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.     

Morphology of complex lymphoepithelial organs of the anal canal (“anal tonsil”) in the bottlenose dolphin,Tursiops truncatus

A complex of lymphoepithelial organs, the “anal tonsils,” is a consistent structure in the anal canal of the bottlenose dolphin, Tursiops truncatus. This complex occurs as a circumferential cluster of discrete tonsil like aggregations of lymphoid tissues, together with epithelial ducts (“crypts”) and occasional mucus secretory units in the extreme lower portion of the intestinal tract. These structures are concentrated in the segment lined by stratified squamous epithelium and extend for a variable distance cephalad from the anal aperture. The tonsils appear to be most active, judged by the amount of lymphoid tissue present, in young animals. Depletion of lymphocytes and cystic enlargement of the crypts, probably representing functional as well as morphological involution, is a consistent feature of older animals. © 1995 Wiley-Liss, Inc.     

The chemical fractionation of lymphatic organs

A method for chemically fractionating lymphatic organs has been described. The method has been shown to be applicable to bovine palatine tonsils, sheep palatine tonsils, and bovine thymus. Approximately 50 per cent of the dry weight of tonsils and about 30 per cent of thymus has been found to be soluble in the 0.15 M NaCl extract. Four components have been isolated which together account for 65 per cent by weight of the material in the extracts. Four other components have been identified and partially defined by means of electrophoretic mobility, solubility, or some other chemical or physical property.     

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.     

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.     

Rabbit tonsil-associated M-cells express cytokeratin 20 and take up particulate antigen.

M-cells are believed to play a pivotal role in initiation of the immune response. These cells, located in the epithelia that overlie mucosal lymphoid follicles, are responsible for the active uptake of particulate antigens and for their translocation to the underlying lymphoid tissue. The identification of reliable markers for M-cells is therefore extremely important for the study of the initial steps that lead to the immune response. For this purpose, we studied cytokeratin 20 (CK20) expression in the epithelium of rabbit palatine tonsils by immunofluorescence, confocal microscopy, and Western blotting. CK20+ cells were observed in all rabbit palatine tonsils examined. By Western blotting, one CK20-immunoreactive band was identified at 46 kD on samples of proteins from the intermediate filament-enriched cytoskeletal fraction of tonsil epithelium. Double labeling of CK20+ cells with cell-specific markers confirmed that such cells were actually M-cells. Moreover, CK20+ M-cells displayed a mature phenotype (they formed pockets harboring lymphoid cells) and were functionally competent because they could take up particulate antigens from the pharyngeal lumen. We conclude that CK20 is an M-cell marker for rabbit palatine tonsils. Moreover, we can hypothesize the use of M-cells as a possible site for antigen delivery of particle-based vaccines.     

Comparison of lymphocyte production in lymphoid organs and their compartments using the metaphase-arrest technique

Summary Lymphocyte proliferation was studied in normal young anesthetized pigs by the metaphase-arrest technique using vincristine (VCR). In each animal biopsies were taken simultaneously from the thymus, mesenteric lymph nodes, spleen, palatine tonsil and Peyer's patches from the ileum and jejunum. After taking the first samples, 0.25 mg VCR/ kg body weight was injected i.v. and then four more biopsies were excised for up to 3.5 h after VCR. Imprints of the lymphoid organs were evaluated as an overall index for each organ, and histological sections were used to determine the mitotic index in typical B-and T-lymphocyte areas in these organs. In follicles of mesenteric lymph nodes, tonsils and the two types of Peyer's patches a comparable increase in the mitotic index was found, 3.62% per hour. In the corona the increase was also comparable but much lower, 0.43% per hour and in the interfollicular area similarly 0.38% per hour. In the spleen the mitotic rate was 0.69% for the white pulp and 0.42% per hour for the red pulp. In the thymic cortex the mitotic index increased by 0.49% and in the medulla by a surprisingly high value of 0.32% per hour. The metaphase-arrest technique in larger animals enables a comparison of lymphocyte production among organs and their different compartments, and demonstrates the important contribution of peripheral lymphoid organs to the renewal of the lymphocyte pools.List of Abbreviations DNA deoxyribonucleic acid - ¹⁴ C-TdR thymidine labelled with ¹⁴C - ³ H-TdR tritiated thymidine - r _m rate of entry of cells in mitosis per hour - VCR vincristine sulphate Partly presented at the XII Int. Anat. Congress in London, August 1985     

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.     

