Thymic hormone containing cells--IX. Steroids in vitro modulate thymulin secretion by human and murine thymic epithelial cells

We investigated the in vitro effects (kinetics and dose-response) of adrenal and sexual steroid hormones on the secretion of thymulin, a thymic hormone, by human thymic epithelial cells in primary cultures as well as in a rat epithelial cell line. We demonstrated that all steroids tested, in a range of physiological doses, stimulated thymulin production to various extents. Progesterone and estradiol, however, were revealed to be the most efficient. Specific steroid antagonists abrogated the steroid-induced stimulation of thymulin production. These findings confirm our previous in vivo results and demonstrate that steroid hormones can act directly on thymic epithelial cells to modulate their endocrine production.     

Thymic hormone-containing cells. VIII. Effects of colchicine, cytochalasin B, and monensin on secretion of thymulin by cultured human thymic epithelial cells

The intracellular pathway for secretion of thymulin, a thymic hormone, was studied in primary cultures of human thymic epithelial cells by experimentally blocking the movement of secretory vesicles within these cells. These cultures were subjected to cytoskeleton inhibitors, such as colchicine and/or cytochalasin B, that block the directed movement of secretory vesicles, or to monensin, an ionophore that specifically perturbs the traffic of Golgi-derived vesicles. Both cytoskeleton inhibitors partially prevented thymulin secretion into the culture supernatants, and their effects were dose-dependent. Moreover, the percentage of thymulin-containing cells (evaluated by immunofluorescence with a zinc-specific anti-thymulin monoclonal antibody), as well as the fluorescence intensity within these cells, was significantly higher than observed in control cultures, suggesting that the hormone was accumulated in the cytoplasm, thus facilitating its detection. Similar results were obtained with monensin. These results, together with the recent identification of high molecular weight proteins reacting with anti-thymulin antibodies, suggest that thymulin is secreted via the following intracellular pathway: a precursor is synthesized at the level of the granular endoplasmic reticulum; it migrates to the Golgi complex, from which it is released within hormone-containing vesicles; the vesicles incorporate zinc, move towards the cell membrane, and eventually fuse with it. This sequence of events characterizes the classical phenomenon of exocytosis.     

Effects of cytokines on human thymic epithelial cells in culture. II. Recombinant IL 1 stimulates thymic epithelial cells to produce IL6 and GM-CSF

Our earlier study reported the ability of interleukin 1 (IL1) to promote proliferation and to induce morphological changes of human thymic epithelial cells (TEC) in culture. The present study was undertaken to examine the effects of IL1 on the secretory function of TEC. Both human recombinant IL1 alpha and IL1 beta induced TEC to produce molecules in the culture supernatant fluids (TES) which displayed marked thymocyte proliferative capacities. This activity was specifically induced by IL1 since other TEC growth factors such as epidermal growth factor and a bovine pituitary extract had no effect on promoting secretion of T cell-activating molecules by TEC. Using specific radioimmunoassays for both forms of IL1, we found that unstimulated TEC produced negligible amounts of IL1 alpha and IL1 beta in TES, which were not increased by IL1 stimulation, and we concluded that the IL1-induced TES molecules were not IL1. IL1 induced TEC to produce IL6, as detected by the hybridoma growth factor biological activity. Neutralizing anti-IL6 antibodies completely blocked the thymocyte activating capacities of the IL1-induced TES thus implying a major role for IL6 in TEC-derived T cell activation. IL1 also induced TEC to produce GM-CSF as measured by bioassay and confirmed by an immunoenzymetric assay. Our results confirm that TEC are a source of cytokines and show that TEC respond to IL1 by producing cytokines with consequences on the thymic lymphoid population. This further emphasizes the importance and complexity of paracrine molecular interactions involved in intrathymic development.     

