Immuno-electron microscopy of the thymic epithelial microenvironment.

Normal T cell development depends upon interactions between progenitor cells and the thymic microenvironment. Monoclonal antibodies (Mabs) have been used to define subtypes of thymic epithelium by light microscopy (clusters of thymic epithelial staining [CTES]). We have now used a range of these Mabs together with gold-coupled reagents in immuno-electron microscopy to study the fine cellular distribution of the molecules to which the antibodies bind. Anti-cytokeratin antibodies were used to identify all thymic epithelial cells, while the distribution of MHC class II molecules was revealed with Mabs to shared nonpolymorphic determinants. MR6, a CTES III Mab, shows strong surface labelling of cortical epithelial cells and thymic nurse cells and very weak surface staining of thymocytes, medullary macrophages, and interdigitating cells. Mab 8.18 (CTES V) also labels a cell surface molecule; this is present on Hassall's corpuscles and associated medullary epithelial cells. The molecules detected by Mabs MR6 and 8.18 are therefore located in a position where they are available to interact with external cellular and soluble signals within the thymus. In contrast, Mabs MR10 and MR19 (CTES II) recognise intracellular molecules within subcapsular, perivascular, and medullary epithelium. A similar distribution was seen with Mab 4beta, directed against the thymic hormone thymulin, although, in addition to the expected intracellular epithelial staining, large lymphoblasts in the subcapsular zone showed surface positivity, indicating the presence of thymulin bound to surface receptors on these early lymphoid cells. As expected, MHC class II molecules were expressed on some medullary and essentially all cortical epithelial cells. However, although most subcapsular epithelium was class II-negative, some cells did express these MHC molecules on their apical surface and on the surface of their cytoplasmic extensions into the cortex. Interestingly, some cortical epithelial cells surrounding capillaries were positive for both MR6 (CTES III) and for MR10, MR19, and 4beta (CTES II). Double-labelling experiments, using MR6 and MR19 simultaneously, revealed a double-positive perivascular epithelial cell population in the thymic cortex. The possibility that these cells represent a thymic epithelial progenitor population is discussed.

Beta-adrenoceptor-mediated effects in rat cultured thymic epithelial cells.

1. Sympathetic nerves were visualized in sections from rat thymus by immunostaining of tyrosine hydroxylase, the rate-limiting enzyme of catecholamine biosynthesis, and by glyoxylic acid-induced fluorescence of catecholamines. Catecholaminergic nerve fibres were detected in close connection to thymic epithelial cells which therefore might be preferred target cells. To evaluate this, rat immunocytochemically defined, cultured thymic epithelial cells were investigated for adrenoceptors and adrenergic effects. 2. In rat cultured thymic epithelial cells mRNA for beta 1- and beta 2-adrenoceptors was detected by reverse transcription-polymerase chain reaction by use of sequence-specific primers. Specific, saturable binding to the cultivated cells was observed with the beta-adrenoceptor agonist CGP 12177. 3. Adrenaline, noradrenaline or the beta-adrenoceptor agonist, isoprenaline, increased intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels in cultivated thymic epithelial cells dose-dependently about 25 fold. The pharmacological properties revealed that this response was mediated by receptors of the beta 1- and the beta 2-subtypes. The selective beta 3-adrenoceptor agonist BRL 37344 had no effect on cyclic AMP levels. The increase in cyclic AMP was downregulated by preincubation with glucocorticoids like dexamethasone or cortisol which also changed the relative importance of beta 1-/beta 2-adrenoceptors to the response. 4. Incubation with isoprenaline or the adenylate cyclase activator forskolin decreased basal and serum-stimulated proliferation of thymic epithelial cells. However, adrenergic stimulation of thymic epithelial cells did not induce interleukin 1 production. Since thymic epithelial cells create a microenvironment which influences the maturation and differentiation of thymocytes to T-lymphocytes, their observed capacity to respond to catecholamines provides novel evidence for the suggestion that adrenergic stimulation may interfere with the regulation of immune functions.

Studies on rat and human thymus to demonstrate immunoreactivity of calcitonin gene-related peptide, tyrosine hydroxylase and neuropeptide Y.

