Expression of nerve growth factor is upregulated in the rat thymic epithelial cells during thymus regeneration following acute thymic involution

Neuroimmune networks in the thymic microenvironment are thought to be involved in the regulation of T cell development. Nerve growth factor (NGF) is increasingly recognized as a potent immunomodulator, promoting "cross-talk" between various types of immune system cells. The present study describes the expression of NGF during thymus regeneration following acute involution induced by cyclophosphamide in the rat. Immunohistochemical stain demonstrated not only the presence of NGF but also its upregulated expression mainly in the subcapsular, paraseptal, and perivascular epithelial cells, and medullary epithelial cells including Hassall's corpuscles in both the normal and regenerating thymus. Biochemical data obtained using Western blot and RT-PCR supported these results and showed that thymic extracts contain NGF protein and mRNA, at higher levels during thymus regeneration. Thus, our results suggest that NGF expressed in these thymic epithelial cells plays a role in the T lymphopoiesis associated with thymus regeneration during recovery from acute thymic involution.     

Upregulation of receptor activator of nuclear factor-κB ligand expression in the thymic subcapsular, paraseptal, perivascular, and medullary epithelial cells during thymus regeneration

The receptor activator of nuclear factor (NF)-B ligand (RANKL; also termed TRANCE/OPGL/ODF/TNFSF11), a new member of the tumor-necrosis factor (TNF) superfamily, was identified as a key cytokine involved in the differentiation of the immune system and the regulation of immunity as well as in bone metabolism. In particular, RANKL-deficient mice showed defects in the early differentiation of T lymphocytes, suggesting that RANKL is a novel regulator of early thymocyte development. Here, we describe the expression of RANKL during regeneration following acute involution induced by cyclophosphamide in the rat thymus. The present study demonstrates the presence and upregulated expression of the RANKL in thymic subcapsular, paraseptal, perivascular, and medullary epithelial cells during thymus regeneration. Our results suggest that the RANKL expressed in these thymic epithelial cells plays a role in the development of T cells during thymic regeneration.     

Up-regulation of VEGF expression by NGF that enhances reparative angiogenesis during thymic regeneration in adult rat

Angiogenesis is important for adult tissue regeneration as well as normal development. Vascular endothelial growth factor (VEGF) is a unique potent angiogenic factor, and plays an essential role in regulating angiogenesis during embryonic development, normal tissue growth, and tissue regeneration. Recent evidence shows that nerve growth factor (NGF) also plays a role as an angiogenic regulator as well as a well-known neurotrophic factor. The aim of this study was to investigate whether thymus regeneration accompanies reparative angiogenesis and also to evaluate whether the thymic expression of VEGF is regulated by NGF in vivo and in vitro. Here, we show that high VEGF mRNA and protein levels are concomitant with reparative angiogenesis that occurs dramatically during regeneration following acute involution induced by cyclophosphamide (CY) in the rat thymus. Fluorescent thymus angiography using FITC-dextran showed that thymic regeneration is associated with a much denser capillary network compared with normal control thymus. Furthermore, the expressions of NGF and TrkA were highly increased during thymic regeneration. We also show that NGF mediates thymic epithelial induction of VEGF expression in vitro and in vivo. Taken together, our results suggest that NGF-mediated VEGF up-regulation in thymic epithelial cells may contribute to reparative angiogenesis during thymic regeneration in adult.     

Changes in the expression of the nerve growth factor receptors TrkA and p75LNGR in the rat thymus with ageing and increased nerve growth factor plasma levels

The nerve growth factor (NGF) receptors p75LNGR and TrkA are expressed by thymic epithelial cells. Presumably, the NGF-TrkA system is involved in the paracrine communication between thymic epithelial cells and thymocytes, whereas the functional role of p75LNGR is still unknown. The thymus of vertebrates undergoes age-related changes that in part depend on hormonal factors. In order to find out whether thymic epithelial cells are responsive to NGF during the whole lifespan of the rat, we studied NGF receptor expression in the thymus from birth to 2 years of age, using immunohistochemistry. Furthermore, to evaluate whether increased plasma levels of NGF affected the ageing process, either NGF or 4-methylcatechol (4MC), an inductor of NGF synthesis, was administered. Both TrkA and p75LNGR were expressed by a subpopulation of thymic epithelial cells during the whole age range studied and their expression peaked at around 3 months. TrkA was primarily found in subcortical and medullary epithelial cells, whereas p75LNGR was seen in a subpopulation of medullary cells. Cortical epithelial cells, neural crest-derived cells, other stromal cells and thymocytes were not immunoreactive for NGF receptors. Neither the administration of NGF nor the increased NGF plasma levels obtained after 4MC treatment seemed to affect the ageing of the thymus as assessed by morphological and immunohistochemical criteria, but this increase in NGF levels did produce a shift in the expression of p75LNGR from epithelial cells to ED1-positive macrophages in animals of 6 months and older. Present results indicate that the expression of p75LNGR and TrkA in the rat thymus undergoes age-dependent changes that parallel those of epithelial cells. NGF could therefore be important for thymus homeostasis, possibly acting on epithelial cells. Nevertheless, NGF did not seem to be able to prevent the involution of this organ, although it produced a switch in the expression of p75LNGR, the significance of which remains to be established.     

