Lymphocyte populations and adhesion molecule expression in bovine tonsils

Bovine tonsils are mucosal-associated lymphoid tissue (MALT) located at the entry of the pharynx where both inhaled and ingested antigens can induce an immune response. This study was conducted to determine the lymphocyte populations and adhesion molecule expression in the palatine tonsil (PT) and pharyngeal tonsil (PhT) of adult cattle and compare them with typical MALT (discrete Peyer's patches, PP) and a peripheral lymph node (parotid lymph node, PLN). The distribution of various lymphocyte subsets was determined in situ by immunofluorescence, and their proportions were determined by multicolor flow cytometry. The tonsils were similar to PP in the proportions of B- and T-cells (25-32% T-cells, 39-45% B-cells), and T cell subpopulations (CD4, CD8, and gammadelta). The PP contained the highest proportion of memory T-helper cells with beta7 integrin (30.3%+/-5.4), the tonsils intermediate (PT: 19.8%+/-4.4 and PhT: 19.7%+/-4.9), and the PLN had the lowest proportion (15.4%+/-3.1). The opposite relationship was observed with CD62L on na?ve T- helper cells as PP had the lowest proportion (14.2%+/-6.4), the tonsils intermediate (PT: 17.4%+/-2.5 and PhT: 24.3%+/-7.3), and the PLN the highest proportion (45.3%+/-6.5). MAdCAM-1 was highly expressed in the high endothelial venules (HEV) of PP, with variable and weak expression in the tonsils and PLN. PNAd, on the other hand, was highly expressed in HEV of tonsils and PLN, and weakly expressed in the PP. These results indicate that the bovine tonsils share characteristics with both PP and PLN. The alpha4beta7/MadCAM-land CD62L/PNAd interaction may be involved in lymphocyte migration to the tonsils, but it is likely that other adhesion molecules participate as well. Similarities between the human and bovine tonsils suggest that cattle may provide a good model to study the role of the tonsil in the respiratory immune response.     

Expression of potential lymphocyte trafficking mediator molecules in the mammary gland

The mammary gland performs a variety of immunological functions, including protecting itself from mastitis and protecting neonates from infectious agents. Several molecules that mediate lymphocyte trafficking in the immune system are also expressed in the mammary gland. This review is focused on the immunological function of these molecules, especially glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in the mammary gland. GlyCAM-1 is expressed in the lactating mouse mammary gland. Endothelial cells produce this protein and secrete it into milk. The glycosylated modification of mammary gland GlyCAM-1 is different from that of the lymph nodes, and lacks the binding ability for L-selectin on lymphocytes. GlyCAM-1 in the mammary gland is not involved in lymphocyte migration, and probably has another function besides that of the lymph nodes. MAdCAM-1 is expressed on endothelial cells of small venules around mouse mammary lobules during lactation. This molecule has the ability to interact with alpha4beta7 integrin on lymphocytes and mediates lymphocyte recruitment to the mammary gland. The density of beta7+/CD3+ T-cells is correlated with the density of the MAdCAM-1-stained area, suggesting that MAdCAM-1 may mediate the migration of these cells. In contrast, there is no relationship between MAdCAM-1 expression and the number of beta7+/c-IgA+ B-cells, implying that some other factor is involved in lymphocyte migration to the mammary gland. Chemokines, such as IL-8, GRO-alpha, MCP-1, RANTES and MEC, have been detected in human and mouse mammary glands. Although little information is available, these molecules may contribute to lymphocyte migration to the mammary gland.     