Palatine tonsils and immunity. VI. Effect of thymosin and palatine tonsil extract on the immunologic reactivity of neonatally thymectomized and intact mice]

Experiments were conducted on Wistar rats, intact and neonatally thymectomized CBA mice; a study was made of the immunological activity of the extracts of the thymus and the palatine tonsils of calves obtained by the method of Goldstein et al (1966). The extract of the palatine tonsils obtained proved to contain a small amount of thymosin at whose expense an insignificant restoration of immunological reactivity occurs in its administration neonatally to thymectomized mice.     

Effect of probiotics on intestinal mucosal immunity and ultrastructure of cecal tonsils of chickens

Abstract

Sixty chickens were randomly divided into two groups to determine the effect of oral administration of probiotics on the intestinal mucosal immune response and ultrastructure of cecal tonsils. The first group (control) was fed with a basic diet without antibiotic or probiotics. The second group was fed with the same diet as the control, except they received drinking water with probiotics (4×10⁹ cfu per chicken and day) from posthatch to day 3 of age. The probiotic preparation was composed of Bacillus subtilis Bs964, Candida utilis BKM-Y74 and Lactobacillus acidophilus LH1F. Intestinal fluid, Peyer's Patch and cecal tonsils were taken at day 1, 4, 7, 10 and 18 after administration of probiotics. The results showed: (i) Compared to the control, probiotics enhanced the content of following items: immunglobulin (Ig)A in the intestinal fluid at day 7 (p < 0.01), the IgG-forming cells at day 10 (p < 0.05), IgM-forming cells in the Peyer's Patch at day 7 (p < 0.05), IgA-forming cells at day 7–10 (p < 0.05), IgG-forming cells at day 7 (p < 0.05) and IgM-forming cells in cecal tonsils diffuse area at day 4–7 (p < 0.05). (ii) T lymphocytes in cecal tonsils were enhanced at day 7 (p < 0.01) after orally fed with probiotics. (iii) The density of microvilli and length of cecal tonsils increased after probiotics were administrated at day 3. With chicken ageing, the efficiency of probiotics would decrease. These results suggested that probiotcs enhance intestinal mucosal immunity of chicken at the early age.     

Comparative analysis of apoptotic changes in peripheral immune organs and lungs following experimental infection of piglets with highly pathogenic and classical porcine reproductive and respiratory syndrome virus

Background

Our previous studies have demonstrated that piglets infected with highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) may develop significant thymus atrophy, which related to thymocytes apoptosis. However, apart from that detected in the thymus, there are no reports describing cell apoptosis induced by HP-PRRSV infection. In this study, we analyzed comparatively the pathological changes, cell apoptosis and viral load in peripheral immune organs including tonsil, inguinal lymph nodes (ILNs) and spleen and lungs following experimental infection of piglets with HP-PRRSV HuN4 and classical PRRSV CH-1a.

Findings

HP-PRRSV HuN4 exhibited much stronger cell tropism than CH-1a in immune organs and lungs of piglets. HuN4 infection led to the serious injuries in tonsils, ILNs, spleens and lungs, especially apoptosis in these organs was significant.

Conclusions

HuN4 infection induced severe lesions (gross pathology, histopathology and cell apoptosis) in the peripheral immune organs and lungs of infected piglets. Large numbers of apoptotic cells in immune organs and lung induced by HuN4 may play a role in the pathogenesis of the HP-PRRS and the distinct injuries caused by HuN4 infection may be associated with the high mortality rate of HP-PRRS in pigs.     

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.     

Expression and function of tight junctions in the crypt epithelium of human palatine tonsils.

The human palatine tonsils have surface and crypt stratified epithelium and may be initiated via the epithelium to mount immune responses to various presenting antigens. Here we investigated the expression and function of tight junctions in the epithelium of human palatine tonsils from patients with tonsillar hypertrophy or recurrent tonsillitis. Occludin, ZO-1, JAM-1, and claudin-1, -3, -4, -7, -8, and -14 mRNAs were detected in tonsillar hypertrophy. Occludin and claudin-14 were expressed in the uppermost layer of the tonsil surface epithelium, whereas ZO-1, JAM-1, and claudin-1, -4, and -7 were found throughout the epithelium. In the crypt epithelium, claudin-4 was preferentially expressed in the upper layers. In freeze-fracture replicas, short fragments of continuous tight junction strands were observed but never formed networks. In the crypt epithelium of recurrent tonsillitis, the tracer was leaked from the surface regions where occludin and claudin-4 disappeared. Occludin, ZO-1, JAM-1, and claudin-1, -3, -4, and -14, but not claudin-7, mRNAs were decreased in recurrent tonsillitis compared with those of tonsillar hypertrophy. These studies suggest unique expression of tight junctions in human palatine tonsillar epithelium, and the crypt epithelium may possess an epithelial barrier different from that of the surface epithelium.     