The peptidergic and noradrenergic innervation of rat and human thymus was investigated by immunohistochemistry at the light and electron microscopical level (avidin-biotin-complex, sucrose-phosphate-glyoxylic-acid, and immunogold techniques). The distribution of noradrenergic neural profiles, and positive immunoreactivity for calcitonin gene-related peptide (CGRP), tyrosine hydroxylase (TH) and neuropeptide Y (NPY) is described in female rats during ageing, and in human children. In the neonatal rat thymus, the arteries and septa are well supplied by fine varicose nerves. In older animals (2 wk-1 y) the number of septa and blood vessels increase and consequently also the innervation. No nerves were found in the cortex. Apart from the innervation of the septal areas, immunoreactivity for CGRP and TH was present in thymic cells. Except for the young rats (neonatal-14 d), all rats showed CGRP positivity in subcapsular/perivascular epithelial cells (type 1 cells). All rat thymuses also contained a few TH positive cells in the medulla, which could only be confirmed as epithelial cells (type 6 cells) in children. Type 1 cells in the human thymus were not CGRP positive, but as in the rat, there were similar TH positive cells in the medulla. It was concluded that in addition to nerves containing CGRP, noradrenaline or dopamine, epithelial cells also contain these transmitters. They could therefore act on different cells (compared with neural targets) in a paracrine manner.

Rat thymic epithelial cells in vitro and in situ: characterization by immunocytochemistry and morphology.

A new method for the long-term culture of pure rat thymic epithelial cells was established. The cultures were characterized by immunocytochemistry, electron microscopy and proliferation assays. Non-epithelial thymic cells were eliminated with a reliable and reproducible pre-plating method, by differential trypsin treatment of the cultures and by addition of horse serum to the culture medium instead of fetal calf serum. The final cultures contained more than 95% pure epithelial cells as evidenced by immunostaining for cytokeratin. Ultrastructural studies indicated that these cells are physiologically active epithelial cells with tonofilaments, desmosomes and filopods. The subsets of the thymic epithelial cells in vitro were investigated by comparing their staining pattern with that obtained in situ using several subtype-selective antibodies. Thymic epithelial cells in vitro showed a preferential expression of subcapsular/perivascular and medullary markers. Only few cultivated cells were of cortical origin. In the first to the fourth subcultures, some cells were immunopositive for the thymus hormone/factor thymulin. The proliferation of thymic epithelial cells was stimulated by horse serum and to a lesser extend by fetal calf serum. The adenylate cyclase activators isoproterenol and forskolin, and the glucocorticoid cortisol inhibited the proliferation.

Immunohistochemical localization of cytokines and receptor-associated molecules in human tonsils and skin.

Human tonsilla palatina and skin were investigated by means of light microscopical and electron microscopical immunohistochemistry using monoclonal antibodies (Mab) against some cytokines. In both tonsils and skin we found intracellular immunoreactivity for interleukin-1-beta in macrophages and interdigitating cells. Also some, but not all crypt-epithelial cells were positive, while keratinocytes in the skin were negative. Interleukin-2-immunoreactivity was found in a subpopulation of lymphocytes (probably T-cells) and unexpectedly also in some antigen-presenting cells (APCs). A Mab against interleukin-4 revealed weak labelling of lymphatic cells in the T-cell area of tonsils. The human skin was negative. A Mab that recognizes a molecule associated with the interleukin-4-receptor gave strong surface labelling in tonsils and skin on APCs and weak immunoreactivity on lymphoid cells. Frequently these APCs formed rosettes with weakly labeled lymphocytes. Mabs against interleukin-8 stained starry sky macrophages in the germinal centers of the tonsil and different APCs in the T-cell region. IL-8 is stored in keratinocytes of normal skin, but becomes mobilized under inflammatory conditions. Our results expand the understanding of cell cell-interactions under normal and inflammatory conditions in tonsil and skin.

Calcitonin gene-related peptide and its receptor in the thymus.

Calcitonin gene-related peptide (CGRP), a 37-amino acid residue neuropeptide, was immunostained in rat thymus at two sites: a subpopulation of thymic epithelial cells, namely subcapsular/perivascular cells, were heavily stained besides some nerve fibers surrounding arteries and arterioles. The administration of nanomolar concentrations of rat alpha-CGRP dose-dependently raised intracellular cyclic adenosine monophosphate (cAMP) levels in isolated rat thymocytes (half-maximum stimulation 1 nM) but not in cultured rat thymic epithelial cells. Peptides structurally related to CGRP (i.e., rat calcitonin or amylin) had no effect. CGRP(8-37), an N-terminally truncated form, acted as an antagonist. Peripheral blood lymphocytes did not respond to CGRP, suggesting that receptors are present only on a subpopulation of thymocytes but not on mature T cells. This was substantiated by visualization of CGRP receptors on single cells by use of CGRP-gold and -biotin conjugates of established biological activity: only a small proportion of isolated thymocytes was surface labeled. In situ, the CGRP conjugates labeled receptors on large thymocytes residing in the outer cortical region of rat thymus pseudolobules. Thus, immunoreactive CGRP is found in subcapsular/perivascular thymic epithelial cells and acts via specific CGRP receptors on thymocytes by raising their intracellular cAMP level. It is suggested that CGRP is a paracrine thymic mediator that might influence the differentiation, maturation, and proliferation of thymocytes.