Differences between the ovine tonsils based on an immunohistochemical quantification of the lymphocyte subpopulations

In sheep, the pharyngeal first defence line against oral and inhaled antigens is organized in six tonsils. Since tonsils are regarded as secondary lymphoid tissue and part of the acquired immune system which is subjected to induction through contact with antigens, an evaluation of the different lymphocyte populations in tonsils is useful to determine a tendency of the specific tonsils to more inductive or more effective immunity. By means of immunohistochemistry, different lymphocyte populations were quantified and localized using a panel of eight antibodies, i.e. anti-CD45, anti-CD21, anti-CD2, anti-CD3, anti-CD4, anti-CD8, anti-WC1 and anti-Ki67. The CD21+ B lymphocytes were localized within the tonsillar lymphoid follicles. The CD2+/CD3+ T lymphocytes were numerous in the interfollicular regions and were aligned underneath and within the epithelium but were also observed at the CD21+ pole of the lymphoid follicles. Near the lingual and tubal tonsils, and the tonsil of the soft palate, the CD45+ cells around the seromucous glands and in the lamina propria were mainly CD3+ T cells. In all tonsils, the WC1+ gamma delta T cells formed a small lymphocyte population which harboured the lamina propria and the interfollicular region. The relative percentages of the different lymphocyte populations of the large palatine and pharyngeal tonsils, which are macroscopically the most developed, were comparable. In contrast, the lingual tonsil was significantly different from the other tonsils not only by its small size and lack of lymphoid follicles, but also by the lymphocyte populations. Based on the lymphocyte populations, the ovine tonsils can be divided in three groups with the tonsil of the soft palate, the tubal and paraepiglottic tonsil forming an intermediate between the palatine and pharyngeal tonsils as true tonsils on the one side, and the lingual tonsil as a scattered lymphocyte aggregation on the other side.     

Histological studies on the ontogeny of bovine palatine and pharyngeal tonsil: germinal center formation, IgG, and IgA mRNA expression

The development and distribution of lymphocyte subsets in calf palatine and pharyngeal tonsil were examined. During prenatal development, B cells were distributed in the subepithelial area, and T cells and MHC class II⁺ cells were found in the deep layer of B-cell area, respectively, in both tonsils. At neonatal stage, lymphoid follicle containing a few CD4⁺ cells have been formed in both tonsils. IgG⁺ and IgA⁺ cells were found in the parafollicular and epithelial area. At 3 months old, many germinal centers were recognized in both tonsils. CD4⁺ cells and IgG mRNA expression were detected in light zone of germinal centers. Many IgG, and IgA mRNA expressions also could be detected in the parafollicular and subepithelial area of both tonsils. The data suggest that both tonsils have an important role of local immune defense against invading antigen after birth. The comparison of the histological characteristics of tonsil and Peyer's patch during ontogeny is also discussed.     

An Immunohistochemical Study of Bovine Palatine and Pharyngeal Tonsils at 21, 60 and 300 Days of Age

An immunohistochemical study was performed on three groups of young cattle (21, 60 and 300 days of age). Tonsils (palatine and pharyngeal) and mucosae (nasal and oral) were removed. Eight monoclonal antibodies (specific for CD3, CD2, CD4, CD8, WC1, cell-surface IgM, cell-surface IgG and MHC class II molecules) and an avidin/biotin complex method on frozen sections were used. The immunological cytoarchitecture of bovine tonsils is similar to that of human tonsils. Nevertheless, these lymphoid tissues are not fully developed during the first weeks of life: T and B dependent areas not well-differentiated, few germinal centres, few intra-epithelial WC1 + T lymphocytes. In contrast, at 2 months, tonsils possess all the elements of a mucosa-associated lymphoid tissue (MALT). Tonsillar or mucosal epithelium is infiltrated by a large number of CD8+, WC1+ T lymphocytes and cells which express MHC class II molecules. Between 21 and 60 days, the number of WC1+ T lymphocytes increase markedly in the tonsillar epithelium. These results accredit the hypothesis that the presence of antigens has an effect on the localization of these lymphocytes at these sites.     