Effect of High Dietary Manganese on the Immune Responses of Broilers Following Oral Salmonella typhimurium Inoculation

Manganese (Mn) is an essential nutrient for both host and pathogen. Recent studies have demonstrated the nutritional immunity of Mn against Salmonella infection in mammals. To investigate the effect of high dietary Mn on immune responses of broilers following Salmonella challenge, 144 1-day-old male broilers were fed a basal diet (containing 20.04 mg Mn/kg) plus an additional 40 (the control group) or 400 mg Mn/kg (the H-Mn group) for 7 days. The 72 broilers in each group were then orally inoculated with 5 × 10⁷ CFUs of Salmonella typhimurium (ATCC#14028) or phosphate-buffered saline. Peripheral blood, spleens, cecal tonsils, and bursa of Fabricius were collected from Salmonella-inoculated and Salmonella-noninoculated broilers (n = 6) at 2 days post inoculation (2 DPI) and 7 days post inoculation (7 DPI). Peripheral blood lymphocyte subpopulations were determined by flow cytometry. The messenger RNA (mRNA) abundance of genes was determined by quantitative real-time polymerase chain reaction. Salmonella counts were higher (P < 0.05) in the H-Mn group than that in the control group at 2 DPI in the cecal contents of Salmonella-inoculated broilers. High dietary Mn increased CD3⁺CD4⁺ and CD3⁺CD8⁺ percentages in the peripheral blood of Salmonella-inoculated broilers at 2 DPI. Salmonella inoculation increased interleukin (IL)-6 mRNA expression in spleens and bursa of Fabricius at 2 DPI and increased IL-1β and IL-6 mRNA expression in cecal tonsils at 7 DPI in the H-Mn group. These changes were not observed in the control group. High dietary Mn increased interferon-γ (IFN-γ) in spleens and decreased IFN-γ and IL-12 mRNA expression in cecal tonsils of Salmonella-inoculated broilers at 2 DPI. High dietary Mn decreased IL-17 mRNA expression in the bursa of Fabricius at 7 DPI, but increased this expression in cecal tonsils at 2 and 7 DPI in Salmonella-inoculated broilers. These results suggested that dietary Mn level affected T helper (Th) 1-cytokine reaction in spleens and cecal tonsils, and Th17-mediated immunity in cecal tonsils and the bursa of Fabricius of broilers when challenged with Salmonella.

     

Functional morphology of the palato‐maxillary apparatus in “Palatine dragging” snakes (Serpentes: Elapidae: Acanthophis,Oxyuranus)

Elapid snakes have previously been divided into two groups (palatine erectors and palatine draggers) based on the morphology and inferred movements of their palatine bone during prey transport (swallowing). We investigated the morphology and the functioning of the feeding apparatus of several palatine draggers (Acanthophis antarcticus, Oxyuranus scutellatus, Pseudechis australis) and compared them to published records of palatine erectors. We found that the palatine in draggers does not move as a straight extension of the pterygoid as originally proposed. The dragger palato‐pterygoid joint flexes laterally with maxillary rotation when the mouth opens and the jaw apparatus is protracted and slightly ventrally during mouth closing. In contrast, in palatine erectors, the palato‐pterygoid joint flexes ventrally during upper jaw protraction. In draggers, the anterior end of the palatine also projects rostrally during protraction, unlike the stability of the anterior end seen in erectors. Palatine draggers differ from palatine erectors in four structural features of the palatine and its relationships to surrounding elements. The function of the palato‐pterygoid bar in both draggers and erectors can be explained by a typical colubroid muscle contraction pattern, which acts on a set of core characters shared among all derived snakes. Although palatine dragging elapids share a fundamental design of the palato‐maxillary apparatus with all higher snakes, they provide yet another demonstration of minor structural modifications producing functional variants. J. Morphol. 2010. © 2009 Wiley‐Liss, Inc.     

