⁺ B cells are present in the tonsils of all the subjects studied at a frequency ranging between ~0.2% and ~0.4% of the conventional CD19⁺CD20⁺GM-CSF^- B cells. These cells reside within the B cell follicles, are mostly IgM⁺IgD⁺, express CD5 and show phagocytic activity. Our results support a role for hIRA B cells in the effector immune response to infections in tonsils.     

Differential cytokeratin and glycoconjugate expression by the surface and crypt epithelia of human palatine tonsils

Human palatine tonsils are clinically important due to their susceptibility to tonsillitis and association with other local and systemic diseases. Paradoxically, the tonsils function as antigen sampling sites of the mucosal immune system and, consequently, the tonsil epithelia perform both protective and antigen sampling roles. These epithelia are divided into stratified squamous epithelium overlying the tonsil surface and crypt epithelium lining the tonsil crypts, the latter of which includes reticular areas which are infiltrated by lymphocytes and are responsible for antigen sampling. In this study we characterised cytokeratin and glycoconjugate expression by healthy epithelia of human palatine tonsils. We identified pan-epithelial tonsil markers and also demonstrated that the surface and reticular crypt epithelia are differentiated by the expression of multiple cytokeratins. The latter finding supports the hypothesis that these epithelia undergo alternate differentiation pathways and possess different functional roles. In addition, we identified cell subpopulations in the tonsil epithelia which may represent distinct cell subtypes including specialised antigen sampling cells. These findings establish a basis for future studies to investigate histochemical changes in tonsil epithelia that are associated with or predispose to local and/or systemic disease.     

Helicobacter pylori: immunoproteomics related to different pathologies

Helicobacter pylori is a Gram-negative bacterium that causes ulcer, atrophic gastritis, adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. Moreover, an ongoing controversial role of this bacterium infection has been suggested in the etiopathogenesis of some extradigestive diseases. The humoral response to H. pylori during a natural infection can be used for diagnostic purposes and as a basis for vaccine development. Host-pathogen interactions may be investigated by means of immunoproteomics, which provides global information about relevant specific and nonspecific antigens, and thus might be suitable to identify novel vaccine candidates or serological markers of H. pylori infection as well as of different related diseases. In this review, we describe how several research groups used H. pylori proteomics combined with western blotting analysis, using sera from patients affected with different H. pylori-related pathologies, to investigate potential associations between host immune response and clinical outcomes of H. pylori infection, resulting in the rapid identification of novel, highly immunoreactive antigens.     

Isolation and characterization of lymphatic microvascular endothelial cells from human tonsils

Human lymphatic endothelial cells (LECs) have isolated prevalently from human derma and tumors. As specialized lymphatic organs within the oropharynx, palatine tonsils are easily obtained and rich in lymphatic venules. Using a two-step purification method based on the sorting of endothelial cells with Ulex Europaeus Agglutinin 1 (UEA-1)-coated beads, followed by purification with monoclonal antibody D2-40, we successfully purified LECs from human palatine tonsils. The LECs were expanded on flasks coated with collagen type 1 and fibronectin for up to 8-10 passages and then analyzed for phenotypic and functional properties. Cultured cells retained the phenotypic pattern of the lymphatic endothelium of palatine tonsils and expressed functional VEGFR-3 molecules. In fact, stimulation with VEGFR-3 ligand, the vascular endothelium grow factor C, induced a marked increase in cell proliferation. Similarly to blood endothelial cells (BECs), LECs were able to form tube-like structure when seeded in Cultrex basement membrane extract. Comparative studies performed on LECs derived from palatine tonsils and iliac lymphatic vessels (ILVs), obtained with the same procedures, showed substantial discrepancies in the expression of various lymphatic markers. This points to the existence of micro- and macrovessel-derived LECs with different phenotypes, possibly involving different biological activities and functions. Palatine tonsil- and ILV-derived LECs may, therefore, represent new models for investigating function and biochemical properties of these lymphatic endothelia.     

Progression from papilloma to carcinoma is accompanied by changes in antibody response to papillomavirus proteins.

Cottontail rabbit papillomavirus induces benign tumors, papillomas, in rabbits which progress at a high frequency to malignant tumors, carcinomas. Cottontail rabbit papillomavirus therefore provides an experimental model for oncogenic human papillomaviruses. The nature of the antigens recognized by the host has not been identified at any stage of tumor development. Here, we characterized the humoral immune response to viral antigens in cottontail and domestic rabbits at the papilloma stage, in domestic rabbits at the carcinoma stage, and in animals in which papillomas had regressed. Antibodies to linear epitopes were identified by Western blotting (immunoblotting) with bacterial fusion proteins, and evidence for recognition of conformational epitopes was obtained by immunoprecipitation. An immune response to the early proteins E1, E2, E6, and E7 was detected only in a fraction of the animals, and all animals were negative for E4 and E5. The response to E6 and E7 peaked around 7 months and then decreased, while that to E1 and E2 remained level after an initial raise. The antibody response to structural proteins was low at the papilloma stage, and antibodies to L1 recognized predominantly conformational epitopes. As papillomas progressed to carcinomas, there was a drastic increase in the response to L1 and L2, suggesting a change in interaction between virus-infected host cells and the host's immune system.     

Vimentin antibodies stain membranous epithelial cells in the rabbit bronchus-associated lymphoid tissue (BALT)

Summary The lymphoepithelium covering the bronchus-associated lymphoid tissue (BALT) of the rabbit lung was studied with monoclonal antibodies against vimentin, using the indirect immunoperoxidase technique. In the lymphoepithelium single cells which had a membranous apical cytoplasm and engulfed intraepithelial lymphocytes were vimentin-immunoreactive. All other epithelial cells of the lymphoepithelium and of the surrounding airway epithelium did not bind vimentin antibodies. The results support the hypothesis that the membranous epithelial cells in the lymphoepithelium of rabbit BALT are analogous with intestinal M-cells, which in rabbit Peyer's patches and appendix are selectively labelled by vimentin antibodies.