DEC-205/CD205+ dendritic cells are abundant in the white pulp of the human spleen, including the border region between the red and white pulp

The distribution of dendritic cells (DCs) and macrophages in the human spleen has received less attention than that of lymphocytes. Here we have addressed this problem with the human DEC-205/CD205 marker ('DEC'), which is an endocytic receptor on DCs that mediates efficient presentation of antigens. DEC was abundant on dendritic profiles in the white pulp but absent from the red pulp, the latter defined with antibodies to two antigens, mannose receptor/CD206 on sinusoidal lining cells, and macrosialin/CD68 on macrophages. Double staining with anti-DEC and anti-CD3 showed the expected concentration of DEC+ cells in the relatively small T-cell areas of the human spleen. DEC+ cells were also found in other regions of the white pulp. In all regions, the DEC+ cells were positive for major histocompatibility complex (MHC) class II and the CD11c integrin but largely immature, with low expression of B7-2/CD86 costimulator and DC-lysosome-associated membrane protein (LAMP)/CD208. When we concentrated on the perifollicular region between the red pulp and the marginal zone, we found macrophages that stained with antibodies to sialoadhesin/CD169 and DC-specific ICAM-3 grabbing non-integrin (SIGN)/CD209, and just inside these cells were DEC+ profiles. The DEC+ DCs were intertwined with cells that stained for the vascular addressin mucosal addressin cell adhesion molecule (MAdCAM). Therefore, anti-DEC-205/CD205 antibodies are useful for identifying DCs in human splenic white pulp and its border region with the red pulp.     

Haematopoietic antigen-presenting cells in the human thymic cortex: evidence for a role in selection and removal of apoptotic thymocytes

Only a small proportion of thymocytes survive T cell selection in the thymus and leave the thymus as mature T cells. The vast majority of thymocytes undergo cell death during selection, either due to failure to undergo positive selection on self peptide-MHC presented by thymic antigen presenting cells (APC) or due to negative selection. In the murine thymus it has been shown that most thymocytes that fail selection undergo apoptosis in the thymic cortex and are removed by cortical macrophages. However, it is unknown how apoptotic thymocytes are cleared from the cortex of the human thymus. Here we report the identification of antigen-presenting cells of haematopoietic origin (hAPCs) by expression of dendritic cell (DC) specific C-type lectin DC-SIGN (CD209) in the cortex of the human thymus, and show that these cells exhibit features of both immature DCs and macrophages. The analysis of cellular markers, in particular the expression of the molecular chaperone HLA-DM, on cortical hAPCs further suggests that these hAPCs may participate in selection of thymocytes in the cortex. Using in situ terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), we demonstrated that these cortical hAPCs are surrounded by apoptotic, TUNEL(+) thymocytes in situ. Futhermore, in situ immuno-cryo-electron microscopy suggests that cortical hAPCs take up and remove apoptotic thymocytes. Thus, DC-SIGN(+) hAPCs in the human thymic cortex appear to function in thymocyte selection and removal of apoptotic thymocytes from the thymic cortex.     

Extracellular matrix components of the mouse thymic microenvironment. III. Thymic epithelial cells express the VLA6 complex that is involved in laminin-mediated interactions with thymocytes

We describe herein the expression of the VLA6 complex by murinethymic epithelial cells (TEC). The immunohistochemical distributionrevealed that VLA6 is found in both thymic medullary and subcapsullaryareas. Moreover, studies by immunoelectron microscopy revealeda membrane labeling of the VLA6 molecule, including at desmosomalsites. By means of immunoblottlng, immunoprecipitation, andaffinity chromatography of extracts from a mouse TEC line, wefurther demonstrated that VLA6 is a laminin (LN) receptor inthese cells. In keeping with this finding, we showed that TECadhesion, spreading, and proliferation were enhanced in vitroby LN. The fact that VLA6 is also expressed by the large majorityof thymocytes raised the hypothesis that it might be involvedin LN-mediated TEC—thymocyte interactions. Interestingly,in vitro experiments showed that there is an increase in theTEC—thymocyte adhesion upon glucocorticold hormone treatment,a situation in which the expression of VLA6 as well as LN isenhanced. Most importantly, this adhesion can be reversed bypre-treating TEC with an anti-6 integrin mAb. Additionally,spontaneous in vitro thymocyte release by thymic nurse cellcomplexes was enhanced by LN and partially blocked by anti-6or anti-ß1 antibodies. Our results suggest that VLA6is involved in LN-mediated TEC—thymocyte interactionsthat can be relevant for thymic microenvironmental cell physiologyand intrathymic T cell differentiation events.     

Analysis of thymic epithelial cell proliferation in vitro by combining bromodeoxyuridine and keratin labeling in an immunofluorescence assay

A simple method of analyzing thymic epithelial cell (TEC) proliferation has been developed by combining bromodeoxyuridine (BrDU) and keratin labeling in an immunofluorescence assay. The first reagent specifically visualizes the cells entering the S phase of the cell cycle, whereas the second immunostaining reveals which of the proliferating BrDU-positive cells actually belong to the epithelial lineage. This method, besides being rapid and free of radioactivity, appears to be reliable in view of the minor variations in the percentages of BrDU+ TEC observed in several distinct experiments. Thus, BrDU/keratin immunolabeling appears to represent a useful tool for the analysis of in vitro TEC proliferation.     

