Modulation of Cytokeratin Expression in The Hamster Thymus: Evidence For a Plasticity of The Thymic Epithelium

Cytokeratin (CK) expression was investigated, by means of immunocytochemistry, in the hamster thymic epithelium during ontogeny, as well as in primary cultures and upon glucocorticoid hormone treatment in vivo. As compared to the distribution pattern of distinct monoclonal antibody-defined cytokeratins in the normal adult thymus, CK modulation was evidenced in the three situations studied. During thymus ontogeny, both cytokeratins of simple lining epithelia, as CK8 and CK18, as well as the CK1/CK10 pair (typical marker of terminal stage of keratinization), were expressed since early stages of thymus development. They were located in the central region of thymic lobules preceding the cortical-medullary distinctions. This differed from what had been previously shown for mouse thymus ontogeny, revealing that the interspecific diversity in the distribution pattern of thymic cytokeratins occurred early in fetal life. A modulation of CK expression was also detected when hamster thymic epithelial cells (TEC) were led to grow in culture, with a down-regulation of CK19 contrasting with an enhancement of CK18 expression. This diverged from the maintenance of the in situ pattern when human TEC were cultured. Last, in vivo hydrocortisone treatment, known to increase the numbers of KL1⁺ cells in the mouse thymus medulla, promoted a cortical expression of the CK1/CK10 pair in the hamster thymus. Taken together, our findings demonstrate a continuous plasticity of the thymic epithelium, at least regarding cytokeratin expression, and enlarge the concept of interspecific diversity of intrathymic CK distribution in conditions as morphogenesis, in vitro system, and responsiveness to glucocorticoid hormone treatment.     

Ontogeny of rat thymic epithelium defined by monoclonal anticytokeratin antibodies.

Ontogenetic study on the expression of cytokeratin (CK) polypeptides within particular subsets of rat thymic epithelial cells (TEC) has been performed by a large panel of anti-CK monoclonal antibodies (mAbs) using the streptavidin-biotin immunoperoxidase method. Simultaneous presence of two or more CK subunits in the same TEC has been demonstrated by double immunofluorescence labeling. The obtained results showed that the expression of CK polypeptides in fetal and neonatal thymus differed from the adult patterns. The main difference was observed in expression of CK10, 18, and 19 polypeptides. During fetal ontogeny, CK10 and 18 are markers for most medullary TEC or a subset of medullary TEC, respectively, whereas CK19 is mainly a pan-TEC marker. In the adult animals, they are localized in the cortical and a subset of medullary TEC (CK18), subcapsular/perivascular and some medullary TEC (CK19), or in a subset of medullary TEC and Hasall's corpuscles (HC) (CK10). The switch in their expression in the cortex was observed during the first two weeks of postnatal life.     

Ontogeny of Rat Thymic Epithelium Defined by Monoclonal Anticytokeratin Antibodies

Ontogenetic study on the expression of cytokeratin (CK) polypeptides within particular subsets of rat thymic epithelial cells (TEC) has been performed by a large panel of anti-CK monoclonal antibodies (mAbs) using the streptavidin-biotin immunoperoxidase method. Simultaneous presence of two or more CK subunits in the same TEC has been demonstrated by double immunoflouorescence labeling. The obtained results showed that the expression of CK polypeptides in fetal and neonatal thymus differed from the adult patterns. The main difference was observed in expression of CK10, 18, and 19 polypeptides. During fetal ontogeny, CK10 and 18 are markers for most medullary TEC or a subset of medullary TEC, respectively, whereas CK19 is mainly a pan-TEC marker. In the adult animals, they are localized in the cortical and a subset of medullary TEC (CK18), subcapsular/perivascular and some medullary TEC (CK19), or in a subset of medullary TEC and Hasall’s corpuscles (HC) (CK10). The switch in their expression in the cortex was observed during the first two weeks of postnatal life.     

Cells reacting with the monoclonal anti-IL-2 receptor antibody AMT-13 in the regenerating thymus of irradiated mice.