Effects of infection by HIV-1, cytomegalovirus, and human measles virus on cultured human thymic epithelial cells

A tissue culture system for the growth of human fetal and infantile thymic epithelial (TE) cells has been established and characterized. We have investigated the effects of infection of these cells by human cytomegalovirus (CMV), measles virus, and human immunodeficiency virus type-1 (HIV-1). In the case of CMV, morphological changes were apparent by 2-4 days after viral inoculation of infantile TE cells. CMV-related antigens were detected by immunofluorescence after 12 days, and progeny infectious CMV was recovered from culture media after 18 days. Following infection by measles virus, distinctive, multinucleated giant TE cells appeared in both cultures of fetal and infantile TE cells. Measles virus-inoculated TE cells displayed an altered phenotype, as revealed by reaction with monoclonal antibodies with specificity for a variety of TE markers. Finally, infection of TE cells by HIV-1 resulted in cellular disarrangement, increased numbers of Hassall's corpuscles, and multinucleated giant cells. An increase in the number of cells reactive with monoclonal antibodies, specific for Hassall's corpuscles, was observed in the case of cells infected by either measles virus or HIV-1. These findings suggest that a variety of different viruses can successfully infect thymic epithelial tissue. Because of the important role of the thymus in development of the immune system, it is reasonable to conclude that viral infection of thymic tissue might play an important role in virus-mediated suppression of immune responsiveness.     

Stimulatory effect of thymic factor(s) on steroidogenesis in cultured rat granulosa cells.

Thymic cells from immature female rats were isolated and used for production of thymic cell culture conditioned medium (TCM). Granulosa cells were obtained from immature diethylstilbestrol (DES)-treated rats. TCM stimulated basal progesterone and estradiol secretion from the granulosa cells in a dose and time dependent manner. Maximal stimulation of progesterone production occurred at 48 hours of incubation, during which period TCM caused approximately 5 times more progesterone secretion than heart cell conditioned medium (HCM) or mock extract (ME). The maximum progesterone secretion by granulosa cells occurred when they were exposed to 48% TCM causing 7 times more progesterone secretion than controls. Under the same maximum stimulatory conditions, however, TCM only approximately doubled estradiol secretion compared to concentrations secreted in the presence of HCM or ME. Thus, the effect of TCM on progesterone secretion was more prominent than its effect on estradiol secretion. The stimulatory action of TCM was not mimicked by HCM, thymosin-alpha 1 or thymulin. Furthermore, the stimulatory action of TCM on steroidogenesis did not appear to be mediated by the cAMP system. The stimulatory factor(s) in TCM were heat, acid and acetone labile, but could not be sedimented by activated charcoal. Thus, the present study demonstrates that the secretory product(s) of thymic epithelial cells can stimulate steroidogenesis in cultured rat granulosa cells. Our data imply that thymic factor(s) may have a direct effect on ovarian function.     

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.     

Prostaglandin production by type II alveolar epithelial cells.

Prostaglandin production was studied in fetal and adult type II alveolar epithelial cells. Two culture systems were employed, fetal rat lung organotypic cultures consisting of fetal type II cells and monolayer cultures of adult lung type II cells. Dexamethasone, thyroxine, prolactin and insulin, hormones which influence lung development, each reduced the production of prostaglandin E and F alpha by the organotypic cultures. The fetal cultures produced relatively large quantities of prostaglandin E and F alpha and smaller quantities of 6-keto-prostaglandin F1 alpha and thromboxane B2. However, prostaglandin E2 production was predominant. In contrast, the adult type II cells in monolayer culture produced predominantly prostacyclin (6-keto-prostaglandin F1 alpha) along with smaller quantities of prostaglandin E2 and F2 alpha. The type II cells were relatively unresponsive to prostaglandins. Exogenously added prostaglandin E, had no effect on cell growth, and only a minimal effect on cyclic AMP levels in the monolayer cultures.     

IL-1, IL-4, and IFN-gamma differentially regulate cytokine production and cell surface molecule expression in cultured human thymic epithelial cells.