The noradrenergic innervation of the rat thymus during pregnancy and in the post partum period.

The noradrenergic innervation of the rat thymus during pregnancy and the post partum period was examined by a sucrose glyoxylic acid method for catecholamines, and by high pressure liquid chromatography. Fluorescent nerves decreased in number throughout pregnancy when there was an overall loss in thymic weight due to cortical involution. These changes are maximal by parturition. There was a dramatic increase in nerves between d 21 of pregnancy and d 1 after parturition, especially in the capsule and around blood vessels in the connective tissue septa. The neonates were removed at parturition and thymic weight was rapidly regained. The increased numbers of nerves remained throughout this post partum period. Noradrenaline levels in the thymus altered in a similar pattern throughout pregnancy and the post partum period, but did not parallel thymic weight changes. The mean noradrenaline concentration in the virgin thymus was 1063 +/- 107 pg/mg protein. Levels remained similar during early pregnancy and increased significantly at d 16. Virgin levels were regained by d 21. Values peaked after parturition but rapidly decreased over the next 3 days, and remained at or below virgin levels to d 28 except for a transient rise at d 10 post partum. Adrenaline values were consistently below detection levels. This study shows that there are variations in both nerves visualised, and in neurotransmitter (noradrenaline) content in the thymus during the course of pregnancy and the post partum period. Thus thymic function could be influenced by central events (levels of catecholamines in peripheral blood) as well as local events mediated by transmitter changes in nerves.

Ultrastructural identification and distribution of the adhesion molecules ICAM-1 and LFA-1 in the vascular and extravascular compartments of the human palatine tonsil.

Immunohistological analysis of sections prepared from human palatine tonsils revealed marked differences in the distribution of the adhesion molecule, leucocyte function antigen-1 (LFA-1) and its counter receptor, intercellular adhesion molecule-1 (ICAM-1). Light microscopy showed that LFA-1 was restricted to the leucocytes, particularly the lymphocytes. In contrast, staining of ICAM-1 was predominantly confined to the vascular endothelium with the greatest expression seen on the morphologically distinct high endothelial venules in the parafollicular areas; these are the sites that appear to support lymphocyte migration. Electron microscopy revealed that ICAM-1 was present on the luminal and lateral surfaces of the high endothelium and absent from the abluminal surface supported by basal lamina. The ICAM-1 was also absent from those surfaces of the endothelium that were in close contact with intravascular lymphocytes. Other cells stained by the anti-ICM-1 antibody included dendritic cells, plasma cells and epithelial cells in the reticulated crypt epithelium and in the upper strata of the non-keratinised stratified squamous epithelium. The high expression of LFA-1 was most prominent on lymphocytes, low on antigen-presenting cells and activated lymphoid cells, and not detectable on plasma cells, epithelial and endothelial cells. We propose that LFA-1/ICAM-1 binding participates in mediating the transendothelial migration of lymphocytes across the high endothelial venules of palatine tonsil.

Infiltration of both T cells and neutrophils in the skin is accompanied by the expression of endothelial leukocyte adhesion molecule-1 (ELAM-1): an immunohistochemical and ultrastructural study.

Various inducible adhesion molecules on human endothelial cells like the endothelial leukocyte adhesion molecule-1 (ELAM-1) seem to be the basis of mechanisms that allow peripheral blood leukocytes to enter precisely areas of inflamed tissue. Because in vitro data had shown that ELAM-1 plays a central role in neutrophil as well as memory T-cell endothelium interactions, we analyzed in vivo at the light and electron microscopic level its expression in various benign and malignant skin diseases, which differ in the composition of the cellular infiltrates. The expression of ELAM-1 on endothelial cells at different anatomical sites could be demonstrated independently from the cell type (neutrophils/memory T cells) infiltrating the surrounding tissue. On the ultrastructural level we demonstrate that the expression of ELAM-1 is restricted to certain segments of post-capillary venules exhibiting distinctive morphologic features. The ELAM-1-positive endothelia are identical to those vessels that are currently described to be the preferred sites of lymphocyte trafficking in diseased skin.

