Differential expression and regulation of cyclooxygenase isozymes in thymic stromal cells

Prostaglandins (PGs) are lipid-derived mediators of rapid and localized cellular responses. Given the role of PG in supporting thymic T cell development, we investigated the expression of the PG synthases, also known as cyclooxygenases (COX)-1 and -2, in the biosynthesis of PGs in thymic stromal cell lines. The predominant isozyme expressed in cortical thymic epithelial cells was COX-1, while COX-2 predominated in the medulla. IFN-gamma up-regulated expression and activity of COX-2 in medullary cells, in which COX-2 was expressed constitutively. In contrast, IFN-gamma down-regulated COX-1 activity, but not expression, in cortical cells. Stromal cells support T cell development in the thymus, although the mediators of this effect are unknown. Selective inhibition of COX-2, but not COX-1, blocked the adhesion of CD4+CD8+ and CD4+CD8- thymocytes to medullary cell lines. No effect of the inhibitors was observed on the interactions of thymocytes with cortical epithelial lines. These data further support the differential regulation of COX-1 and COX-2 expression and function in thymic stromal cells. PGs produced by COX-2 in the medullary thymic stroma may regulate the development of thymocytes by modulating their interaction with stromal cells.     

Biochemical and functional characterization of a molecule expressed by a subset of thymic medullary epithelial cells.

A monoclonal antibody (mAb), named TE-4F 10, was produced by fusing P3X-Ag8 myeloma cells with splenocytes of BALB/c mice immunized with a rat medullary thymic epithelial cell (TEC) line, (TE-R 2.5), previously established in our Institute. Flow cytometry showed that 85-95% TE-R 2.5 cells expressed the TE-4F10 antigen. The mAb immunoprecipitated a 29 kDa molecule from the TE-R2.5 cell lysate. Immunohistochemical analysis using single and double staining of the thymus with anti-cytokeratin (CK) mAb, showed that TE- 4F10 mAb selectively stains a subpopulation of medullary TEC. Hematopoietic and lymphoid cells were negative. The expression of the TE-4F10 antigen on TE-R 2.5 cells in vitro was significantly upregulated by interleukin 1 (IL-1) and tumor necrosis factor (TNFalpha). Other cytokines IL-4, IL-6, IL-10 and granulocyte - macrophage colony stimulating factor (GM-CSF) showed lesser stimulation on its expression, whereas interferon gamma (IFN) and dexamethasone were without significant effect. The TE-R 2.5 cell line strongly bound and induced apoptosis of a rat / mouse thymocyte heterohybridoma (BWRT8), phenotypically alphabetaTCRhiCD4hiCD8lo. TE-4F10 mAb significantly inhibited binding (40-50%) of both BWRT8 cells and the BWRT8 - MDP.1 subclone to TE-R 2.5 cells. The inhibition was enhanced when TEC were stimulated with IL-1 + TNFalpha. The mAb also significantly blocked apoptosis of BWRT8 but did not modulate cell death of the BWRT8 - MDP.1 subclone, which was resistant to TEC-induced apoptosis. These findings indicate that the TE-4F10 antigen might be selectively involved in adhesion and selection processes in the medullary thymic microenvironment. The mAb of the same characteristics has not been described so far.     

The human thymic microenvironment: cortical thymic epithelium is an antigenically distinct region of the thymic microenvironment

