Macrophages and epithelial cells of the thymus gland. An ultrastructural study in the natterjack, Bufo calamita

In the present study, the ultrastructure of the stromal components, basically epithelial elements and macrophages, of the thymus of adult natterjacks, Bufo calamita has been analyzed. A network of stellate epithelial-reticular cells joined together by desmosomes, constitutes the main component of the thymic parenchyma in both cortex and medulla. In the medulla pale, electron-lucent epithelial cells, sometimes showing surface interdigitations, are striking elements. Moreover, uni- and multicellular epithelial cysts appear in the thymic medulla as well as granulated cells of possible endocrine significance. Remarkably, isolated or grouped gland cells whose morphology and cytoplasmic content resemble that of the skin glands, were occasionally found. Finally, macrophages, multinucleated giant cells and dendritic-like cells, the latter intimately associated to lymphocytes, occur in the thymus of Bufo calamita. The most remarkable morphologic characteristics of all those non-lymphoid cell types, as well as their possible functional significance are comparatively discussed with available information on the amphibian and higher vertebrate thymic cytoarchitecture.     

The thymus microenvironment in regulating thymocyte differentiation

The thymus plays a crucial role in the development of T lymphocytes providing an inductive microenvironment in which committed progenitors undergo proliferation, T-cell receptor gene rearrangements and thymocyte differentiation into mature T-cells. The thymus microenvironment forms a complex network of interaction that comprises non lymphoid cells (e.g., thymic epithelial cells, TEC), cytokines, chemokines, extracellular matrix elements (ECM), matrix metalloproteinases and other soluble proteins. The thymic epithelial meshwork is the major component of thymic microenvironment, both morphologically and phenotypically limiting heterogeneous regions in thymic lobules and fulfilling an important role during specific stages of T-cell maturation. The process starts when bone marrow–derived lymphocyte precursors arrive at the outer cortical region of the thymic gland and begin to mature into functional T lymphocytes that will finally exit the thymus and populate the peripheral lymphoid organs. During their journey inside the thymus, thymocytes must interact with stromal cells (and their soluble products) and extracellular matrix proteins to receive appropriate signals for survival, proliferation and differentiation. The crucial components of the thymus microenvironment and their complex interactions during the T-cell maturation process with the objective of contributing to a better understanding of the function of the thymus as well as assist in the search for new therapeutic approaches to improve the immune response in various pathological conditions are summarized here.     

A scanning electron-microscopic study of the rat thymus with special reference to cell types and migration of lymphocytes into the general circulation

Summary The three-dimensional structure of the rat thymus was studied by combined scanning and transmission electron microscopy. The thymus consists mainly of four types of cells: epithelial cells, lymphocytes, macrophages, and interdigitating cells (IDCs).The epithelial cells form a meshwork in the thymus parenchyma. Cortical epithelial cells are stellate in shape, while the medullary cells comprise two types: stellate and large vacuolated elements. A continuous single layer of epithelial cells separates the parenchyma from connective tissue formations of the capsule, septa and vessels. Surrounding the blood vessels, this epithelial sheath is continuous in the cortex, while it is partly interrupted in the medulla, suggesting that the blood-thymus barrier might function more completely in the cortex.Cortical lymphocytes are round and vary in size, whereas medullary lymphocytes are mainly small, although they vary considerably in surface morphology.Two types of large wandering cells, macrophages and IDCs, could be distinguished, as well as intermediate forms. IDCs sometimes embraced or contacted lymphocytes, suggesting their role in the differentiation of the latter cells.Perivascular channels were present around venules and some arterioles in the cortico-medullary region and in the medulla. A few lymphatic vessels were present in extended perivascular spaces.The present study suggests the possible existence of two routes of passage of lymphocytes into the general circulation. One is via the lymphatics, while the other is through the postcapillary venules into the blood circulation. Our SEM images give evidence that lymphocytes use an intracellular route, i.e., the endothelium of venules.     

p73 is expressed in human thymic epithelial cells.

The thymus is a heterogeneous immune organ in which immature T-cells develop and eventually specialize to make certain immune responses of their own. Among various types of stromal cells in the thymus, thymic epithelial cells (TECs) have a crucially important function for presenting self-antigens and secreting cytokines to thymocytes for their maturation into T-cells. In this study we show that the p73 gene, a homologue of the tumor suppressor gene p53, was expressed in the nucleus of the human TEC in vivo and in TEC lines in vitro. Because p73 has the capacity to be a transactivator like p53, it may contribute to T-cell development in the context of TEC biology as regulated in the cell cycle and apoptosis.     

