Thymic hormone-containing cells. V. Immunohistological detection of metallothionein within the cells bearing thymulin (a zinc-containing hormone) in human and mouse thymuses

Using an immunofluorescence (IF) assay, the presence of metallothionein (MT) was investigated in sections of normal and pathologic human thymuses as well as in cultures of thymic epithelial cells. This protein, known to have a high binding affinity for class II B transitional metals, such as zinc, was detected in the epithelial component of the thymus. Moreover, double labeling experiments with the anti-MT and an anti-thymulin monoclonal antibody showed that all cells containing thymulin, a thymic hormone whose active structure is known to contain zinc, also exhibited large amounts of metallothionein. These results, together with the fact that zinc and thymulin have been detected in the same type of cell organelles, lead to the conclusion that the MT present in thymic epithelial cells might be involved in the mechanism of zinc storage in these cells, thus favoring the secretion of thymulin in its biologically active, zinc-containing form.     

Localization of thymulin (FTS-Zn) in mouse thymus. Comparative data using monoclonal antibodies following different plastic embedding procedures

The distribution of thymulin (FTS-Zn) was studied in thymuses from normal mice (OF1) or autoimmune mice (NZB). Thymulin localization was investigated using immunocytochemical techniques on sections of GMA and epon-embedded mouse thymuses. Two monoclonal antibodies were used: anti-synthetic thymulin and anti-intracellular thymulin. In the immunofluorescence assay, GMA sections allowed a more subtle localization of thymulin in the cytoplasm of epithelial cells (with a vacuolar pattern) compared to the epon sections (with a homogeneous fluorescence in the cytoplasm). In both cases, the number of labeled cells was greater in the medullary region than in the cortex of the thymus. At the electron microscopic level, immunolabeling of epon ultrathin sections showed ferritin distributed in some of the epithelial cell vacuoles. The two monoclonal antibodies revealed similar distributions of thymulin in the thymus. The results obtained in this study confirm that the amount of thymulin is greater in the epithelial cells of normal compared to autoimmune thymuses.     

Localization of thymulin (FTS-Zn) in mouse thymus. Comparative data using monoclonal antibodies following different plastic embedding procedures

The distribution of thymulin (FTS-Zn) was studied in thymuses from normal mice (OF1) or autoimmune mice (NZB). Thymulin localization was investigated using immunocytochemical techniques on sections of GMA and epon-embedded mouse thymuses. Two monoclonal antibodies were used: anti-synthetic thymulin and anti-intracellular thymulin. In the immunofluorescence assay, GMA sections allowed a more subtle localization of thymulin in the cytoplasm of epithelial cells (with a vacuolar pattern) compared to the epon sections (with a homogeneous fluorescence in the cytoplasm). In both cases, the number of labeled cells was greater in the medullary region than in the cortex of the thymus. At the electron microscopic level, immunolabeling of epon ultrathin sections showed ferritin distributed in some of the epithelial cell vacuoles. The two monoclonal antibodies revealed similar distributions of thymulin in the thymus. The results obtained in this study confirm that the amount of thymulin is greater in the epithelial cells of normal compared to autoimmune thymuses.     

Cytotoxic T cell response and thymic hormonal dysfunction in graft-vs-host mice

As an approach to dissect complex mechanisms that lead to graft-vs-host (GvH)-associated immune disorders, we have compared the splenic cytotoxic T lymphocyte (CTL) response and thymic hormonal function in nonirradiated F1 hybrid mice injected with parental spleen cells. Thymic secretory function was studied by the determination of serum thymulin levels, and the number of thymic epithelial cells containing thymulin as assessed by indirect immunofluorescence with the use of an anti-thymulin monoclonal antibody. In addition, the epithelial cell network was analyzed with an anti-keratin serum, and the general histology pattern was studied by conventional histologic methods. An initial analysis was performed on day 15, which was characterized by CTL suppression mediated by parental suppressor T cells. No thymic abnormalities were detected at this time. By day 45 after GvH induction, active CTL suppression had decreased, and GvH was associated with a progressive decline in thymic hormonal function. Finally, by day 60 and thereafter, F1 GvH mice recovered normal in vitro CTL responsiveness, which contrasted with profound alterations of the epithelial cell network and severely reduced serum thymulin levels. This hormonal dysfunction was shown to be directly associated with a reduction in the number of thymulin-containing cells. Moreover, no anti-thymulin auto-antibodies could be detected. The results are discussed with respect to the role of thymic hormonal dysfunction in the modulation of F1 CTL responses observed during the course of a GvH reaction, and the additional analogy of this GvH model with human immunodeficiency.     