The thymic microenvironment is a complex tissue essential for normal T cell maturation. Prothymocytes in the subcapsular cortical (SCC) region of the thymus undergo cell division and migrate to the inner cortex. The majority of cortical thymocytes cease dividing and die, but a minority are exported to the periphery. We have previously shown thymic hormones in SCC and medullary thymic epithelium and have identified a monoclonal antibody (TE-4) that defines human endocrine thymic epithelium. However, no marker that selectively defines cortical thymic epithelium has been available. In this study, we have produced two monoclonal antibodies, TE-3A and TE-3B, raised against human thymic stroma that bind to an intracellular antigen in cortical but not medullary thymic epithelium. In double immunofluorescence assays in which we used anti-keratin, anti-thymosin alpha 1, and anti-endocrine thymic epithelium antibodies (TE-4, A2B5), TE-3+ SCC epithelium was TE-4+ and contained keratin and thymosin. alpha 1. In contrast, TE-3+ inner cortical epithelium was TE-4/A2B5 nonreactive and did not contain thymosin alpha 1. An ontogeny study of seven fetal and five neonatal thymuses demonstrated that expression of the TE-3 antigen was acquired at 10 wk fetal gestation. Using TE-3 antibody, we observed sequential stages of separation of cortical and medullary epithelium from 12 to 20 wk fetal gestation. In dysplastic (severe combined immunodeficiency disease) thymuses, strands of TE-3+ nonendocrine cells encircled nests of TE-4+ endocrine epithelium. Thus, human cortical thymic epithelium is antigenically distinct from endocrine medullary epithelium. Antibodies against the TE-3 antigen define an intracellular molecule that may reflect a specialized function of cortical thymic epithelium.     

Cross-reactivity of antibodies on thymic epithelial cells from humans and marmosets by flow-cytometry

Callithrix jacchus, the common marmoset, is particularly suitable for immunological studies in vivo and in vitro since many antibodies directed against epitopes of human cells do also react with their analogues from this non-human primate. We studied the reactivity of antibodies against human epitopes on primary cultures of thymic epithelial cells from marmosets and humans by flow-cytometry after different culture periods. The antibodies against integrins, including CD61, reacted with thymic epithelial cells from both humans and marmosets, as did anti-CD44 and anti-CD106. Antibodies specific for thymic epithelial cells (TE-3, TE-4, TE-8, TE-15, TE-16, TE-19) also bound to cells from marmosets but expression of all epitopes was not observed in all cultures studied. The expression of CD51, CD54, CD58 and CD106 on human cells declined after 4 weeks of culture. Our findings indicate that marmosets are a valuable model for immunological studies of effects of xenobiotics on the thymic epithelium.     

The human thymic microenvironment. Phenotypic characterization of Hassall's bodies with the use of monoclonal antibodies

The human thymic microenvironment is important in promotion of T cell maturation, particularly during early stages of thymic ontogeny. Hassall's bodies (HB) are epithelial swirls in the human thymic medulla that are thought to be derived from endocrine medullary thymic epithelium. To study the ontogeny and function of various components of the human thymic microenvironment, we have produced four monoclonal antibodies (TE-8, TE-15, TE-16, and TE-19) that selectively reacted in thymus with HB. Antibodies TE-8 and TE-16 reacted with the cells forming the outer rim of the HB swirl. Antibody TE-19 reacted with the entire cellular portion of HB and with epithelial cells immediately surrounding HB. Granular foci in the cellular swirls of greater than 90% of HB reacted with antibody TE-15. During thymic ontogeny, the antigens defined by antibodies TE-8, TE-15, TE-16, and TE-19 were first detected in fetal thymus on HB beginning at 16 wk gestation, the age when HB morphologically appear in the thymus. Aberrant expression of the antigens corresponding to antibodies TE-8, TE-15, TE-16, and TE-19 was observed on thymic tissue from individuals with severe cellular immunodeficiency disease. In human skin, antibodies TE-8, TE-16, and TE-19 reacted with the stratum granulosum; antibody TE-15 reacted with the stratum corneum. Thus, with the use of antibodies TE-8, TE-15, TE-16, and TE-19, we have identified HB as antigenically distinct regions of endocrine thymic epithelium. Furthermore, we have shown that these anti-HB reagents also selectively react with epidermal keratinocytes in the terminal stages of keratinocyte maturation.     