We investigated the response of purified and cloned human thymic epithelial cells (TEC) to IL-1, IL-4, and IFN-gamma stimulation in vitro. IL-1 alpha strongly up-regulated the production of granulocyte-macrophage CSF (GM-CSF), granulocyte CSF (G-CSF), IL-6, and IL-8, as measured by specific immunoenzymetric assays and by increased steady state mRNA levels. IL-4 or IFN-gamma did not induce these cytokines in TEC but in a sustained and dose-dependent manner down-regulated the IL-1-induced GM-CSF protein and mRNA levels. Only IFN-gamma, and not IL-4, suppressed the IL-1-induced G-CSF and IL-8 production, as shown at both the protein and mRNA levels. The inhibition was dose dependent, sustained for at least 96 h, and more pronounced for G-CSF than for IL-8. In contrast, both IL-4 and IFN-gamma enhanced the IL-1-induced IL-6 production. IL-4 and IFN-gamma had additive effects to increase IL-6 secretion and to more completely suppress the IL-1-induced GM-CSF. Analyses of cell surface molecules showed that intercellular adhesion molecule 1 (ICAM-1) expression on TEC was increased by IL-1 or IFN-gamma. IL-4 slightly down-regulated constitutive ICAM-1 levels but did not significantly modify the levels of expression induced by either IL-1 or IFN-gamma. MHC class II expression was induced by IFN-gamma but not by IL-1 or IL-4. The combination of IL-1 and IL-4 with IFN-gamma did not alter the levels of class II MHC Ag induced by IFN-gamma. In conclusion, TEC cytokine production and cell surface molecule expression are differentially regulated via a complex cytokine network. Our data suggest that developing T cells provide, in part, the signals controlling the function of their supporting stroma.     

Synergistic induction of interleukin-6 production and gene expression in human thymic epithelial cells by LPS and cytokines

We examined the ability of LPS and several cytokines (TNF-alpha, IL-1-beta, IFN-gamma, IL-4) to modulate IL-6 production by cultured human thymic epithelial cells (TEC). IL-6 activity was measured by the hybridoma growth factor biological activity. Moderate but detectable IL-6 activity was spontaneously produced in the presence of serum proteins. LPS as well as the cytokines TNF-alpha and IL-1-beta was a potent inducer of IL-6, increasing, respectively, IL-6 levels by 9-, 28-, and 75-fold (mean values) while IL-4 and IFN-gamma provoked no significant effect. Interestingly, clearly different kinetics were observed for IL-6 induction by the various activation agents, the maximal effect being reached at 24, 48, and 72 hr, respectively for LPS, TNF-alpha, and IL-1-beta. Moreover, a synergistic effect of TNF-alpha and either LPS or IL-1-beta was observed. Indeed, TEC incubated with the cytokines in combination at optimal doses produced 5- to 170-fold more IL-6 than TEC stimulated with the cytokines individually. Neutralizing anti-IL-6 polyclonal and monoclonal antibodies completely blocked hybridoma proliferation stimulating activity of TEC supernatants; thus, implying that this activity is essentially due to IL-6. In situ hybridization analysis of cytocentrifuged TEC with an mRNA antisense probe specific for human IL-6 and labeled with 35S demonstrated that up to 90% of TEC could be induced to express the IL-6 gene. Computer-aided quantification of IL-6 mRNA levels indicated that upon stimulation with TNF-alpha combined to LPS, both the numbers of cells expressing IL-6 mRNA and the amounts of cytoplasmic IL-6 mRNA per cell were increased. Taken altogether these results demonstrate that LPS and/or cytokines can modulate and synergistically stimulate IL-6 production. In addition to a possible role in regulating normal thymic T cell activation, the IL-6 produced by TEC could be of pathophysiological relevance in disregulated situations such as in hyperplastic thymuses from patients with myasthenia gravis.     

Ligand binding to the LFA-3 cell adhesion molecule induces IL-1 production by human thymic epithelial cells.