The glycoconjugate sugar residues of the sessile and motile cells in the thymus of normal and cyclosporin-A-treated rats: lectin histochemistry

It is well known that cell surface glycoconjugates play a determinant role in cellular recognition, cell-to-cell adhesion and serve as receptor molecules. T-lymphocytes are in strict contact with the thymic epithelial cells, which control their process of maturation and proliferation. On the other hand the normal maturation of the epithelial cells is believed to be induced by T-lymphocytes. For these reasons we have studied the glycoconjugates saccharidic moieties of the sessile and motile cells in the thymus of normal male albino Wistar rats and their changes following cyclosporin-A treatment, using a battery of seven HRP-lectins. Cytochemical controls were performed for specificity of lectin-sugar reaction. Some sections were pre-treated with neuraminidase prior to staining with HRP-lectins. Our results have demonstrated, in the control rats, a large amount and a variety of terminal and subterminal oligosaccharides within and/or on the epithelial thymic cells and in macrophages. After cyclosporin-A treatment, among the thymic epithelial cells, the subcapsular, paraseptal and perivascular cells showed the loss of some sugar residues, which characterized the same cells in the intact thymus. Some hypotheses are reported on the role played by the glycoconjugate sugar residues in control and cyclosporin-A treated rats.     

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 -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.     

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.     

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. Received: 12 May 1995 / Accepted: 13 October 1995     

Culture and characterization of thymic epithelium from autoimmune NZB and NZB/W mice

Autoimmune NZB and NZB/W mice display early abnormalities in thymus histology, T cell development, and mature T cell function. Abnormalities in the subcapsular/medullary thymic epithelium (TE) can also be inferred from the early disappearance of thymulin from NZB. It has also been reported that NZB thymic epithelial cells do not grow in culture conditions that support the growth of these cells from other strains of mice. In order to study the contribution of TE to the abnormal T cell development and function in NZB and NZB/W mice, we have devised a culture system which supports the growth of TE cells from these mice. The method involves the use of culture vessels coated with extracellular matrix produced by a rat thymic epithelial cell line. TEA3A1, and selective low-calcium, low-serum medium. In addition TEA3A1 cells have been used as an antigen to generate monoclonal antibodies specific for subcapsular/medullary TE. These antibodies, as well as others already available, have been used to show that the culture conditions described here select for cells displaying subcapsular/medullary TE markers, whereas markers for cortical TE and macrophages are absent.     

P2 receptors in the thymus: expression of P2X and P2Y receptors in adult rats, an immunohistochemical and in situ hybridisation study

The expression of the seven P2X receptor subtypes and of two P2Y receptors was examined immunohistochemically and by in situ hybridisation in thymi of adult male rats. P2X4, P2Y2 and 4 receptor mRNA colocalisation studies combining in situ hybridisation and immunohistochemistry were also carried out. P2X and P2Y receptors were found on thymocytes. P2X receptors were also abundant in cells of the thymic microenvironment, involved in control of T-cell maturation in vivo. We are the first to describe the expression of P2X4 receptors on thymocytes and confirm the finding of P2X1 and P2Y2 receptors on subpopulations of lymphocytes. P2X1,2,3,4 and 5 receptors were present in blood vessels of the thymus. P2X1,2 and 4 receptors were detected in vascular smooth muscle, while P2X3 receptors appeared to be associated with endothelial cells; some small arteries were positive for P2X5, possibly labelling vascular smooth muscle or fibroblasts in the adventitia. P2X2,3,6 and 7 receptors were found on thymic epithelial cells. P2X2 and 3 receptors were abundant on medullary epithelial cells, whilst P2X6 receptors were prominent in Hassall's corpuscles. P2X2 receptors were found on subcapsular and perivascular epithelial cells. P2X2,6 and 7 receptors were detected in epithelial cells along the thymic septa. Expression of P2X receptors was also investigated by Western blotting of crude thymic tissue extracts under reducing conditions. All seven P2X receptor subtypes were found to be dimers of approximately 70 kDa and 140 kDa molecular weight. ATP-mediated apoptosis and cell proliferation of thymocytes are discussed.     