The thymus microenvironment in regulating thymocyte differentiation

The thymus plays a crucial role in the development of T lymphocytes by providing an inductive microenvironment in which committed progenitors undergo proliferation, T-cell receptor gene rearrangements and thymocyte differentiate into mature T cells. The thymus microenvironment forms a complex network of interaction that comprises non lymphoid cells (e.g., thymic epithelial cells, TEC), cytokines, chemokines, extracellular matrix elements (ECM), matrix metalloproteinases and other soluble proteins. The thymic epithelial meshwork is the major component of the thymic microenvironment, both morphologically and phenotypically limiting heterogeneous regions in thymic lobules and fulfilling an important role during specific stages of T-cell maturation. The process starts when bone marrow-derived lymphocyte precursors arrive at the outer cortical region of the thymic gland and begin to mature into functional T lymphocytes that will finally exit the thymus and populate the peripheral lymphoid organs. During their journey inside the thymus, thymocytes must interact with stromal cells (and their soluble products) and extracellular matrix proteins to receive appropriate signals for survival, proliferation and differentiation. The crucial components of the thymus microenvironment, and their complex interactions during the T-cell maturation process are summarized here with the objective of contributing to a better understanding of the function of the thymus, as well as assisting in the search for new therapeutic approaches to improve the immune response in various pathological conditions.Key words: thymus, T-cell maturation, thymic microenvironment, thymocyte differantiation, chemokines, extracellular matrix, thymic nurse cells, metalloproteinases     

Repopulation of the mouse thymus after sublethal fission neutron irradiation. II. Sequential changes in the thymic microenvironment

The stromal cells of the thymus of sham-irradiated and sublethal fission neutron-irradiated CBA/H mice were analyzed with immunohistology, using monoclonal antibodies directed to I-A and H-2K antigens as well as specific determinants for cortical and medullary stromal elements. In the control thymuses, I-A expression in the thymus shows a reticular staining pattern in the cortex and a confluent staining pattern in the medulla. In contrast, H-2K expression is mainly confluently located in the medulla. Whole body irradiation with 2.5 Gy fission neutrons reduces within 24 hr the cortex to a rim of vacuolized "nurse cell-like" epithelial cells, largely depleted of lymphoid cells. The localization of I-A antigens changes in the cortex and I-A determinants are no longer associated with or localized on epithelial reticular cells. Medullary stromal cells, however, are more or less unaffected. A high rate of phagocytosis is observed during the first 3 days after irradiation. About 5 days after irradiation, the thymus becomes highly vascularized and lymphoid cells repopulate the cortex. The repopulation of the thymic cortex coincides with the appearance of a bright H-2K expression in the cortex which is associated with both stromal cells as well as lymphoid blasts. During the regeneration of the thymus, the thymic stromal architecture is restored before the expression of cell surface-associated reticular MHC staining patterns. The observed sequential changes in the thymic microenvironment are related to the lymphoid repopulation of the thymus.     

Characterization of an antigenic determinant preferentially expressed by type I epithelial cells in the murine thymus.

A hamster monoclonal antibody (MAb), designated 8.1.1, was raised against murine thymic stromal cell lines and was found to react with cell surface molecules expressed by a morphologically distinct population of epithelial cells of the murine thymus comprising the subcapsular environment, cells investing vascular structures throughout the thymus, and some of the cellular elements in the medulla. The epithelial nature of the labeled cells was confirmed with immunoelectron microscopy. Reactivity with MAb 8.1.1 was associated with thymic epithelial cells in contact with basal laminae. Ontological studies of thymic tissue demonstrated that the epitope recognized by this MAb was expressed before Day 14 of gestation, although the restricted subcapsular and medullar expression of 8.1.1 was not apparent until sometime after birth. MAb 8.1.1 also reacted with a number of extra-thymic tissues, including lamina propria of gut, glomeruli and tubules in the kidney, mesothelia covering a number of organs, and the dermis and epidermis of skin. Within the epidermis, reactivity of MAb 8.1.1 was largely restricted to basal epithelial cells. Immunochemical analysis of 8.1.1 reactivity with detergent-soluble extracts of thymic stromal cell lines and thymus tissue indicated that detergent-soluble extracts of thymic stromal cell lines and thymus tissue indicated that the epitope recognized by this MAb was associated with a glycoprotein bearing terminal N-acetylglucosamine residues and possessing an Mr of approximately 36-38 KD under reducing or non-reducing conditions.     