We have shown that human thymic epithelial (TE) cells produce IL-1 alpha, IL-1 beta, and TE cells bind to thymocytes by CD2 and LFA-1 molecules on thymocytes and LFA-3, ICAM-1 on TE cells. We investigated whether ligand binding to LFA-3 on human TE cells can modulate TE cell IL-1 production. First, we investigated the ability of human thymocytes to regulate IL-1 release by TE cells. Both autologous and allogenic emetine-treated thymocytes when cultured with TE cells augmented IL-1 release by TE cells. The augmentation of IL-1 release was cell density dependent. Inasmuch as the interaction between thymocytes and TE cells is mediated in part by CD2 molecules on thymocytes and LFA-3 molecules on TE cells we next determined the effect on IL-1 release of ligand binding (anti-LFA-3 mAb TS2/9) to TE cell surface LFA-3. Purified anti-LFA-3 mAb augmented IL-1 release in a concentration-dependent fashion. The anti-LFA-3-mediated augmentation of IL-1 release required both new protein and RNA synthesis as shown by the ability of cycloheximide and actinomycin-D to inhibit augmentation of IL-1 production by TE cells, and by direct quantitation of IL-1 alpha and IL-1 beta mRNA by Northern blot analysis. Both F(ab)'2 and Fab' fragments of anti-LFA-3 mAb augmented IL-1 alpha and IL-1 beta mRNA production, indicating that monovalent binding to cell surface LFA-3 was sufficient to provide the inducing signal. The identification of LFA-3, the cell surface ligand for thymocyte CD2 molecules, as a molecule via which TE cell-derived cytokine production may be regulated suggests a mechanism at the cell surface by which direct TE cell-thymocyte interaction might result in the triggering of local IL-1 release within the human thymic microenvironment.     

Development of dendritic cells in culture from human and murine thymic precursor cells.

The earliest T-precursor population in the adult murine thymus can give rise to dendritic cells (DC) in culture if stimulated with a cocktail of cytokines that includes interleukin (IL)-3, but not with cytokine mixes based on granulocyte-macrophage colony stimulating factor (GM-CSF), normally used to generate myeloid-derived DC. This and other evidence led to the proposal that two different lineages of DC exist, one lymphoid-related and the other myeloid-related. To determine whether this selective response to cytokines was restricted to murine DC, early human thymic T-precursors were isolated and their capacity to generate DC in response to various cytokines directly compared to their murine counterparts. In contrast to cultures of murine thymic precursors, CD34+CD1a- lineage marker negative (Lin-) precursor cells from the human thymus proliferated and generated DC with both the IL-3-containing cytokine mix lacking GM-CSF and with GM-CSF based cytokine mixes. These CD34+CD1a-Lin- human precursor cells also gave rise to NK cells under appropriate culture conditions, but produced no granulocyte, monocyte, eosinophil, megakaryocyte or erythroid cells in standard soft-agar colony-forming cell assays. Thus, although apparently lymphoid-restricted, the human thymic DC precursors responded to the myeloid factor GM-CSF as well as to the cytokines selective for murine lymphoid-related DC.     

Pertussis toxin-dependent and -independent hormonal effects on cultured renal epithelioid cells

The present study has been performed to test for the involvement of pertussis toxin-sensitive GTP-binding proteins (G-proteins) in the cellular transduction of hormone-induced activation of potassium channels. In Madin Darby canine kidney (MDCK) cells, a permanent cell line from dog kidney, epinephrine, acetylcholine, bradykinin, serotonin and ATP hyperpolarize the cell membrane by activation of potassium channels. In cells pretreated with pertussis toxin the hyperpolarizations elicited by either acetylcholine or serotonin are completely abolished; that following epinephrine is blunted and only transient. The hyperpolarizing effects of ATP or bradykinin are not affected by pertussis toxin. Thus, in MDCK cells both pertussis toxin-dependent and -independent mechanisms operate in parallel to enhance the potassium conductance of the cell membrane.     

Thymocyte LFA-1 and thymic epithelial cell ICAM-1 molecules mediate binding of activated human thymocytes to thymic epithelial cells.