The proportion and anatomical localization in murine thymus of T cell subpopulations, including those which are defined by the monoclonal anti-interleukin 2 (anti-IL-2) receptor antibody AMT-13, were studied by flow cytofluorometry and by immunohistochemical methods, both in irradiated and in normal mice. As a consequence of irradiation the proportion of AMT-13 positive cells and that of Lyt-1 positive cells were markedly enhanced, while the proportion of Lyt-2 positive cells was reduced. The vast majority of the AMT-13 positive cells both in normal and in irradiated thymi were located in the subcapsular area of the thymic cortex, whereas the irradiation resistant Lyt-1 positive cells were located in the medulla. These findings are compatible with the view that, similar to the developing thymus in the mouse embryo, in the regenerating adult thymus, AMT-13+ cells include the activated pro-thymocytes that repopulate the irradiated thymus.     

Morphological and immunohistochemical changes to thymic epithelial cells in the irradiated and recovering rat thymus

The present study analyzed morphological and immunohistochemical changes of thymic epithelial cells in the irradiated and recovering rat thymus. Observations showed the number of thymocytes was initially severely reduced after irradiation but abruptly increased on days 3 to 5 after 6 Gy and on days 7 to 11 after 8 Gy irradiation. To analyse the mechanisms for this abrupt recovery of the thymocytes after irradiation, the expression of p63 in the normal and irradiated thymus was immunohistochemically studied as the expression of this antigen may be related to the proliferation of epithelial cells. In the fetal thymus tissue, thymic epithelial cells were the principal cell type that stained strongly positive for p63. The sporadic expression of p63 was also observed in the normal adult thymus tissue, especially in the subcapsular region. An increased number of p63- positive cells in the thymus after irradiation indicates that repair or renewal of the thymic epithelial cells may be taking place because p63 is more specific to transient amplifying thymic epithelial cells. A RT-PCR analysis of p63 expression in irradiated and regenerating thymus tissue also showed an increased expression of p63 after irradiation compared with that of the normal thymus. These results suggest that changes in the thymic microenvironment-especially in relation to the repair and renewal of thymic epithelial cells- may have an important influence on thymocyte proliferation in the normal thymus as well as in the irradiated and recovering thymus.     

Pregnancy‐induced thymic involution is associated with suppression of chemokines essential for T‐lymphoid progenitor homing

During normal pregnancy, the thymus undergoes a severe reduction in size and thymocyte output, which may contribute to maternal–fetal tolerance. It is presently unknown whether the pregnancy‐induced thymic involution also affects nonlymphoid thymic cell populations and whether these changes in stromal cells play a role in the reduction in thymocyte numbers. Here, we characterize the changes in thymic lymphoid and nonlymphoid cells and show that pregnancy results in a reduction of all major thymic lymphoid cell populations, including the early T‐lymphoid progenitors (TLPs) and thymic regulatory T cells. In addition to the thymocytes, the thymic involution also includes all major nonlymphoid cell populations, which show a profound reduction in cell numbers. We also show that during pregnancy, the thymic nonlymphoid cells exhibit decreased expression of chemokines that are essential for TLP homing: CCL25, CXCL12, CCL21, and CCL19. In addition, the expression of these chemokines was substantially downregulated by short‐term treatment with progesterone but not estrogen. Collectively, these findings suggest a novel mechanism for the pregnancy‐induced reduction in TLP homing and the resulting thymic involution.     

Intermediate filaments as differentiation markers of normal pancreas and pancreas cancer.

Expression of intermediate filaments (IF) is regulated during development and differentiation. The authors have studied the expression of vimentin and cytokeratins (CK) 4, 7, 8, 13, 18, 19 in normal pancreas, chronic pancreatitis, and pancreas cancer using monoclonal antibodies. Immunohistochemical assays were performed on fresh frozen tissue sections and on cultured pancreas cancer cells using the streptavidin-peroxidase method. In normal pancreas, acinar cells expressed CK 8 and 18, whereas ductal cells expressed CK 7, 8, 18, and 19. CK 4 was expressed by 5-10% of pancreas duct cells in all specimens of normal pancreas. CK 13 was not detected in any epithelial cells of normal pancreas or pancreatitis. CK 7, 8, 18, and 19 were homogeneously expressed in all pancreas cancers, whereas CK 4 was expressed only in 5-50% of cells in 10/16 tumors. Foci of squamous metaplasia expressed CK 13 but showed partial loss of expression of CK 7, 8, 18, and 19. Thirteen pancreas cancer cell lines examined showed homogeneous expression of CK 7, 8, 18, and 19; 2/11 lines expressed CK 4 weakly, and 6/11 expressed vimentin. CK 13 was not detected in any of the lines. These results indicate that pancreas cancer cells consistently express cytokeratin polypeptides characteristic of ductal epithelial cells and that this phenotype is retained in pancreas cancer cell lines. In addition, squamous metaplasia is associated with a coordinate change in the expression of CK polypeptides.     