Immunochemical characterization of molecules identified in human thymic epithelium by anti-thymulin monoclonal antibodies

Four high molecular weight molecules (ranging from 59 to 48 kd) were evidenced in the human thymic epithelium, after one and two dimensional immunoblot analyses, using monoclonal antibodies directed against synthetic thymulin. One of these immunoreactive proteins might correspond to the intra-thymic precursor of the hormone.     

Neuroendocrine control of thymic hormonal production. II. Stimulatory effects of endogenous opioids on thymulin production by cultured human and murine thymic epithelial cells

Data have now accumulated to strongly demonstrate that several neuropeptides, including endogenous opioids, can have immunomodulatory functions. Most of the studies have so far focused on the direct action of these substances on lymphocytes. We decided to investigate whether thymic epithelial cells (TEC) - the major component of the thymic microenvironment - could also be modulated by endogenous opioids. Primary cultures of human and murine TEC were subjected to several opioids (alpha-beta- or gamma-endorphins, as well as met- or leuenkephalins) applied in concentrations ranging from 10(-6) to 10(-9) M. On the following days we measured the levels of thymulin (a chemically-defined thymic hormone known to stimulate some steps of T-cell differentiation) in the culture supernatants, as well as the numbers of thymulin containing cells, evaluated by immunofluorescence with an anti-thymulin monoclonal antibody. After treatment of TEC cultures with beta-endorphin or leu-enkephalin a significant increase in the levels of thymulin in the culture media was observed, paralleled by a rise in the percentage of thymulin containing cells. In addition, this stimulatory effect was dose-dependent. Preincubation of the opioids with the specific antibodies abrogated the opioid-induced stimulatory effect on TEC. Moreover, naloxone, an opioid receptor antagonist, blocked the effect of beta-endorphin on thymulin production, suggesting that the effect of this neuropeptide on epithelial cells was mediated by an opioid receptor. Importantly, no effect on thymulin production was observed with the other opioids used, whatever the dose. These results suggest that, at least in vitro, beta-endorphin and leu-enkephalin stimulate the hormonal function of the thymic epithelium. These findings lead to the general concept that the modulatory role of endogenous opioids on the immune system is not restricted to lymphocytes but can also take place at the level of cells belonging to T-cell differentiating microenvironments.     

Induction of thymocyte proliferation by supernatants from a mouse thymic epithelial cell line

The thymic stroma plays a critical role in the generation of T lymphocytes by direct cell-to-cell contacts as well as by secreting growth factors or hormones. The thymic epithelial cells, responsible for thymic hormone secretion, include morphologically and antigenically distinct subpopulations that may exert different roles in thymocyte maturation. The recent development of thymic epithelial cell lines provided an interesting model for studying thymic epithelial influences on T cell differentiation. Treating mouse thymocytes by supernatants from one of TEC line (IT-76M1), we observed an induction of thymocyte proliferation and an increase in the percentages of CD4-/CD8- thymocytes. This proliferation was largely inhibited when thymocytes were incubated with IT-76M1 supernatants together with an anti-thymulin monoclonal antibody, but could be enhanced by pretreating growing epithelial cells by triiodothyronine. We suggest that among the target cells for thymulin within the thymus, some putative precursors of early phenotype might be included.     

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.     

Heterogeneity of thymic epithelial cell (TEC) keratins--immunohistochemical and biochemical evidence for a subset of highly differentiated TEC in the mouse

The mouse thymic epithelial network was studied using three different anti-keratin antibodies. One of these antibodies, KL1, exclusively recognized a small subset of medullary epithelial cells characterized by its content of a high molecular weight keratin (63 kD). Since epithelial differentiation is known to be associated with the acquisition of high molecular weight keratins, KL1-positive cells, which express the Ia antigen and secrete thymulin, may represent a subset of highly differentiated cells among mouse thymic epithelial cells (TEC). These data reflect the heterogeneity of the thymic epithelium and support the concept that distinct TEC subsets might provide the thymus with different microenvironments.     