Effects on adhesion molecule expression and lymphocytes in the bovine mammary gland following intra-mammary immunisation

Changes to adhesion molecule expression and lymphocyte populations were evaluated in alveolar mammary tissue collected from cows following an immunisation protocol that involved intra-mammary inoculation to induce an IgA response in mammary secretions. The right quarters of the udder were immunised; the left side acted as a control. Antibody titres in secretions showed that at least two animals responded with antigen-specific IgA. Numbers of T-lymphocytes were 4-fold higher in immunised glands compared with controls (P < 0.05). IgA-, IgM- and IgG-positive cell numbers were significantly higher (P < 0.01) in immunised glands compared with controls in three of the four cows. No mucosal addressin molecule-1 (MAdCAM-1), vascular cell-adhesion molecule-1 (VCAM-1) or peripheral node addressin (PNAd) protein expression was detected on smaller venules that stained positively for von Willebrand factor in alveolar mammary tissues, from either immunised or control glands. Both VCAM-1 and PNAd were detected on smaller venules in supramammary lymph nodes, however, there was no significant difference between immunised and control glands. Quantification of MAdCAM-1 mRNA showed very low expression in both immunised and control alveolar tissue compared with Peyer's patch positive-control tissue. These findings suggest that the bovine mammary gland is capable of a mucosal antibody response; however, MAdCAM-1 is not involved with lymphocyte homing to the mammary gland in this species.     

Stereological assessment of the epithelial surface area of the ovine palatine and pharyngeal tonsils

Although ovine tonsils play a major role in prion diseases, their morphology is poorly documented and morphometric data are sparse. Therefore, a stereological assessment of the surface area of the palatine and pharyngeal tonsils of five sheep was performed. The epithelial surface area of both tonsils is considerably increased by the presence of tonsillar crypts and folds. The mean epithelial surface area of the paired palatine tonsil was 7.14 cm(2) and that of the pharyngeal tonsil was 23.5 cm(2). In comparison with the paired palatine tonsil, the volume of the pharyngeal tonsil was almost two times larger, whilst its surface area was more than three times larger.     

Histology, immunohistochemistry and ultrastructure of the tonsil of the soft palate of the horse

The tonsil of the soft palate was an oval, flat structure located centro-rostrally on the oral surface of the soft palate. Its stratified squamous non-keratinized epithelium was perforated by holes or small crypts the deeper parts of which were loosely spongiform inter-digitated with lymphoid tissue. These unusual features have not previously been reported in tonsils of any species. Crypts and reticulated epithelium as found in the lingual and palatine tonsils were not observed. Lectins showed varying affinities for specific layers of the epithelium. M cells were not observed. A few Langerhans cells were distributed among surface epithelial cells. Lymphoid tissue was arranged loosely and in isolated lymphoid follicles in the subepithelial lamina propria mucosae. Although IgA+ cells and macrophages were proportionately more numerous the amount of lymphoid tissue was much less than in the lingual and palatine tonsils. Most of the follicular germinal centres lacked a darkly stained corona. CD4 positive were more numerous than CD8+ lymphocytes and were distributed in the parafollicular and inter-follicular areas. Large clusters of mucus acini positive for glycogen, acidic and neutral mucopolysaccharides separated lymphoid tissue from deeply placed striated muscle. Only a few high endothelial venules were observed in the parafollicular and inter-follicular areas. These had relatively few vesiculo vacuolar or other organelles in their high endothelial cells and few lymphocytes attaching to their walls.     

Regulated expression of peripheral node addressin-positive high endothelial venules controls seeding of B lymphocytes into developing neonatal rabbit appendix