Functional histology of the human thymus.

The thymus develops from a paired epithelial anlage in the neck. This review considers how ectoderm (vesicula cervicalis) and endoderm (third pharyngeal pouch) contribute to the epithelial stroma of the thymus. Stromal elements of mesodermal origin are capillaries, septae and perivascular spaces and single invading cells. These elements separate the thymus into pseudolobuli. The thymus epithelial space and the perivascular spaces are always separated from each other by a closed, flat epithelial cell layer, with a basal lamina which contributes to the blood-thymus barrier. From the 9th gestational week, prethymic precursor cells from hemopoietic centers, begin to invade the thymus anlage. There they finally mature to committed post-thymic T cells. The thymus microenvironment of postnatal thymus is composed of six different types of epithelial cells and several stromal cells of mesodermal origin. The location of these diverse stationary cells is described, and their functional significance is discussed. Obviously these stromal cell types have a special function in providing the proper environment for T-cell maturation. The function of the thymus includes the maturation and/or selection of antigen specific T-cells. The main issue of intra-thymic T-cell differentiation is the development and expression of T-cell-antigen receptors. The great diversity of these receptors is generated by a rearrangement of the T-cell-receptor-genes in order to furnish the host with a mature T-cell repertoire that is capable of recognizing the world of extrinsic antigens. In a synopsis the manyfold interrelationships between the thymus microenvironment and the developing thymocytes are summarised.

Crystalline and amorphous gold in chrysiasis.

Skin biopsy specimens from five patients (three females and two males) treated parenterally with gold were investigated using transmission electron microscopy. X-ray microanalysis and electron diffraction were used to determine the dermal heavy metal content. Additional sections were stained for light microscopic examination. The amount of elemental gold administered to the patients over a period of years to alleviate rheumatoid arthritis lay between a minimum of 4.0 g and a maximum of 10.0 g. In one and the same patient dermal histiocytic gold aggregations in sun-exposed areas of skin displayed a different pattern and divergent physiochemical states from the gold deposits in non-UV-exposed skin, where aurosome-like amorphous formations are found in the cells of the upper dermis. Additional spherical particles are associated predominantly with phagolysosomes in melanophages beneath solar-irradiated epidermis. Convergent beam electron diffraction proves the crystalline nature of the spherical auriferous deposits. The occurrence of skin rash was not related to different physicochemical states of the precious metal.

Immunoelectron microscopic localization of MHC structures in isolated pancreatic rat islets.

An immunogold-silver enhancement technique, which combines effective labeling of viable isolated islets with the ultrastructural resolution of cytological details, was applied in electron microscopy to identify major histocompatibility complex (MHC) structures on islet cells. Incubation of freshly isolated islets from CAP (RT1c) and LEW (RT1l) rats with OX18, an MHC class I antibody, showed strong positive reactivity in macrophages and/or dendritic-like cells (M0-DCs) and vascular endothelial cells (VEs) and a comparatively weaker reactivity in endocrine alpha-, beta-, and delta-cells. With MHC class II antibody OX6 (anti-I-A), M0-DCs were strongly labeled in both rat strains on the surface and on internal structures. Three of five particularly high titered batches of OX6 revealed MHC class II expression on VE and beta-cells. Four days of in vitro culture in combination with a high concentration of glucose and interferon-gamma induced strong enhancement of MHC class I structures and, to a lesser extent, class II structures on beta-cells.

Analysis of the fine distribution of thymic epithelial microenvironmental molecules by immuno-electron microscopy.

Normal T cell development depends upon an interaction between progenitor cells and their microenvironment. Previously raised monoclonal antibodies to subtypes of human thymic epithelium were used with gold-coupled reagents in immuno-electron microscopy to study the fine cellular distribution of the molecules to which these Mabs bind. Mab MR6, thought to recognize the human IL-4 receptor, shows strong surface labeling of cortical epithelial cells and thymic nurse cells, and very weak staining of thymocytes, medullary macrophages and interdigitating cells. Mab 1st 8.18 labels the surface of Hassall's corpuscles and associated medullary epithelial cells. The molecules detected by these two antibodies are therefore located in a position where they are available to interact with external cellular and soluble signals within the thymus. In contrast Mabs MR10 and 19 recognize Intracellular molecules within subcapsular, perivascular and some medullary epithelium. These molecules may represent soluble material awaiting secretion from the cell; alternatively, they may be internal structural proteins.