Epithelial cell-specific laminin 5 is required for survival of early thymocytes

The gene LamC2 encoding the gamma2 chain of laminin 5, an epithelial cell-specific extracellular matrix protein, was identified in a PCR-based subtracted cDNA library from mouse thymic stromal cells. The mRNA existed in two alternative forms (5.1 and 2.4 kb). The full-length message was highly expressed in SCID thymus and in a nurse cell line, but not in other thymic epithelial cell lines, while the short form was more widely expressed. In situ hybridization and immunohistochemical staining revealed laminin 5 expression mostly in the subcapsular region of the adult thymus. Addition to fetal thymic organ cultures of a cell adhesion-blocking mAb to the alpha3 chain of laminin 5 interrupted T cell development. There was a 40% reduction in the total yield of thymocytes, and the most profound decrease (75-90%) was seen in the CD25+CD44+ and CD25+CD44-subsets of the CD4-CD8- double negative fraction. Most of the surviving double negative thymocytes expressed Sca-1, and there were significant increases in the number of cells with CD69 expression and in the fraction of annexin V-stained cells. None of these changes were observed with a nonblocking anti-laminin alpha3 chain mAb. These results suggest that the interaction between double negative thymoctyes and laminin 5 made by subcapsular epithelial cells is required for the survival and differentiation of mouse thymocytes.     

Parathyroid hormone-related peptide (hPTHrP) and parathyroid hormone-related peptide receptor type 1 (PTHR1) expression in human thymus.

Parathyroid hormone-related peptide (hPTHrP) is expressed in human tissues and regulates cellular proliferation, differentiation, and apoptosis by an autocrine/paracrine loop. In rodent thymus, both parathormone and parathyroid hormone-related peptide (PTHrP) are expressed by thymic epithelial cells (TECs). The present study demonstrated by RT-PCR and immunohistochemistry that hPTHrP and parathyroid hormone-related peptide receptor type 1 (PTHR1) were expressed in human thymus at both RNA and protein levels. hPTHrP was expressed mainly in the thymic medulla by epithelial (cytokeratin-positive), mature dendritic (CD40+/86+) and plasmacytoid interleukin (IL)-3Ralpha1 cells. This protein was also present in some cells forming Hassall's bodies and a few subcapsular and cortical TECs. PTHR1 was expressed by scattered subcapsular and cortical TECs and by rare TECs in the medulla. Thymocytes did not express either hPTHrP or PTHR1. Primary cultures of human TECs revealed the presence of both hPTHrP and PTHR1 mRNAs, confirming the capacity of TECs to synthesize both peptides. Moreover, synthetic (1-39) hPTHrP peptide administered on cultured TECs induced the expression of IL-6 mRNA, suggesting that hPTHrP can regulate thymic functions by inducing in TECs the expression of IL-6, which is involved in the development and maturation of thymocytes.     

Nerve growth factor stimulates proliferation, adhesion and thymopoietic cytokine expression in mouse thymic epithelial cells in vitro

Thymic epithelial cells, which constitute a major component of the thymic microenvironment, provide a crucial signal for intrathymic T cell development and selection. Neuroimmune networks in the thymic microenvironment are thought to be involved in the regulation of T cell development. NGF is increasingly recognized as a potent immunomodulator, promoting “cross-talk” between various types of immune system cells. The present study clearly shows that NGF stimulates mouse thymic epithelial cell activities in vitro including cell proliferation, thymocyte adhesion to thymic epithelial cells, and the expression of cell adhesion molecules such as ICAM-1 and VCAM-1, and thymopoietic factors including IL-7, GM-CSF, SDF-1, TARC and TECK. Thus, our data are of considerable clinical importance showing that trophic NGF activity could be used to enhance the thymus regeneration and develop methods to improve host immunity when the immune function is depressed due to thymic involution.     