Young rabbit appendix is a homologue of chicken bursa of Fabricius; both are crucial sites for preimmune B-cell repertoire diversification. Here, we report that appendix regulates precursor lymphocyte recruitment for further development by modulating the sites of extravasation. The total area of peripheral node addressin-positive (PNAd(+)) high endothelial venules (HEVs) increased from 1 day to 1 week after birth, remained constant up to 2 weeks and declined to a low and persistent amount by 3 weeks. In normal 1-week and manipulated 5-week appendix where growth of follicles was retarded, PNAd(+) HEVs were present in the basolateral sides of B-cell follicles whereas, in normal 5-wk-appendix these were restricted to T-cell areas. The PNAd was expressed on the lumenal surface of HEVs. The proportions of CD62L(+) B cells in appendix declined from approximately 40% at 3 days to 2-3% at 4 weeks. In lymphocyte transfer experiments, CD62L(+) B cells were preferentially recruited compared with CD62L(-) B cells, anti-PNAd antibody blocked migration of B cells by approximately 50%, and 100 times more B cells were recruited in 1-week compared to 6-week appendix. Thus, a unique spatiotemporal expression pattern of PNAd(+) HEVs is associated with development of B-cell follicles. This regulates migration of blood-borne B-lymphocytes into developing appendix by interacting with CD62L.     

Preliminary age-related histological studies of the lymphoid tissues of the respiratory tract and associated structures of the brushtail possum

Extract

Lymphoepithelium was observed in palatine and pharyngeal tonsils in possums from 2 months of age, but was not seen in the lung. Morphological maturity of lymphoid tissue occurred at about 4 months of age, coinciding with first emergence of young from the pouch. Peribronchiolar and/or perivascular lymphoid aggregations were first observed in the lung of a 3.5-month-old possum, and subpleural lymphoid aggregations were first seen at 7 months of age.     

Preliminary age-related histological studies of the lymphoid tissues of the respiratory tract and associated structures of the brushtail possum

Abstract Extract Lymphoepithelium was observed in palatine and pharyngeal tonsils in possums from 2 months of age, but was not seen in the lung. Morphological maturity of lymphoid tissue occurred at about 4 months of age, coinciding with first emergence of young from the pouch. Peribronchiolar and/or perivascular lymphoid aggregations were first observed in the lung of a 3.5-month-old possum, and subpleural lymphoid aggregations were first seen at 7 months of age.     

Histological characteristics and stereological volume assessment of the ovine tonsils

Tonsils form a first line of defence against foreign antigens and therefore play a key role in immunity. Since documented information about ovine tonsils is limited, a study was performed in which the morphological characteristics and the volume of lymphoid tissue present in each ovine tonsil were determined. The tonsils of five adult healthy sheep were examined histologically and the volumes were estimated using the Cavalieri method. The pharyngeal tonsil had a mean volume of 1296.1 ± 205.9 mm³ and was by far the largest ovine tonsil, followed by the paired palatine tonsil with a mean volume of 715.0 ± 110.5 mm³. The tonsil of the soft palate, the paired tubal and paraepiglottic tonsils and the lingual tonsil were much smaller with a mean volume of, respectively, 90.3 ± 24.9 mm³, 80.1 ± 24.3 mm³, 29.7 ± 11.8 mm³ and 10.1 ± 2.8 mm³. The folds and crypts of the pharyngeal and palatine tonsils were covered by a reticular and a non-reticular epithelium. Both tonsils were mainly composed of primary and secondary lymph follicles. The palatine tonsils contained 1–3 crypts with a few secondary infoldings. Lymphoid tissue in the tonsil of the soft palate was located at the nasopharyngeal (dorsal) side of the soft palate. The tubal tonsil was lined with a pseudostratified columnar ciliated epithelium and consisted of scattered lymphoid cells and lymph follicles. The paraepiglottic tonsil consisted of lymph follicles and aggregated lymphoid cells. Its overlying keratinized stratified squamous epithelium was folded and often heavily infiltrated by lymphocytes. The ovine lingual tonsil was not macroscopically visible and did not contain clearly distinguishable lymph follicles. It consisted of aggregations of lymphoid cells that were mainly located within the vallate lingual papillae.     