Development and functional anatomy of the human tonsilla palatina.

The tonsilla palatina, belonging to the gut-associated lymphoepithelial organs (GALT), shows close morphological and functional correlation between the lymphatic tissue of mesenchymal origin and the endodermal epithelium of the second pharyngeal pouch. During the 4th fetal month epithelial crypts grow down into the connective tissue and are infiltrated by non-epithelial cells. In the underlying lymphoid tissue the first primary follicles with precursors of follicular dendritic reticulum cells can be localized already in the 14th fetal week post conceptionem (p.c.). The parafollicular areas develop into T-cell regions. Interdigitating cells in these regions as well as within the crypt epithelium express the HLA-DR antigen. They were frequently found in close contact with T-helper cells. This fact is discussed as an indication of the tonsillar crypt epithelium partly serving as T-cell region. But also B-cells and macrophages invade the crypt epithelium in great numbers with a distinctly different distribution. Natural killer (NK) cells (Leu-7-positive) are localized within the crypt epithelium. Small Leu-7-positive lymphocytes (probably precursors) have been found to be numerous in the germinal centers. The interaction of specific stationary cells in the T-cell and B-cell regions is obviously an important factor for the maturation of different subpopulations of immunocompetent lymphoid cells.

Interaction of rat peritoneal macrophages with homologous, sialidase-treated lymphocytes in vitro.

The interaction in vitro between rat peritoneal macrophages and homologous, sialidase-treated lymphocytes was investigated. Lymphocytes were isolated from blood, thymus, and spleen on a density gradient. Total sialic acids obtained by acid hydrolysis were 10 nmol/10(8) lymphocytes, composed of 29% N-acetyl-neuraminic acid and 71% N-glycoloylneuraminic acid. Sialidase treatment released maximally 33% of membrane sialic acids. Lymphocytes were bound to peritoneal macrophages to an extent which increased in parallel with the amount of sialic acids released, whereas binding of untreated lymphocytes was not significant. This interaction was inhibited by free galactose and substances containing terminal galactose residues. Asialoorosomucoid with its oligoantennary sugar chains proved to be a 10(5) times more potent inhibitor of the interaction than lactose. The addition of homologous serum had no influence on binding. Electron microscopy revealed that vital lymphocytes were tightly bound to macrophages and only damaged lymphocytes appeared to be phagocytozed. The experiments demonstrate that the interaction between rat peritoneal macrophages and sialidase-treated lymphocytes is mediated by a macrophage receptor specific for galactose. This sugar is demasked on the surface of lymphocytes after the removal of terminal sialic acids. The role of this mechanism in cell recognition, elimination and homing of lymphocytes is discussed.

Immunohistochemical characterization of the thymic microenvironment. A light-microscopic and ultrastructural immunocytochemical study.

The epithelial framework of the human thymus has been studied in parallel by immunohistochemical methods at the light- and electron-microscopic levels. Different monoclonal antibodies were used, reacting with components of the major histocompatibility complex, keratins, thymic hormones and other as yet antigenically undefined substances, which show specific immunoreactivities with human thymus epithelial cells. The electron-microscopic immunocytochemical observations clearly confirm microtopographical differences of epithelial cells not only between the thymic cortex and medulla, but also within the cortex itself. At least four subtypes of epithelial cells could be distinguished: 1) the cortical surface epithelium; 2) the main cortical epithelial cells and thymic nurse cells; 3) the medullary epithelial cells; and 4) the epithelial cells of Hassall's corpuscles. The various epithelial cell types of the thymus display several common features like tonofilaments, desmosomes and some surface antigens as demonstrated by anti-KiM3. In other respects, however, they differ from each other. The cortical subtype of thymic epithelial cells including the thymic nurse cells shows a distinct pattern of surface antigens reacting positively with antibodies against HLA-DR (anti-HLA-DR) and anti-21A62E. Electron-microscopic immunocytochemistry with these antibodies clearly reveals a surface labeling and a narrow contact to cortical thymocytes particularly in the peripheral cortical regions. An alternative staining pattern is realized by antibodies to some antigens associated with other subtypes of thymic epithelial cells. Medullary epithelial cells as well as the cortical surface epithelium react likewise positively with antibodies to special surface antigens (anti-Ep-1), to special epitopes of cytokeratin (anti-IV/82), and to thymic hormones (anti-FTS). The functional significance of distinct microenvironments within the thymus provided by different epithelial cells is discussed in view of the maturation of T-precursor cells.