Assessment of the intensity of lymphocyte blast transformation in the crypts of the palatal tonsils by using DNA cytophotometry

Cell fractions isolated from lacunae of palatine tonsils with different functional activities were taken from 40 children 6-15 years of age. The cell fractions were examined using vital phase-contrast microscopy, light microscopy of Pappenheim-stained smears and the Feulgen cytophotometry. Lymphocytes with signs of immunological involvement were shown to constitute 95 per cent of the whole cell lacunar population of the active functional tonsils. 35 per cent of lymphocytes, being in different stages of blast transformation, were found. The most intense blast transformation was revealed in patients with tonsil inflammation. 5-13 per cent of lacunar nuclei with DNA content above the diploid level were found in these patients. Some multicellular islets accumulating small lymphocytes, lymphocytes at different stages of blast transformation, and macrophages were revealed to imitate the cooperation of immunocompetent cells. The data obtained can be helpful in the practical surgery of palatine tonsils.     

High rates of detection of respiratory viruses in tonsillar tissues from children with chronic adenotonsillar disease

Chronic tonsillar diseases are an important health problem, leading to large numbers of surgical procedures worldwide. Little is known about pathogenesis of these diseases. In order to investigate the role of respiratory viruses in chronic adenotonsillar diseases, we developed a cross-sectional study to determine the rates of viral detections of common respiratory viruses detected by TaqMan real time PCR (qPCR) in nasopharyngeal secretions, tonsillar tissues and peripheral blood from 121 children with chronic tonsillar diseases, without symptoms of acute respiratory infections. At least one respiratory virus was detected in 97.5% of patients. The viral co-infection rate was 69.5%. The most frequently detected viruses were human adenovirus in 47.1%, human enterovirus in 40.5%, human rhinovirus in 38%, human bocavirus in 29.8%, human metapneumovirus in 17.4% and human respiratory syncytial virus in 15.7%. Results of qPCR varied widely between sample sites: human adenovirus, human bocavirus and human enterovirus were predominantly detected in tissues, while human rhinovirus was more frequently detected in secretions. Rates of virus detection were remarkably high in tonsil tissues: over 85% in adenoids and close to 70% in palatine tonsils. In addition, overall virus detection rates were higher in more hypertrophic than in smaller adenoids (p = 0.05), and in the particular case of human enteroviruses, they were detected more frequently (p = 0.05) in larger palatine tonsils than in smaller ones. While persistence/latency of DNA viruses in tonsillar tissues has been documented, such is not the case of RNA viruses. Respiratory viruses are highly prevalent in adenoids and palatine tonsils of patients with chronic tonsillar diseases, and persistence of these viruses in tonsils may stimulate chronic inflammation and play a role in the pathogenesis of these diseases.     

Application of an enzyme‐labeled antigen method for visualizing plasma cells producing antibodies against Strep A,a carbohydrate antigen of Streptococcus pyogenes,in recurrent tonsillitis

Streptococcus pyogenes is the main causative pathogen of recurrent tonsillitis. Histologically, lesions of recurrent tonsillitis contain numerous plasma cells. Strep A is an antigenic carbohydrate molecule on the cell wall of S. pyogenes. As expected, plasma cells in subjects with recurrent tonsillitis secrete antibodies against Strep A. The enzyme‐labeled antigen method is a novel histochemical technique that visualizes specific antibody‐producing cells in tissue sections by employing a biotin‐labeled antigen as a probe. The purpose of the present study was to visualize plasma cells producing antibodies reactive with Strep A in recurrent tonsillitis. Firstly, the lymph nodes of rats immunized with boiled S. pyogenes were paraformaldehyde‐fixed and specific plasma cells localized in frozen sections with biotinylated Strep A. Secondly, an enzyme‐labeled antigen method was used on human tonsil surgically removed from 12 patients with recurrent tonsillitis. S. pyogenes genomes were PCR‐detected in all 12 specimens. The emm genotypes belonged to emm12 in nine specimens and emm1 in three. Plasma cells producing anti‐Strep A antibodies were demonstrated in prefixed frozen sections of rat lymph nodes, 8/12 human specimens from patients with recurrent tonsillitis but not in two control tonsils. In human tonsils, Strep A‐reactive plasma cells were observed within the reticular squamous mucosa and just below the mucosa, and the specific antibodies belonged to either IgA or IgG classes. Our technique is effective in visualizing immunocytes producing specific antibodies against the bacterial carbohydrate antigen, and is thus a novel histochemical tool for analyzing immune reactions in infectious disorders.