Immunohistochemical expression of vascular endothelial growth factor A (VEGF), and its receptors (VEGFR1, 2) in normal and pathologic conditions of the human thymus.

Vascular endothelial growth factor A (VEGF-A) is an angiogenic growth factor that is a primary stimulant of the vascularization of solid tumors. In the tumor microenvironment, an upregulation of both VEGF and its receptors occurs, leading to a high concentration of occupied receptors on tumor vascular endothelium. Also, VEGF is involved in the development of the normal vascular network of the thymus. Little is known about VEGF expression in normal and malignant thymic tissue. Our purpose was to study the pattern and localization of VEGF expression in benign conditions of the thymus and thymoma to determine a possible correlation with VEGF receptors VEGFR1, VEGFR2 and microvascular density. All cases were positive for VEGF and VEGFR1, 2 in the epithelial cells, in a cytoplasmic, granular pattern. In the normal thymus, there were positive epithelial cells with subcapsular distribution and Hassall's corpuscle epithelial cells. In acute thymic involution, the positive fields were correlated with dilation and stasis of blood vessels and lymphocyte depletion. Rare positive cells were found in other types of involution; the myasthenic thymus showed an intense and diffuse reaction in lymphoid follicles of the medulla. A strong reaction for VEGF was observed in type B3 thymomas in neoplastic epithelial cells, normal endothelial cells, plasma within the blood vessels and focally in the stroma adjacent to the tumor. Receptors for VEGF were positive in neoplastic epithelial cells and endothelium. We hypothesized that VEGF acts as an immunoregulatory factor in the normal thymus and as proangiogenic and autocrine factor in thymomas.     

Developmentally regulated interactions of human thymocytes with different laminin isoforms

The gene family of heterotrimeric laminin molecules consists of at least 15 naturally occurring isoforms which are formed by five different alpha, three beta and three gamma subunits. The expression pattern of the individual laminin chains in the human thymus was comprehensively analysed in the present study. Whereas laminin isoforms containing the laminin alpha1 chain (e.g. LN-1) were not present in the human thymus, laminin isoforms containing the alpha2 chain (LN-2/4) or the alpha5 chain (LN-10/11) were expressed in the subcapsular epithelium and in thymic blood vessels. Expression of the laminin alpha4 chain seemed to be restricted to endothelial cells of the thymus, whereas the LN-5 isoform containing the alpha3 chain could be detected on medullary thymic epithelial cells and weakly in the subcapsular epithelium. As revealed by cell attachment assays, early CD4- CD8- thymocytes which are localized in the thymus beneath the subcapsular epithelium adhered strongly to LN-10/11, but not to LN-1, LN-2/4 or LN-5. Adhesion of these thymocytes to LN-10/11 was mediated by the integrin alpha6beta1. During further development, the cortically localized CD4+ CD8+ thymocytes have lost the capacity to adhere to laminin-10/11. Neither do these cells adhere to any other laminin isoform tested. However, the more differentiated single positive CD8+ thymocytes which were mainly found in the medulla were able to bind to LN-5 which is expressed by medullary epithelial cells. Interactions of CD8+ thymocytes with LN-5 were integrin alpha6beta4-dependent. These results show that interactions of developing human thymocytes with different laminin isoforms are spatially and developmentally regulated.     