Role of beta1 integrins in adhesion of canine mastocytoma cells to extracellular matrix proteins

The interactions of tumor cells with the extracellular matrix (ECM) are a crucial step in invasion and metastasis. Integrins are adhesive molecules forming heterodimers with alpha and beta subunits that play a definitive role in these interactions. In this study, mastocytoma (mast cell tumor: MCT) cell-ECM interaction was investigated using 3 canine MCT cell lines: CM-MC (originating from cutaneous MCT), VI-MC (originating from intestinal MCT), and CoMS (originating from oral MCT). Flow cytometric analysis showed that all cells highly expressed the integrin beta1 and alpha1 through alpha5 subunits and that they moderately expressed the alpha6 subunit. In adhesion studies, CoMS weakly but spontaneously adhered to fibronectin (FN), which was enhanced by phorbol ester (TPA), while CM-MC and VI-MC required cell activation by TPA to adhere to FN. Anti-beta1 and alpha5 integrin antibodies strongly inhibited cell adhesion to FN in CM-MC and CoMS and moderately inhibited cell adhesion in VI-MC. Only VI-MC adhered to laminin (LN) under activation by TPA. Anti-beta1 integrin antibodies strongly inhibited cell adhesion to LN, but all anti-alpha integrin antibodies failed to inhibit cell adhesion to LN. No cells adhered to collagen types I and IV. Canine MCT cells from different origins expressed similar integrin patterns; however, there were some differences in adhesive behavior in response to various ECM proteins and activating stimuli.     

Histology, immunohistochemistry and ultrastructure of the equine palatine tonsil

The palatine tonsils of five young horses formed 10-12 cm elongated follicular structures extending from the root of the tongue on either side to the base of the epiglottis and lateral to the glossoepiglottic fold. The stratified squamous non-keratinized epithelium of the outer surface was modified into crypts as reticular epithelium by heavy infiltration of lymphoid cells from underlying lymphoid follicles. In places, lymphoid tissue reaching almost to the surface and with only one to two cell layers intact was identified as the lymphoepithelium. Langerhans cells with Birbeck granules were interspersed between epithelial cells. Lymphoid tissue organized in lymphoid follicles constituted the parenchyma of the palatine tonsil. CD4-positive cells were more numerous and CD8-positive lymphocytes less numerous compared with their distribution in the lingual tonsil. B cells and macrophages were also more numerous than in the lingual tonsil and lectins showed a different pattern of attachment. M cells were not observed. High endothelial venules with well-developed vesiculo-vacuolar organelle had structural evidence of transendothelial and interendothelial migration of lymphocytes. Striated muscles as seen in the deeper lamina propria mucosae of the lingual tonsil were absent. The immunohistological and ultrastructural characteristics of the equine palatine tonsil are similar to those of humans but differ from those of the lingual tonsil and are consistent with a role as an effector and inductor immunological organ.     

Anatomical localisation and histology of the ovine tonsils

The topography and histologic structure of the various tonsils were studied anatomically and microscopically in 15 sheep aged between 9 and 15 months. The palatine, pharyngeal and paraepiglottic tonsils were readily visible macroscopically. They consisted mainly of secondary lymph nodules and were encapsulated in dense connective tissues. The epithelium covering the tonsils and their crypts was frequently infiltrated heavily by lymphocytes. The tubal tonsil and the tonsil of the soft palate were macroscopically visible after fixation in 2% acetic acid. These tonsils consisted of scattered lymph nodules, aggregations of lymphocytes and diffuse lymphoid tissue. They were not encapsulated, and therefore the borders of these tonsils could not be clearly delineated. The lingual tonsil was not macroscopically visible in sheep and consisted of scattered small aggregations of lymphocytes.     

Immunopathology of Bartonella vinsonii (berkhoffii) in experimentally infected dogs.