In vitro negative selection of αβ T cell receptor transgenic thymocytes by conditionally immortalized thymic cortical epithelial cell lines and dendritic cells

We have established conditionally immortalized thymic cortical epithelial cell lines from transgenic mice carrying a temperature-sensitive SV40 large Tantigen. One of these cell lines expresses cortical markers and produces IL-1α, IL-6, IL-7, and TGF-β1. These cells express class I major histocompatibility complex (MHC) constitutively and class II MHC upon induction with IFN-μ. The cells appear to have a normal class I antigen presenting pathway since messages for both peptide transporter genes (TAP1, TAP2) were detected. The ability of these cortical epithelial cells to present peptide antigen was compared to that of thymic dendritic cells. In suspension culture with αβ Tcell receptor (TcR) transgenic thymocytes, these epithelial cells and dendritic cells (pre-pulsed with peptide cognate for the transgenic TcR) caused down-regulation of CD4, CD8, and TcR in an antigen dose-dependent and MHC-restricted manner. CD4^dullCD8^dull cells were taken as evidence for negative selection because these cells contained apoptotic DNA. Concentration of peptide required for negative selection of thymocytes was similar between dendritic cells and cortical epithelial cells. In contrast, αβ transgenic spleen cells were activated only by dendritic cells but not by cortical epithelial cells.     

DNA methylation signatures of the AIRE promoter in thymic epithelial cells, thymomas and normal tissues

Mutations in the AIRE gene cause autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), which is associated with autoimmunity towards several peripheral organs. The AIRE protein is almost exclusively expressed in medullary thymic epithelial cells (mTEC) and CpG methylation in the promoter of the AIRE gene has been suggested to control its tissue-specific expression pattern. We found that in human AIRE-positive medullary and AIRE-negative cortical epithelium, the AIRE promoter is hypomethylated, whereas in thymocytes, the promoter had high level of CpG methylation. Likewise, in mouse mTECs the AIRE promoter was uniformly hypomethylated. In the same vein, the AIRE promoter was hypomethylated in AIRE-negative thymic epithelial tumors (thymomas) and in several peripheral tissues. Our data are compatible with the notion that promoter hypomethylation is necessary but not sufficient for tissue-specific regulation of the AIRE gene. In contrast, a positive correlation between AIRE expression and histone H3 lysine 4 trimethylation, an active chromatin mark, was found in the AIRE promoter in human and mouse TECs.     

Immunohistochemical evidence for the expression of the carcinoembryonic antigen by human thymic epithelial cells in vitro and in neoplastic conditions.

Thymic epithelial cells (TECs), which are known to influence T-cell differentiation, may undergo phenotypic changes and lose some differentiation antigens (for example, the HLA-DR complex) in neoplastic conditions and when they are grown in culture. Using an indirect immunofluorescence assay, the authors investigated the expression of the carcinoembryonic antigen (CEA) by normal cultured or pathologic human TECs. This antigen, which can be regarded as a marker of undifferentiation, disappears during the normal development of epithelial tissues and reappears in neoplastic conditions. In normal as well as hyperplastic (myasthenia gravis-associated) thymuses, the epithelial network (revealed in double-labeling experiments by an anti-keratin monoclonal antibody) is virtually CEA-negative, except for the specific labeling observed on some cells of Hassall's corpuscles. In thymomatous epithelial cells, however, a strong and specific fluorescent labeling was consistently detected in all thymomas studied. Thymic epithelial cells grown in cultures from fragments of normal thymuses also expressed CEA on their cell membranes. Interestingly, the relative number of CEA-positive cells increased as a function of the age of the primary culture and reached virtually 100% when monolayers became confluent (Days 12-14). Moreover, using an ELISA assay, the authors demonstrated the presence of CEA in supernatants from TEC cultures. Interestingly, the amount of CEA in these supernatants decreased as a function of the age of the culture. In addition, a marked inhibition of TEC proliferation was observed after treating the cultures with an anti-CEA serum. Our results demonstrate that CEA is expressed not only in situ by differentiated neoplastic TECs but also by normal TECs cultured in vitro. In addition, the inhibitory action of the anti-CEA serum on TEC proliferation suggests that CEA may act physiologically as a growth factor for proliferating epithelial cells. In this respect, cultures of human TECs represent a good model for further studies.     