Thymic microenvironment reconstitution after postnatal human thymus transplantation

A functional thymus develops after cultured thymus tissue is transplanted into subjects with complete DiGeorge anomaly. To gain insight into how the process occurs, 7 post-transplantation thymus biopsy tissues were evaluated. In 5 of 7 biopsies, the thymus appeared to be predominantly cortex with thymocytes expressing cortical markers. Unexpectedly, the epithelium expressed both cortical [cortical dendritic reticulum antigen 2 (CDR2)] and medullary [cytokeratin (CK) 14] markers. Early medullary development was suggested by epithelial cell adhesion molecule (EpCAM) reactivity in small areas of biopsies. Two other biopsies had distinct mature cortex and medulla with normal restriction of CK14 to the medulla and subcapsular cortex, and of CDR2 to cortex. These data are consistent with a model in which thymic epithelium contains CK14+ "progenitor epithelial cells". After transplantation these cells proliferate as CK14+CDR2+ thymic epithelial cells that are associated with cortical thymocytes. Later these cells differentiate into distinct cortical and medullary epithelia.     

Essential microenvironment for thymopoiesis is preserved in human adult and aged thymus

Normal human thymuses at various ages were immunohistologically examined in order to determine whether adult or aged thymus maintained the microenvironment for the T cell development and thymopoiesis was really ongoing. To analyze the thymic microenvironment, two monoclonal antibodies (MoAb) were employed. One is MoAb to IL-1 receptor (IL-1R) recognizing medullary and subcapsular cortical epithelial cells of normal infant human thymus. The other is UH-1 MoAb recognizing thymic epithelial cells within the cortex, which are negative with IL-1R-MoAb. Thymus of subjects over 20 years of age was split into many fragments and dispersed in the fatty tissue. However, the microenvironment of each fragment was composed of both IL-1R positive and UH-1 positive epithelial cells, and the UH-1 positive portion was populated with lymphocytes showing a follicle-like appearance. Lymphocytes in these follicle-like portions were mostly CD4+CD8+ double positive cells and contained many proliferating cells as well as apoptotic cells. Thus these follicle-like portions in adult and aged thymus were considered to be functioning as cortex as in infant thymus. Proliferative activity of thymocytes in the thymic cortex and the follicle-like portions definitely declined with advance of age, while incidence of apoptotic thymocytes increased with aging.     

Chemokine-mediated thymopoiesis is regulated by a mammalian Polycomb group gene, mel-18.

Multiple chemokines are made in the thymus, and they are likely to function in the fine control of cellular migration and regulation of thymic T cell development. Mice lacking the gene mel-18, a member of the mammalian Polycomb group genes, displayed impaired thymic T cell development. Here we report that expression of chemokine receptors CXCR4 and CCR9 are regulated by mel-18 and that CXCL12/SDF-1- and CCL25/TECK-mediated chemotactic activities are also affected by the loss of mel-18. In mel-18-/- mice, high expression of CXCR4 on CD4-CD8- cells might lead to trapping in the SDF-1 rich subcapsular region, while low expression of CCR9 on CD4+CD8+ cells might reduce cell migration to the medulla. Therefore, this member of the Polycomb group genes plays a role in thymic T cell migration and differentiation via the chemokine system.     

Detection and localization by the monoclonal anti-interleukin 2 receptor antibody AMT-13 of IL 2 receptor-bearing cells in the developing thymus of the mouse embryo and in the thymus of cortisone-treated mice

During embryonic development of the mouse, before expressing classical T cell markers, the blast cells colonizing the thymus react with the monoclonal antibody AMT-13 shown previously to detect interleukin 2 receptors. The proportion of AMT-13+ cells decreases as gestation time increases. On the other hand, the proportion of Thy-1+, Lyt-1+ and Lyt-2+ cells increases during ontogenesis. On the 19th day of gestation when the thymus architecture is comparable to the adult thymus, the AMT-13+ cells become localized in the subcapsular area of the cortex. In the adult thymus after cortison treatment the regenerating cells express the AMT-13 antigen. The AMT-13 antigen presumably the interleukin 2 receptor is the first marker of the early embryonic thymocytes reported until now that may be related to cellular function.     

Differential effect of cyclosporin application on epithelial cells of the rat thymus. Immunohistochemical study.