The fine structure of the thymus of the fetal and neonatal monkey (Macaca mulatta)

Summary The morphologic features of the fetal and neonatal thymus were investigated by light and electron microscopy to determine developmental changes. Primitive epithelial cells differentiate into reticular epithelial cells, medullary epithelial cells, elongated epithelial cells, Hassall's corpuscles and cysts. Thymocytes first appear at 50 days fetal age and the number of thymocytes is amplified from 75–150 days fetal age. Minor differences between the fetal thymus of the monkey and that of other species were observed. Possible functions for the various cellular components of the fetal monkey thymus are discussed.This research was supported in part by grants 5-F3-CA-28, 793-02 and FR-0167 from the National Institutes of Health.     

Distribution of different cell types within the rat thymus in the neonatal period of life

Summary This study attempted to define reciprocal positions of cell types within the thymus. Random or non-random contacts between specific cell types were analyzed by means of graph theory. For analysis, thymus blocks were sectioned serially and, then, thymus cells were categorized into types, based on morphological criteria. The distribution of individual cell types within the cortex, cortico-medullary zone and medulla was presented in form of a map. In the analysis, three types of epithelial cell, characteristic of each thymus zone, macrophages, Langerhans-like cells and lymphocytes were found in non-random relations to one another. Moreover, characteristic groups of cells associated with one another were also demonstrated.This study was supported by the Polish Academy of Sciences     

Phagocytosis of Protein Coated Colloidal-Gold-Agarose-Gelatin Microbeads by Cultured Uterine Glandular Epithelial and Stromal Cells

A procedure for fixing and immunostaining whole cells from primary cultures of ovine and bovine uterine gland fragments was used to identify keratin in intermediate filaments of epithelial cells to distinguish them from stromal cells. Colloidal gold encapsulated aga-rose-gelatin microbeads were coated with different proteins and used to investigate uptake by epithelial and stromal cells in culture. Micro-beads were taken up by stromal cells and by epithelial cells on the outskirts of colonies. These cells formed ridges where they contacted and grew above stromal cells. Electron microscopy demonstrated that the microbeads had been internalized and appeared to be nontoxic. Individual cells could harbor more than 90 microbeads within their cytoplasm for at least seven to ten days with no apparent harm. Some cells with microbeads were seen to divide.     

Immunohistochemical detection of progesterone receptors in the canine uterus and their relation to sex steroid hormone levels

The aim of this immunohistochemical study is to describe the normal distribution of progesterone receptors in the various cell types of the canine uterine horns, body and cervix. The results can be used for research on uterine and endocrinological pathology, since the impact of progesterone on different uterine cell types is partly determined by the receptor availability. Nuclear staining for progesterone receptors was observed in epithelial cells of the surface epithelium, glandular ducts and basal glands of the endometrium, in endometrial stroma cells and in myometrial smooth muscle cells. This staining was positively correlated with the estradiol-17 beta:progesterone ratio, and reflects the positive effect of estradiol-17 beta and the negative influence of progesterone on the receptors. Staining scores were high during proestrus and decreased through estrus to early metestrus. In late metestrus, staining scores of the stromal and smooth muscle cells increased again. In anestrus, high scores of the surface-epithelial cells contrasted with minimal scores of the basal glands. This finding suggests a different hormonal regulation of the progesterone receptor expression in both epithelial cell groups. The higher staining intensities for progesterone receptors in stromal cells compared with epithelial cells might be explained by the fact that stromal cells mediate some effects of steroid hormones on the epithelial cells in the genital tract. Therefore, the role of stromal cells in regulation of the cyclic endometrial changes and in pathologic changes of uterine tissue should not be underestimated.     

Isobaric labeling-based quantitative proteomics of FACS-purified immune cells and epithelial cells from the intestine of Crohn's disease patients reveals proteome changes of potential importance in disease pathogenesis

Crohn's disease (CD) is a chronic condition characterized by recurrent flares of inflammation in the gastrointestinal tract. Disease etiology is poorly understood and is characterized by dysregulated immune activation that progressively destroys intestinal tissue. Key cellular compartments in disease pathogenesis are the intestinal epithelial layer and its underlying lamina propria. While the epithelium contains predominantly epithelial cells, the lamina propria is enriched in immune cells. Deciphering proteome changes in different cell populations is important to understand CD pathogenesis. Here, using isobaric labeling-based quantitative proteomics, we perform an exploratory study to analyze in-depth proteome changes in epithelial cells, immune cells and stromal cells in CD patients compared to controls using cells purified by FACS. Our study revealed increased proteins associated with neutrophil degranulation and mitochondrial metabolism in immune cells of CD intestinal mucosa. We also found upregulation of proteins involved in glycosylation and secretory pathways in epithelial cells of CD patients, while proteins involved in mitochondrial metabolism were reduced. The distinct alterations in protein levels in immune- versus epithelial cells underscores the utility of proteome analysis of defined cell types. Moreover, our workflow allowing concomitant assessment of cell-type specific changes on an individual basis enables deeper insight into disease pathogenesis.     