Neoplastic thymic epithelial cells of human thymoma support T cell development from CD4−CD8− cells to CD4+CD8+ cells in vitro

Human thymoma is a thymic epithelial cell tumour which often contains a large number of immature T cells and is frequently associated with autoimmune diseases. Since thymic epithelial cells play key roles in the development and selection of T cells in the normal thymus, we hypothesized that the neoplastic thymic epithelial cells of thymoma may support T cell differentiation in the tumour. We characterized CD4^?CD8^? cells in thymoma and applied an in vitro reconstitution culture system using the CD4^?CD8^? cells and the neoplastic epithelial cells isolated from thymoma. CD34, a stem cell marker, was expressed on 29.9 ± 12.2% of CD4^?CD8^? cells in thymoma. TCRγδ was expressed on 27.4 ± 15.1% of CD4^?CD8^? cells and CD19, a B cell marker, was expressed on 14.1 ± 23.1% of CD4^?CD8^? cells. CD4^?CD8^? cells expressed both IL-7R α-chain and common γ-chain. Purified CD4^?CD8^? cells from thymomas were cultured with the neoplastic epithelial cells, and their differentiation into CD4⁺CD8⁺ cells via CD4 single-positive intermediates was observed within 9 days' co-culture in the presence of recombinant IL-7. Furthermore, we examined the reconstitution culture using CD34⁺CD4^?CD8^? cells purified from normal infant thymus. The CD34⁺CD4^?CD8^? cells in normal thymus also differentiated to CD4⁺CD8⁺ cells in the allogeneic co-culture with the neoplastic epithelial cells of thymoma. These results indicate that the tumour cells of thymoma retain the function of thymic epithelial cells and can induce differentiation of T cells in thymoma.     

Analysis of thymic stromal cell subpopulations grown in vitro on extracellular matrix in defined medium. IV. Cytokines secreted by human thymic epithelial cells in culture and their activities on murine thymocytes and bone marrow cells.

In previous reports we described our approach to the cultivation of murine and human thymic epithelial cells in primary cultures, using defined, serum-free growth factor-supplemented medium and extracellular matrix-coated culture plates. The cells in these cultures displayed high metabolic activity and their supernatant was highly active on thymocytes. In the study reported here we analysed cytokine activities in the supernatant of human thymic epithelial cell cultures (HTES), by using the respective cytokine-dependent cell lines and by neutralization with specific monoclonal antibodies. Three cytokine activities were detected--interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF) and macrophage (M)-CSF. Other cytokine activities tested for [IL-1, IL-2, IL-7, interferon (IFN) and tumour necrosis factor (TNF)] were negative. The effect of HTES on concanavalin A (Con A)-induced proliferation of murine thymocytes could be completely abolished by anti-IL-6 antibodies, but not by antibodies to CSF, whereas enhancement of bone marrow cell proliferation by HTES was partially inhibited by either anti-G-CSF or anti-M-CSF antibodies and completely inhibited by both antibodies, but not at all by anti-IL-6. We can thus distinguish between thymocyte-related cytokines (IL-6) and bone marrow (myeloid/monocyte) related ones (G-CSF, M-CSF) in HTES.     

Construction of glomerular epithelial cells in vitro for removal of immune complex

Patients with systemic lupus erythematosus (SLE), an autoimmune disease caused by excessive amounts of immune complex in the serum, have a loss of complement receptor type 1 (CR1), a receptor of complements C3b and C4b, on glomerular epithelial cells. In order to calrify the biological function of CR1 on renal glomeruli and the correlation with clearance of immune complex, a full length of human tonsil CR1 cDNA was transfected to normal rat glomerular epithelial cells (SGE1) to enable CR1 expression in the long term (more than 1 year). As compared with the non-CR1-expressing cells, the CR1-expressing cells showed a higher binding effect to immune complex in 60 min, and the binding effect was mediated by complement. Blocking of the binding effect by anti-CR1 antibody indicates that CR1 plays an important role in removal of immune complex. Results from microscopy showing that the constructed cell has an enhanced phagocytic ability in the presence of complement suggest that the mechanism of removing immune complex by CR1-expressing cells has three steps: opsonization of immune complex by complement, the ligand-receptor interaction between opsonized complex and CR1 on the cell, and phagocytosis of complex by CR1-expressing cells.     

Use of the Comet test and micronucleus assay on human white blood cells for in vitro assessment of genotoxicity induced by different drinking water disinfection protocols

Surface water disinfection can lead to the formation of mutagenic/carcinogenic by-products derived from reactions with naturally occurring inorganic compounds. We investigated the feasibility and potential usefulness of an integrated approach to genotoxicity analysis of drinking water. The approach employed the Comet and micronucleus (MN) assays to evaluate the DNA and chromosomal damage produced by water extracts in human blood cells. Surface water samples from Lago Trasimeno (Italy) were collected in different seasons (July 2000, October 2000, February 2001, and June 2001), and samples were disinfected with sodium hypochloride (NaClO), chlorine dioxide (ClO(2)), or peracetic acid (PAA). Extracts of untreated and treated water were incubated with primary human leukocytes. The Comet assay revealed both strong seasonal variations and differences between samples processed by the three disinfection protocols. The three disinfectants increased the genotoxicity of the water collected in July 2000 and October 2000, with PAA producing the greatest amount of DNA damage. Extracts of raw water collected in February 2001 produced so much DNA damage that the relative genotoxic potentials of the three disinfectants could not be evaluated. No increase in MN frequency was detected in any of the samples. The multi-endpoint MN assay indicated, however, that our study samples (especially the sample collected in the February 2001) were cytotoxic. We conclude that this integrated approach to genotoxicity assessment may be useful both for the quality control of raw drinking water and to help compare the potential health risks associated with alternative disinfection processes.