Young adult male Wistar rats were given 30 mg per kg of cyclosporin (CS) for 21 consecutive days. A panel of monoclonal antibodies was used to study the phenotype of thymic epithelial cells. After treatment with CS, subcapsular epithelial cells, although phenotypically similar to medullary epithelial cells, were changed in a similar manner to phenotypically distinct epithelial cells of the deep cortex. These cells became enlarged, stockier and their cytoplasmic prolongations were thicker and coarser compared with control cells and their number was not decreased. In contrast, the number of medullary epithelial cells was markedly reduced, whereby the cells with the most mature phenotype (CK8+10-19- and CK8+10+19-) were the most prominently depleted. No proliferation of thymic epithelial cells was detected as monitored by incorporation of 5-bromodeoxyuridine.     

Epithelial-cell architecture during involution of the human thymus

One hundred thymus glands were assessed histologically as to their degree of involution. Epithelial cells were demonstrated by an immunoperoxidase method using a monoclonal antibody against cytokeratin. The distribution of these cells was studied in the medulla, the cortico-medullary junction, the cortical parenchyma and the subcapsular cortex. As involution proceeds, the loss of cells from the thymus is almost totally confined to the lymphoid-cell elements. The architecture of the epithelial-cell network remains largely intact although there is extensive collapse of the structure due to the loss of the intervening lymphocytes. Even when involution is apparently complete; sheets of epithelial cells can be demonstrated in the thymic remnant.     

CCRL1/ACKR4 is expressed in key thymic microenvironments but is dispensable for T lymphopoiesis at steady state in adult mice

Thymus colonisation and thymocyte positioning are regulated by interactions between CCR7 and CCR9, and their respective ligands, CCL19/CCL21 and CCL25. The ligands of CCR7 and CCR9 also interact with the atypical receptor CCRL1 (also known as ACKR4), which is expressed in the thymus and has recently been reported to play an important role in normal αβT‐cell development. Here, we show that CCRL1 is expressed within the thymic cortex, predominantly by MHC‐II^lowCD40^? cortical thymic epithelial cells and at the subcapsular zone by a population of podoplanin⁺ thymic epithelial cells in mice. Interestingly, CCRL1 is also expressed by stromal cells which surround the pericytes of vessels at the corticomedullary junction, the site for progenitor cell entry and mature thymocyte egress from the thymus. We show that CCRL1 suppresses thymocyte progenitor entry into the thymus, however, the thymus size and cellularity are the same in adult WT and CCRL1^?/? mice. Moreover, CCRL1^?/? mice have no major perturbations in T‐cell populations at different stages of thymic differentiation and development, and have a similar rate of thymocyte migration into the blood. Collectively, our findings argue against a major role for CCRL1 in normal thymus development and function.     

Human intrathymic T cell differentiation

The human thymus develops early on in fetal gestation with morphologic maturity reached by the beginning of the second trimester. Endodermal epithelial tissue from the third pharyngeal pouch gives rise to TE3+ cortical thymic epithelium while ectodermal epithelial tissue from the third pharyngeal cleft invaginates and splits during development to give rise to A2B5/TE4+ medullary and subcapsular cortical thymic epithelium. Fetal liver CD7+ T cell precursors begin to colonize the thymus between 7 and 8 weeks of fetal gestation, followed by rapid expression on thymocytes of other T lineage surface molecules. Human thymic epithelial cells grown in vitro bind to mature and immature thymocytes via CD2 and CD11a/CD18 (LFA-1) molecules on thymocytes and by CD58 (LFA-3) and CD54 (ICAM-1) molecules on thymic epithelial cells. Thymic epithelial cells produce numerous cytokines including IL1, IL6, G-CSF, M-CSF, and GM-CSF--molecules that likely are important in various stages of thymocyte activation and differentiation. Thymocytes can be activated via several cell surface molecules including CD2, CD3/TCR, and CD28 molecules. Finally, CD7+ CD4-CD8- CD3- thymocytes give rise to T cells of both the TCRab+ and TCR gd+ lineages.     

Subcapsular thymic lymphoblasts expose receptors for soy bean lectin.