Development of functional thymic epithelial cells occurs independently of lymphostromal interactions

The thymus provides a specialised microenvironment for the development of T-cell precursors. This developmental programme depends upon interactions with stromal cells such as thymic epithelial cells, which provide signals for proliferation, survival and differentiation. In turn, it has been proposed that development of thymic epithelial cells themselves is regulated by signals produced by developing thymocytes. Evidence in support of this symbiotic relationship, termed thymic crosstalk, comes from studies analysing the thymus of adult mice harbouring blocks at specific stages of thymocyte development, where it is difficult to separate mechanisms regulating the initial development of thymic epithelial cells from those regulating their maintenance. To distinguish between these processes, we have analysed the initial developmental programme of thymic epithelial cells within the embryonic thymus, in either the presence or absence of normal T-cell development. We show that keratin 5+8+ precursor epithelial cells present in the early thymic rudiment differentiate into discrete cortical and medullary epithelial subsets displaying normal gene expression profiles, and acquire functional competence, independently of signals from T-cell precursors. Thus, our findings redefine current models of thymus development and argue against a role for thymocyte-epithelial cell crosstalk in the development of thymic epithelial progenitors.     

Ultrastructural and histochemical features of the thymus glands of the adult lungless salamander, Plethodon glutinosus (Caudata: Plethodontidae).

The thymus glands of adult slimy salamanders (Plethodon glutinosus) were examined by light and electron microscopy with the objective of describing the populations of epithelial cells believed to be secretory. The results of various histochemical procedures designed to demonstrate nucleic acids, proteins, lipids, and mucosubstances were evaluated by light microscopy. Each thymus is incompletely subdivided into a variable number of interconnected lobules by trabeculae extending inward from a thin capsule composed of connective tissues. The thymic parenchyma lacks distinct cortical and medullary regions, although developing lymphocytes and plasma cells tend to accumulate in larger numbers in the outermost portions of the glands. Basophils are found regularly in the capsule and trabeculae, but only very rarely within the thymic parenchyma. The epithelial cells of the thymus can be classified into five categories: epithelial reticular cells; three varieties of granulated cells (types I, II, and III), and myoid cells. Epithelial reticular cells form a three-dimensional network which extends throughout all portions of the thymus. Type I and type II granulated cells can be distinguished from one another by various morphological criteria at the ultrastructural level, but only small differences in the composition of their inclusions can be demonstrated histochemically. Both types of granules are composed principally of a proteinaceous material containing an abundance of primary amino and guanidyl groups. In addition, most type I inclusions possess a lipid component that cannot be demonstrated in type II granules. Type III granulated cells possess very small cytoplasmic inclusions resembling those of gastroenteric endocrine cells. Myoid cells contain concentrically arranged myofibrils composed of sarcomeres. In favorably oriented material, small cysts can be identified whose walls are composed of mixtures of type I cells, type II cells, and epithelial reticular cells. Groups of degenerating epithelial cells form lamellated structures corresponding to Hassall's (thymic) corpuscles.     

Thymic nurse cells: morphological study during their isolation from murine thymus

Summary Thymic nurse cells (TNC), which are multicellular complexes composed of epithelial cells and thymocytes, were obtained from C3H-mice thymuses. They were described by means of light and electron microscopy. The morphology of epithelial cells forming isolated TNC compared to that of small tissue fragments obtained by enzymatic digestion revealed that TNC could be derived from all parts of the thymus: cortex, corticomedullary junction and medulla, the cortex being their principal source. This variety of origin, the presence of several epithelial cells inside a single TNC, the presence of non-lymphoid cells, and the various locations of eleaved desmosomes confirmed that their aspect in vitro as round and sealed structures can be considered to be an artifact due to the isolation technique used. Indeed, during this procedure, they are formed by a process of wrapping of the epithelial cytoplasm around the tightly associated thymocytes. All three epithelial cell types: cortical reticular cells, medullary reticular cells, and medullary globular cells can form TNC.A portion of this work was presented at the first Thymus Workshop. Rolduc, Netherlands, April, 1988     