Immunohistochemical localization of thymopoietin with an antiserum to synthetic Cys-thymopoietin28-39

Thymopoietin-containing cells in the thymus were identified immunohistochemically using murine antiserum generated by immunization with synthetic Cys-thymopoietin28-39 (Cys-TP28-39). human thymopoietin, This antiserum, previously shown to react with both bovine and human thymopoietin, gave reactivity restricted to cortical and medullary epithelial cells of bovine and human thymus. Monoclonal antibodies with reactivity restricted to native bovine thymopoietin did not react with tissue sections of bovine thymus; most likely the epitopes recognized by monoclonal antibodies are not expressed on the inactive precursor forms of thymopoietin within thymic epithelial cells.     

Rat thymic epithelial cells in vitro and in situ: characterization by immunocytochemistry and morphology

A new method for the long-term culture of pure rat thymic epithelial cells was established. The cultures were characterized by immunocytochemistry, electron microscopy and proliferation assays. Non-epithelial thymic cells were eliminated with a reliable and reproducible pre-plating method, by differential trypsin treatment of the cultures and by addition of horse serum to the culture medium instead of fetal calf serum. The final cultures contained more than 95% pure epithelial cells as evidenced by immunostaining for cytokeratin. Ultrastructural studies indicated that these cells are physiologically active epithelial cells with tonofilaments, desmosomes and filopods. The subsets of the thymic epithelial cells in vitro were investigated by comparing their staining pattern with that obtained in situ using several subtype-selective antibodies. Thymic epithelial cells in vitro showed a preferential expression of subcapsular/perivascular and medullary markers. Only few cultivated cells were of cortical origin. In the first to the fourth subcultures, some cells were immunopositive for the thymus hormone/factor thymulin. The proliferation of thymic epithelial cells was stimulated by horse serum and to a lesser extend by fetal calf serum. The adenylate cyclase activators isoproterenol and forskolin, and the glucocorticoid cortisol inhibited the proliferation. Received: 12 May 1995 / Accepted: 13 October 1995     

Monoclonal antibodies to the pituitary growth-hormone receptor by the anti-idiotypic approach. Production and initial characterization.

We obtained 10/192 and 3/384 antibody-secreting hybrids after immunization of Balb/c mice with either human growth hormone or affinity-purified rabbit anti-(human growth hormone) respectively. Radiolabelled rabbit anti-(human growth hormone) antibodies, but not human growth hormone, were specifically bound by supernatants from the 13 hybrids. The binding was completely inhibited by human-growth-hormone serum binding protein. However, anti-(human growth hormone antibodies) were detected in the sera of all the mice immunized with human growth hormone. In an independent fusion, which was carried out after immunization with fewer doses of human growth hormone, anti-(human growth hormone) antibodies were also obtained. Five hybrids, where the starting antigen was human growth hormone, were selected for ascites production, and the corresponding monoclonal antibodies were partially purified and characterized with respect to their immunoglobulin isotype and their interaction with human-growth-hormone receptors. These antibodies were found to enhance the binding of radioiodinated human growth hormone to human-growth-hormone serum binding protein from human and rabbit plasma by 40%. Scatchard analysis of the effect of one of the monoclonal antibodies showed that this enhancement was due to an increased number of binding sites. All of the partially purified antibodies but one (F12) inhibited the binding of human growth hormone to rat but not rabbit, liver microsomes to various extents, as well as to H-4-II-E rat hepatoma cells. Monoclonal antibody F12 enhanced the binding of radiolabelled human growth hormone to rat liver microsomes and H-4-II-E hepatoma cells. This enhancement was found to be due to an increase in the number of binding sites.     

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.     