We have investigated the binding in vitro of activated thymocytes to thymic epithelial (TE) cells, and studied the effect of up-regulation of TE cell surface intracellular adhesion molecule 1 (ICAM-1) and HLA-DR by IFN-gamma on the ability of TE cells to bind to both resting and activated human thymocytes. TE cell binding to activated and resting thymocytes was studied by using our previously described suspension assay of TE-thymocyte conjugate formation. We found that activated mature and immature thymocytes bound maximally at 37 degrees C to IFN-gamma-treated ICAM-1+ and HLA-DR+ TE cells and this TE-activated thymocyte binding was inhibited by antibodies to LFA-1 alpha-chain (CD11a) (68.1 +/- 5.6% inhibition, p less than 0.01) and ICAM-1 (73.9 +/- 7.7% inhibition, p less than 0.05). Neither anti-HLA-DR antibody L243 nor anti-MHC class I antibody 3F10 inhibited IFN-gamma-treated TE binding to activated thymocytes. As with antibodies to LFA-3 and CD2, antibodies to LFA-1 and ICAM-1 also inhibited PHA-induced mature thymocyte activation when accessory signals were provided by TE cells in vitro. Finally, LFA-1 and ICAM-1 were expressed early on in human thymic fetal ontogeny in patterns similar to those seen in postnatal thymus. Taken together, these data suggest that resting mature and immature thymocytes bind to TE cells via the CD2/LFA-3 ligand pair, whereas activated thymocytes bind via both CD2/LFA-3 and LFA-1/ICAM-1 ligand systems. We postulate that IFN-gamma produced intrathymically may regulate TE expression of ICAM-1 and therefore potentially may regulate TE cell binding to activated thymocytes beginning in the earliest stages of human thymic development.     

Homocysteine decreases endothelin-1 production by cultured human endothelial cells.

Hyperhomocysteinemia is believed to be responsible for the development of vascular disease via several mechanisms, including the impairment of endothelial-cell functionality. In-vitro studies have demonstrated that homocysteine decreases the production or bioavailability of vasodilator autacoids, such as prostacyclin and NO. Here, we show that the treatment of human endothelial cells with noncytotoxic homocysteine concentrations leads to a dose-dependent decrease in both the secretion of the vasoconstrictor agent endothelin-1 (ET-1) and the level of its mRNA. Homocysteine had an inhibitory effect at pathophysiological (0.1 and 0.5 mmol.L(-1)) and pharmacological noncytotoxic (1.0 and 2.0 mmol.L(-1)) concentrations. Mean percentage variation from control for ET-1 production was -36. 2 +/- 18.9% for 0.5 mmol.L(-1) homocysteine and -41.5 +/- 26.8% for 1.0 mmol.L(-1) homocysteine, after incubation for 8 h. Mean percentage variation from control for steady-state mRNA was -17.3 +/- 7.1% for 0.5 mmol.L(-1) homocysteine and -46.0 +/- 10.1 for 1.0 mmol.L(-1) homocysteine, after an incubation time of 2 h. ET-1 production was also reduced by incubation with various other thiol compounds containing free thiol groups, but not by incubation with thiol compounds with no free thiol group. Co-incubation of cells with homocysteine and the sulfhydryl inhibitor N-ethylmaleimide prevented the effect of homocysteine on ET-1 production, confirming a sulfhydryl-dependent mechanism. Based on the reciprocal feedback mechanism controlling the synthesis of vasoactive mediators, these preliminary data suggest a mechanism by which homocysteine may selectively impair endothelium-dependent vasodilation by primary inhibition of ET-1 production.     

Secretion of lysosomal hydrolases by cultured human amnion epithelial cells

Primary microcultures of human amnion epithelial cells were established, starting from sterile term placentae. Over a period of 1 week in culture, the epithelial cells release into the extracellular medium substantial amounts of some lysosomal hydrolases, such as sphingomyelinase, N-acetyl-beta-glucosaminidase, alpha-fucosidase, beta-glucuronidase, alpha-mannosidase, and arylsulfatase. Judging from experiments conducted with the protein synthesis inhibitor, cycloheximide, the enzymes released are not newly synthesized forms, but very likely derive from lysosomes. The constitutive secretion of lysosomal enzymes, coupled with lack of immunogenicity, makes amnion epithelial cells a convenient source of enzymes for implantation in attempts of enzyme replacement therapies.