Molecular aspects of stromal-parenchymal interactions in malignant neoplasms

Carcinomas are composed of parenchymal and stromal elements, and the malignant behavior is principally dictated by the cancer cells. However, the malignant tumors not merely grow into a preexisting interstitial tissue, but they actively form a new stroma and modify their composition. Thus, the tumor stroma is significantly different from that of the neighboring tissues. Cancer cells may alter their stroma by cell-to-cell contact, soluble factors or by modification of the extracellular matrix (ECM), they induce myofibroblast differentiation and govern the desmoplastic stroma reaction. On the other hand, the stromal cells (especially the myofibroblasts) are able to modify the phenotype, invasiveness, metastatic capacity of carcinomas, typically promoting the progression. Regarding pancreatic cancer, the pancreatic stellate cells (PSCs) seem to be the key elements in the cross-talk between the parenchymal cells and the desmoplastic stroma. The tumor stroma is also rich in tumor-associated macrophages (TAM), but their role in the malignant process is contradictory and may be different in various tumor types, but most studies suggest a negative impact on the tumor growth. The relationship between the parenchymal and stromal elements is highly complex, they mutually alter their characteristics. Because the neostroma of the carcinomas largely seems to promote the invasiveness of the malignant tumors, novel therapeutic strategies are being evaluated targeting the stromal elements, with some encouraging, but still fragmentary results.     

Distribution and ultrastructure of S-100-immunoreactive cells in the human thymus

Summary The present study deals with the localization and ultrastructure of S-100-immunoreactive cells in the human thymus. These immunoreactive cells are distributed mainly in the medulla with some scattered elements in the cortex. Electron-microscopic observation revealed that the cells are characterized by an irregularly shaped nucleus, tubulovesicular structures in the cytoplasm and characteristic interdigitations of the plasma membrane. The cells often embrace lymphocytes with their branched processes. On the basis of these morphological features, the immunostained elements were identified as interdigitating cells (IDCs). The immunocytochemistry for S-100 visualizes the precise distribution and extension of the IDCs under the light microscope and indicates that the IDCs form no structural networks such as those established by the thymic epithelial cells. Since the IDCs in human lymph nodes have also been reported to contain S-100-like immunoreactivity, S-100 protein can be regarded as a useful marker for identifying the IDCs in the human thymus and other lymphoid organs.     

Establishment of functional epithelial cell lines from a rat thymoma and rat thymus

Summary Seven clonal epithelial cell lines from a thymoma of an (ACI/NMs×BUF/Mna)F1 rat and seven clonal epithelial cell lines from an ACI/NMs rat thymus were established in a medium containing 1 μM dexamethasome (DM) and were characterized cytologically. Long-term treatment of DM stabilized the epithelial nature of these epithelial cells irreversibly. The established cell lines showed a polygonal shape, were positively stained with antikeratin antiserum and had tonofilaments and desmosomes. Species of their keratin paptides were the same as those of normal thymic epithelial cells in primary cultures. The cell lines were positively stained with Th-4 monoclonal antibody which preferentially stains the medullary epithelial cells of the thymus, but not with Th-3 which preferentially stains the subcapsular and cortical epithelial cells of the thymus. The cells from the rat thymoma were much large than those from the normal thymus, as reflected in their primary cultures. No transformed phenotypes, such as high growth rate, high saturation density anchorage independency, low serum dependency and so on, were found on the cell lines from the thymoma as in the cell lines from the normal thymus by in vitro assays. DNA synthesis of the thymic lymphocytes was stimulated by culturing with a line of rat thymoma with no lectins. Thymic lymphocytes strongly bound on the cell lines from the thymoma and changed the shape of the cells. These cell lines may be useful to investigate the mechanism of thymomegenesis and the interactions between epithelial cells and thymocytes in the rat thymoma.     

Loss of Pten Disrupts the Thymic Epithelium and Alters Thymic Function

The thymus is the site of T cell development and selection. In addition to lymphocytes, the thymus is composed of several types of stromal cells that are exquisitely organized to create the appropriate environment and microenvironment to support the development and selection of maturing T cells. Thymic epithelial cells (TECs) are one of the more important cell types in the thymic stroma, and they play a critical role in selecting functional T cell clones and supporting their development. In this study, we used a mouse genetics approach to investigate the consequences of deleting the Pten tumor suppressor gene in the TEC compartment of the developing thymus. We found that PTEN deficiency in TECs results in a smaller thymus with significantly disordered architecture and histology. Accordingly, loss of PTEN function also results in decreased T cells with a shift in the distribution of T cell subtypes towards CD8⁺ T cells. These experiments demonstrate that PTEN is critically required for the development of a functional thymic epithelium in mice. This work may help better understand the effects that certain medical conditions or clinical interventions have upon the thymus and immune function.