Human thymomas: evidence of immunohistologically defined normal and abnormal microenvironmental differentiation

Fifteen human thymomas were analyzed by immunoperoxidase studies on frozen and paraffin-embedded tissue sections in an attempt to identify the existence of immunologically defined microenvironments. All nine lymphocyte predominant thymomas contained a predominance of lymphocytes bearing the phenotype of cortical thymocytes and dendritic Class II major histocompatibility complex antigen-positive epithelial cells, thus defining cortical-like microenvironments. Medullary-like foci were also seen in all of these cases. Minor phenotypic abnormalities in Leu-2 and -3 antigen expression were seen in three cases. In contrast, the two epithelial predominant thymomas and four mixed thymomas all exhibited features of aberrant microenvironmental differentiation, with only two cases showing demarcation into cortical and medullary foci. A lack of Class II major histocompatibility complex antigens was associated with a decrease in the lymphoid populations and an increase in Leu-1 antigen expression by T cells of otherwise normal cortical phenotype when lymphocytes were present. In contrast, lack of Class I antigen on epithelial cells was not associated with any abnormality in lymphocyte phenotype or microenvironmental organization. We document for the first time abnormal microenvironments in thymomas that may offer insights into understanding normal thymic differentiation.     

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.     

Phenotypic and functional characterization of human thymic stromal cell lines.

To establish new tools for studying human thymic stromal cells, we transfected adherent cells from a human postnatal thymus using a plasmid encoding SV40 large T antigen. Among the cell lines obtained, we characterized four epithelial cell lines (LT-TEC1 to LT-TEC4) and one thymic myoid cell line (MITC). Several morphological, functional and phenotypic differences were observed between these 2 cell types. Epithelial cells were heterogeneous and larger than myoid cells. Untreated LT-TEC lines expressed MHC class I, ICAM-1 and LFA-3 antigens and not MHC class II antigens, similarly to primary thymic epithelial cells (PTEC), while MITC line expressed only class I and LFA-3 antigens. After IFN-gamma treatment, MHC class II and ICAM-1 antigens were markedly upregulated in LT-TEC lines but not in MITC, indicating the absence or a dysfunction of regulatory factors in MITC line. Myoid cells expressed mRNA for all the subunits of the acetylcholine receptor (AChR) while epithelial cells expressed only the alpha, beta and epsilon subunits. Strikingly, LT-TEC produced much more C-C chemokines and IL-6 than MITC cells, while these latter produced higher levels of IL-8 and TNF-alpha. Altogether, these results reveal phenotypic and functional differences between these two stromal cell types, suggesting a potential involvement of myoid cells in the thymic function.     

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.     

Epithelial heterogeneity in the murine thymus: a cell surface glycoprotein expressed by subcapsular and medullary epithelium

A monoclonal antibody (MAb), G8.8, was raised against glycoconjugates isolated from a cloned line of murine medullary thymic epithelial cells. Flow cytometric analysis of the reactivity of this MAb with cultured thymic epithelium demonstrated that the ligand was expressed on the cell surface. Immunohistochemical examination of normal murine thymus revealed labeling of cells in the subcapsular and medullary areas. Immunoelectron microscopy revealed surface labeling restricted to cells possessing ultrastructural features of epithelium (desmosomes, tonofilaments, and cytoplasmic cysts). During thymic ontogeny, G8.8+ cells predominated in fetal development at the earliest time point examined (Day 14 of gestation). There was an expansion of the cortical epithelial component so that by Day 18 cortical and medullary compartments could be clearly distinguished. Immunoprecipitation of radioiodinated thymic stroma with MAb G8.8 detected a molecule with an apparent Mr of approximately 38 KD under non-reducing conditions. When reduced, the apparent Mr was slightly increased (42 KD). This MAb also exhibited reactivity with gut and epidermal epithelium and some tubular epithelium in the kidney, but did not react with epithelial parenchymal cells of the liver.     

Establishment of a mouse thymic epithelial cell line, IT-76MHC and a brief review on cultured thymic epithelial cells.