Factors regulating stem cell recruitment to the fetal thymus

Colonization of the thymic rudiment during development is initiated before vascularization so that hemopoietic precursors must leave the pharyngeal vessels and migrate through the perithymic mesenchyme to reach the thymus, suggesting that they may be responding to a gradient of chemoattractant factors. We report that diffusible chemoattractants are produced by MHC class II+ epithelial cells of the fetal thymus, and that the response of precursors to these factors is mediated via a G protein-coupled receptor, consistent with factors being members of the chemokine family. Indeed, a number of chemokine receptors are expressed by thymic precursors, and several chemokines are also expressed by thymic epithelial cells. However, these chemokines are also expressed in a tissue that is unable to attract precursors, although the thymus expressed chemokine, TECK, is expressed at higher levels in thymic epithelial cells and we show that it has chemotactic activity for isolated thymic precursors. Neutralizing Ab to TECK, however, did not prevent thymus recolonization by T cell precursors, suggesting that other novel chemokines might be involved in this process. In addition, we provide evidence for the involvement of matrix metalloproteinases in chemoattractant-mediated T cell precursor recruitment to the thymus during embryogenesis.     

Post-hatching development of the thymic epithelial cells in the rainbow trout Salmo gairdneri: an ultrastructural study

This study reports the ultrastructure of subpopulations of epithelial cells of the thymic parenchyma during the post-hatching development of the rainbow trout, Salmo gairdner, kept at 14 degrees C. At hatching, the thymus contained a small number of medium and large thymocytes interspersed among three different types of epithelial cells: (1) epithelial cells adjacent to the connective tissue capsule; (2) ramified dark epithelial cells with electron-dense cytoplasm; and (3) pale electron-lucent epithelial cells displaying secretory-like features. All these cells types were anchored to one another by desmosomes and had apparently differentiated from the pharyngeal epithelium. At 4 days after hatching, the thymus enlarged, and numerous gaps occurred between the cell processes of contiguous epithelial cells adjacent to the capsular connective tissue. In 21-day-old trout, thymic trabeculae developed carrying blood vessels, and a subcapsular zone became evident containing lymphoblasts and large subcapsular epithelial cells. In 30-day-old trout, an outer thymic zone developed consisting of spindle-shaped epithelial cells which formed a dense network. At this stage, scattered cystic cells, which apparently differentiated from the pale epithelial cells, were present.     

Histological and cytological studies on the developing thymus of mandarin fish Siniperca chuatsi (Perciformes: Teleostei)

The thymus of the mandarin fish, Siniperca chuatsi, was examined by light and transmission electron microscopy to understand its formation and cellular composition. Larvae of the mandarin fish were collected and sectioned from 1 to 35 days post‐hatching (dph). On dph 7 the thymus was packed with lymphocytes. From 12 dph onward, mucous cells were observed on the epithelial layer; from 23 dph, three zones could be differentiated in the thymic parenchyma. The thymus was connected with the extension of the third, fourth and fifth branchial pouches throughout early development, remaining in a superficial position in the adult S. chuatsi. In the thymus of the adult fish, thymic epithelial cells (TECs) characteristic of tonofilaments were observed, with limiting TECs (LECs) found in subcapsular, subseptal, perivascular and nurse‐like TECs containing viable intact lymphocytes inside their vacuoles. In addition, three kinds of granulocytes were observed throughout the thymus, and an incomplete blood–thymus barrier was found in the inner zone. Other cell components such as cystic cells, macrophages and plasma cells, were also described in the thymus of the adult S. chuatsi. The thymus development in mandarin fish agrees, to some extent, with the ontogenetic patterns observed in other fish species.     

Isolation,Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.     

Ontogenesis of rat immune system: Proteasome expression in different cell populations of the developing thymus

Immune proteasomes in thymus are involved in processing of self-antigens, which are presented by MHC class I molecules for rejection of autoreactive thymocytes in adults and probably in perinatal rats. The distribution of immune proteasome subunits LMP7 and LMP2 in thymic cells have been investigated during rat perinatal ontogenesis. Double immunofluorescent labeling revealed LMP7 and LMP2 in thymic epithelial and dendritic cells, as well as in CD68 positive cells - macrophages, monocytes - at all developmental stages. LMP2 and LMP7 were also detected by flow cytometry in almost all thymic CD90 lymphocytes through pre- and postnatal ontogenesis. Our results demonstrate that the immune proteasomes are expressed in all types of thymic antigen presenting cells during perinatal ontogenesis, suggesting the establishment of the negative selection in the thymus at the end of fetal life. The observation of the immune proteasome expression in T lymphocytes suggests their role in thymocyte differentiation besides antigen processing in thymus.