Following natural infection with Bartonella, dogs and humans develop comparable disease manifestations including endocarditis, peliosis hepatis, and granulomatous disease. As the immunologic response to infection in these hosts has not been clearly established, data presented here was derived from the experimental infection of six specific pathogen free (SPF) beagles with a known pathogenic strain of Bartonella. Six dogs were inoculated intravenously with 10(9)cfu of B. vinsonii ssp. berkhoffii and six control dogs were injected intravenously with an equivalent volume of sterile saline. Despite production of substantial levels of specific antibody, blood culture and molecular analyses indicated that Bartonella established chronic infection in these dogs. Flow cytometric analysis of monocytes indicated impaired bacterial phagocytosis during chronic Bartonella infection. There was also a sustained decrease in the percentage of CD8+ lymphocytes in the peripheral blood. Moreover, modulation of adhesion molecule expression (downregulation of L-selectin, VLA-4, and LFA-1) on CD8+ lymphocytes suggested quantitative and qualitative impairment of this cell subset in Bartonella-infected dogs. When compared with control dogs, flow cytometric analysis of lymph node (LN) cells from B. vinsonii infected dogs revealed an expanded population of CD4+ T cells with an apparent na?ve phenotype (CD45RA+/CD62L+/CD49D(dim)). However, fewer B cells from infected dogs expressed cell-surface MHC II, implicating impaired antigen presentation to helper T cells within LN. Taken together, results from this study indicate that B. vinsonii establishes chronic infection in dogs which may result in immune suppression characterized by defects in monocytic phagocytosis, an impaired subset of CD8+ T lymphocytes, and impaired antigen presentation within LN.     

The effect of experimental infectious mastitis on leukocyte subpopulations and cytokine production in non-lactating ewes.

The interactions between leukocytes and cytokines during the acute response to intramammary infections in the dry mammary gland of sheep were studied. Dry ewes were experimentally infected in one udder half with either Staphylococcus aureus or Escherichia coli, or infused with saline as control. Udder secretion samples, blood samples and udder tissue samples were collected before and 4, 8 and 24 h after infections/infusions. Total and differential leukocyte counts were calculated in both blood and mammary secretions, and flow cytometry was used to detect the presence of CD4+, CD8+, WC1+, IL-2R+, CD18+ or L-selectin + lymphocytes, CD18+ or L-selectin + neutrophils, and CD14+ leukocytes. Moreover, the concentrations of interleukin-1 beta (IL-1 beta), interleukin-8 (IL-8) and granulocyte-macrophage colony stimulating factor (GM-CSF) in udder secretions were measured using ELISA, and RT-PCR was used to detect the presence of corresponding cytokine mRNA in udder tissue biopsies. The results suggest an association between the concentrations of IL-1 beta, IL-8 and the intensity of neutrophil infiltration of the infected gland. Immunologically relevant changes in proportions of lymphocyte subpopulations might also occur in the acute phase of the inflammatory reaction of the udder. Greater cellular and cytokine responses to E. coli infection may have contributed to the milder clinical picture and more rapid resolution of infection than that seen for S. aureus. Enhancing the production of pro-inflammatory cytokines may improve defence against bacterial mastitis.     

Feline Langerhans cells migrate from skin and vaginal mucosa to regional lymph nodes during experimental contact sensitization with fluorescein isothiocyanate

Recently, feline Langerhans cells (LC) were immunophenotypically characterized as CD1a+, CD4+, CD18+, CD53+ and MHC II+ cells. In mice, these cells are known to internalize antigens and to migrate to the lymph nodes (LN). In the cat, we have investigated the migration of LC from the skin and vaginal mucosa to regional LN in response to chemical exposure (fluorescein isothiocyanate). Three days after the administration of a FITC solution on the posterior limb of two male cats and in the vagina of one female, a biopsy was carried out on the draining LN of the sensitized zones. Immunostaining with monoclonal antibodies anti-CD79, anti-CD8, and antibodies recognizing LC was performed on cytospins and frozen sections of LN and showed that a majority of FITC+ cells displayed a LC immunophenotype and were localized in T-cell areas, but not in follicular areas. These results are the first evidence of migration of feline LC from skin and vaginal mucosa to the regional LN.