Interactions between thymocytes and thymic stromal cells are essential for thymocyte differentiation, but little evidence has been presented to directly show in vivo functions or interactions of the stromal cells. Among the stromal cells, the thymic epithelial cell has been considered to have profound effect on thymocyte differentiation and maturation. The calcium-depleted medium, originally developed for the culture of mouse epidermal cells, was applied for the culture of the mouse thymic epithelial cells, and successfully, an epithelial cell line, IT-76MHC was obtained from the mouse thymus. IT-76MHC cells were identified as distinct mouse thymic epithelial cells by 1/ mosaic-like arrangement, 2/ presence of well-developed desmosome and 3/ tonofilaments, 4/ positivity for cytokeratin, and 5/ induced expression of MHC class I and II by IFN-gamma treatment. IGF-1, IGF-2, oxytocin and vasopressin were also detected immunohistochemically in IT-76MHC cells. Furthermore, the IT-76MHC thymic epithelial cells, when injected intrathymically in the allogeneic mouse, prolonged the survival of skin graft from the same donor strain that IT-76MHC cells were derived. These results demonstrate that the thymic epithelial cell line IT-76MHC produces modest thymocyte survival factors as well as a growth suppressor, and that IT-76MHC cells have the ability to induce transplantation tolerance probably through their expression of MHC class I and II molecules. Taken altogether, the IT-76MHC thymic epithelial cells have been proved to be useful tools to better understand the in vivo functions of thymic epithelial cells, and to gain a deep insight into their involvement in the critical selection process of thymocytes which still remains obscure. Finally and additionally, literatures so far reported on thymic epithelial cells in culture, especially lines and clones, are reviewed and their identity as well as their functions are discussed.     

Unique subset of thymic epithelial cells defined by the expression of a novel neuroendocrine antigen

Murine monoclonal antibodies (MoAbs) were produced against a rat medullary thyroid carcinoma to identify neuroendocrine differentiation antigens. One of these antibodies (1D4) identified a novel 62- to 69-kDa antigen expressed by subsets of immune system epithelial and neuroendocrine cells. This antigen is expressed by distinct subsets of thymic epithelial and splenic reticular cells and is shared by discrete subsets of anterior pituitary and thyroid neuroendocrine cells. In the thymus, 1D4 expression identified a unique subset of stellate-shaped Ia+ medullary epithelial cells which did not react with thymosin alpha-1 antisera nor with the MoAb A2B5 specific for a GQ ganglioside expressed by thymic hormone-producing cells. The availability of the 1D4 MoAb should facilitate further characterization of 1D4+ immune system epithelial cells and may advance our understanding of neuroendocrine-immune system interactions.     

Analysis of thymic stromal cell subpopulations grown in vitro on extracellular matrix in defined medium. I. Growth conditions and morphology of murine thymic epithelial and mesenchymal cells

We report here the successful selective cultivation of murine thymic mesenchymal reticular cells (MTMC) and murine thymic epithelial cells (MTEC) grown on extracellular matrix in the presence of defined medium. The selective growth of these two cell types was based on 1) conditions of tissue disruption and 2) differential growth requirements. Both cell types were dependent on transferrin, high density lipoproteins, insulin, hydrocortisone, and epidermal growth factor, whereas MTMC was dependent also on selenium and 3,5,3'-triiodothyronine. The elimination of single factors or extracellular matrix resulted in specific and different changes in the growth pattern of each cell subpopulation. Cells of both types exhibited the ultrastructural features of high metabolic activity. The epithelial nature of MTEC cultures was defined by bundles of tonofilaments and desmosomes and by positive staining to keratins and negative to vimentin. In addition MTEC were positively stained with mAb to thymic medullary epithelial cells and by Ulex europeus agglutinin, and were able to form Hassall's corpuscles, suggesting their medullary origin. MTEC were also H-2 and Ia positive. In contrast MTMC were positive for vimentin and periodic acid-Schiff, low positive for H-2, and negative for keratin and Ia. Both cells did not contain nonspecific esterase, nor did they phagocytize latex beads. With the use of all these criteria we classified MTEC as epithelial cells from the medullary compartment of the thymus and MTMC as reticular cells of mesenchymal origin.     