In the course of analysing thymic cellular constituents concerning the expression of lectin receptors and defined lymphocyte differentiation antigens, respectively, a subpopulation of thymic lymphocytes was detected exposing both peanut lectin (PNL) and soy bean lectin (SBL) receptors on their surfaces, while the majority of cortical thymic lymphocytes were found to be PNL positive, but SBL negative. This subset of SBL+PNL+thymic lymphocytes in the adult populates the area beneath the thymic capsule, forming a narrow rim only, while in perinatal thymus a corresponding subcapsular cell layer comprises almost one third of the thymic tissue and in early ontogenesis the vast majority of thymic lymphoid cells are PNL+SBL+. By ultrastructural analysis of double labelled cell suspensions as well as of cells separated by affinity chromatography, it could be shown that PNL/SBL+ cells frequently exhibit morphological features of lymphoblastic cells. Thus this subcapsularly located subset seems to represent a compartment of proliferating immature cells, descended from bone marrow and foetal liver prethymic cells, respectively, and giving rise to PNL/SBL- cortical thymic lymphocytes.     

Cytokeratin (CK5, CK8, CK14) expression and presence of progenitor stem cells in human fetal thymuses

The aim of the current study was to observe the expression of cytokeratins in human fetal thymuses. Specific cytokeratin markers in adult humans and mice have been well described but there has been little similar work on human fetuses. We also aimed to see whether progenitor stem cells that could be harvested to treat various immunodeficiency disorders are present in fetal thymic tissue. Thymuses obtained from 30 aborted human fetuses (12 to 31 weeks) were examined immunohistochemically to investigate changes in cytokeratin expression in the epithelial cells (TEC) at various gestational ages. Before 16 weeks of gestation, cortical (cTEC) and medullary (mTEC) TEC exhibited homogenous staining for cytokeratins CK8 and CK5. After 16 weeks there was differential staining, with cTEC positive for CK8 and mTEC for CK5 and CK14. Interestingly, both CK5 + CK8+ progenitor stem cells were present in the fetal thymic cortex at all gestational ages, with a relatively high number from 12 to 16 weeks. Cytokeratin expression in fetal thymuses was quite different from that in the adult thymus owing to the presence of undifferentiated progenitor stem cells in fetal thymic stroma along with differentiated TEC. The best time to harvest these progenitor stem cells from fetal thymic stroma in order to treat various immune deficiency disorders appears to be 12–16 weeks. Clin. Anat. 29:711–717, 2016. © 2016 Wiley Periodicals, Inc.     

Effects of FK506 on rat thymus: time-course analysis by immunoperoxidase technique and flow cytofluorometry.

The effect of administration of FK506 at 1 mg/kg body weight for 14 days on rat lymphoid tissues, especially the thymus, and recovery after discontinuation of treatment, were investigated by the immunoperoxidase technique and flow cytofluorometry using monoclonal antibodies OX6, OX7, OX8, OX18 and W3/25, reactive with rat lymphocytes. Marked reduction of the thymic medulla upon treatment was clearly demonstrated by staining with OX18 and OX6. Changes produced by FK506 were also observed in the cortical area of the thymus, and were especially marked in the subcapsular area and around blood vessels. Eventually, the thymic cortex appeared patchy, this change being maximal 14 days after the start of administration. Obvious restitution of the thymic medulla was evident about 14 days after withdrawal of FK506. Flow cytometric analysis of the thymus showed that the percentages of cells labelled positively with OX7, OX8 and W3/25 were increased with FK506 treatment, and recovered to the normal level soon after withdrawal. Furthermore, the peak of fluorescence intensity of OX7+, OX8+ and W3/25+ cells showed a temporary shift to the right during FK506 treatment; however, the peak of fluorescence intensity of OX18+ cells showed a temporary shift to the left. Treatment with FK506 also produced a significant change in 3H-thymidine uptake by thymocyte. These results suggest that FK506 may inhibit the proliferation, maturation and differentiation of thymocytes. However, thymocytes prepared from FK506-treated rats and labelled with FITC behaved similarly to rat thymocytes in normal recipient rats. This suggests that during FK506 treatment thymocytes may retain their potential for peripheral mobilization.