Variations in the expression of pili: the effect on adherence of Neisseria meningitidis to human epithelial and endothelial cells

The effect of variations in Neisseria meningitidis pili on bacterial interactions with three epithelial cell lines as well as human umbilical vein endothelial cells was studied using a panel of seven strains expressing Class I or Class II pili. Comparison of adherence of piliated and pilus-deficient variants of each strain to epithelial cells suggested that Class I pili may mediate bacterial adherence with all three epithelial cell lines. In contrast, Class II pili of the strains used did not increase bacterial adherence to Hep-2 larynx carcinoma cells, although an increase in adherence to Chang conjunctival and A549 lung carcinoma epithelial cells was observed in the Class II pili-expressing strains. In addition to these interclass functional variations, differences in adherence to epithelial cells were also observed among Class I and Class II strains. Functionally different pilin variants of one Class I strain, MC58, were obtained by single colony isolation. One piliated variant was identified which had concurrently lost the ability to adhere to both Chang and Hep-2 cells ('non-adherent' phenotype; adherence of less than 2 bacteria per cell). In addition, several adherent pilin variants were isolated from non-adherent Pil- and Pil+ bacteria by selection on Chang cells (adherence of 10-25 bacteria per cell). In contrast to epithelial cells, all variant pili, whether of Class I or Class II, adhered to endothelial cells in substantially larger numbers (greater than 50 bacteria per cell) and therefore implied the existence of distinct mechanisms in pilus-facilitated interactions of N. meningitidis with endothelial and epithelial cells.     

IGFBP3 and BAG1 enhance radiation-induced apoptosis in squamous esophageal cancer cells

Identification of reliable markers of radiosensitivity and the key molecules that enhance the susceptibility of esophageal cancer cells to anticancer treatments would be highly desirable. To identify molecules that confer radiosensitivity to esophageal squamous carcinoma cells, we assessed the radiosensitivities of the TE-5, TE-9 and TE-12 cloneA1 cell lines. TE-12 cloneA1 cells showed significantly greater susceptibility to radiotherapy at 5 and 10 Gy than either TE-5 or TE-9 cells. Consistent with that finding, 24 h after irradiation (5 Gy), TE-12 cloneA1 cells showed higher levels of caspase 3/7 activity than TE-5 or TE-9 cells. When we used DNA microarrays to compare the gene expression profiles of TE-5 and TE-12 cloneA1 cells, we found that the mRNA and protein expression of insulin-like growth factor binding protein 3 (IGFBP3) and Bcl-2-associated athanogene 1 (BAG1) was five or more times higher in TE-12 cloneA1 cells than TE-5 cells. Conversely, knocking down expression of IGFBP3 and BAG1 mRNA in TE-12 cloneA1 cells using small interfering RNA (siRNA) significantly reduced radiosensitivity. These data suggest that IGFBP3 and BAG1 may be key markers of radiosensitivity that enhance the susceptibility of squamous cell esophageal cancer to radiotherapy. IGFBP3 and BAG1 may thus be useful targets for improved and more individualized treatments for patients with esophageal squamous cell carcinoma.     

H2-O inhibits presentation of bacterial superantigens, but not endogenous self antigens

H2-O/HLA-DO are MHC class II accessory molecules that modulate exogenous Ag presentation. Most class II accessory molecules are expressed in all professional APC; however, H2-O is only expressed in B cells and medullary thymic epithelial cells. Because B cells present exogenous Ags and superantigens (SAgs), and medullary thymic epithelial cells are specialized APC for self Ags during negative selection in the thymus, we have hypothesized that H2-O might play a role in MHC class II-restricted SAg and self Ag presentation. In this study, we demonstrate that H2-O expression inhibits presentation of the bacterial SAgs staphylococcal enterotoxins A and B to four SAg-reactive T hybridoma cells. In contrast, H2-O has no effect on presentation of endogenous self Ags, as measured by tumorigenicity in vivo and Ag presentation to three self Ag-specific T hybridoma cells. Additional experiments suggest that H2-O inhibits presentation of exogenous Ags by both newly synthesized and recycling MHC class II molecules. These data suggest H2-O may have a physiological role in tolerance induction and SAg-mediated toxic shock.