Immunohistochemical evidences showing the presence of thymulin containing cells located in involuted thymus and in peripheral lymphoid organs

Thymulin is a well-characterized thymic hormone that exists as a nonapeptide coupled to equimolar amounts of Zn2+. Thymulin is known to have multiple biological roles, including T cell differentiation, immune regulation, and analgesic functions. It has been shown that thymulin is produced by the reticulo-epithelial cells of the thymus, and it circulates in the blood from the moment of birth, maintain its serum level until puberty diminishing thereafter in life. To study the localization of this hormone, we prepared polyclonal and monoclonal antibodies against the commercial peptide and utilized immunocytochemical techniques for visualization. The results indicate that thymulin stains the thymic reticular cells, the outer layers of Hassall's corpuscles and a large round cellular type, which is keratin-negative and does not show affinity for the common leukocyte antigen (CD-45). In mice, this thymulin-positive cell remains in the thymus throughout life and even appears in relatively increased numbers in old involuted thymi. It also appears in thymus-dependent areas of the spleen and lymph nodes, demonstrating that at least one of the thymus cells containing this peptide can be found in peripheral lymphoid tissue.     

Characterization of monoclonal antibodies specific for the Lewis a human blood group determinant

Four hybridoma cell lines were derived from the spleen cells of mice immunized with the neutral glycolipids of human meconium. The antibodies secreted by these lines were specific for the Lewis a antigen of the human Lewis blood group system as determined by solid phase immunoassay using synthetic carbohydrate antigens and by plate binding assay and thin layer chromatography-autoradiography using natural glycolipid antigens. Coating protein A-bearing Staphylococcus aureus with one of the antibodies yielded a stable reagent that produced rapid agglutination of Lewis a positive human erythrocytes. The fine structural specificity of these antibodies was assessed by competition radioimmunoassay using synthetic structural analogs of Lewis a conjugated to bovine serum albumin. One antibody was specific for the Lewis a trisaccharide (Gal beta 1 leads to 3(Fuc alpha 1 leads to 4) beta GlcNAc), while a second recognized the entire Lea (1 leads to 3) beta Gal tetrasaccharide. The third and fourth were directed at topography largely provided by only the alpha Fuc and beta GlcNAc units. These monoclonal antibodies not only represent potentially useful reagents for detecting the Lewis a antigen but also provide a system for studying precise relationships between anticarbohydrate antibody structure and binding specificity.     

Thymic hormone-containing cells. VII. Adrenals and gonads control the in vivo secretion of thymulin and its plasmatic inhibitor

The influence of adrenals and gonads on the intrathymic production and the circulating level of thymulin was evaluated in young adult mice. Adrenalectomy (Adx) and gonadectomy (Cx) induce a temporary decrease of thymulin serum level. One simultaneously notes, as a compensatory phenomenon, an increase in the thymic content of the hormone-producing cells. The decrease of serum thymulin levels after Adx and Cx is at least partially due to the appearance of low m.w. thymulin-inhibitory molecules. The fact that thymectomy prevents the appearance of these inhibitors suggests that the effects of Adx and Cx could be explained by a negative control by sex hormones of the synthesis or activity of thymulin inhibitors produced or controlled by the thymus. Specific hormone replacement therapy of castrated/adrenalectomized animals normalized thymulin serum level and thymic content. Such correction was also spontaneously observed after 4 mo, suggesting that other mechanisms (e.g., an influence of the hypothalamus-hypophysis axis) might be involved in the endocrine control of thymic hormone secretion.     

Neonatal thymulin gene therapy in nude mice: Effects on the morphology of the pituitary corticotrope population

The integrity of the thymus during early life is necessary for a proper maturation of the neuroendocrine system, including the adrenal axis. The thymic metallopeptide thymulin seems to be a central physiologic mediator of thymus-pituitary communication. Furthermore, neonatal thymulin gene therapy has been shown to prevent the typical alterations of gonadotrophic cell number and morphology and serum gonadotropin levels in nude female mice. In the present study we assessed the impact of athymia and the effect of neonatal thymulin gene therapy on the corticotropic cell population in nude mice. The effect of thymulin administration to adult nudes on their hypothalamic content of corticotropin-releasing hormone (CRH) and the adrenal content of corticosterone was also determined. We used an adenoviral vector expressing a synthetic gene for the thymic peptide thymulin (metFTS) termed RAd-FTS. On postnatal day 1 or 2, heterozygous (nu/+) and homozygous (nu/nu) pups of both sexes received a single bilateral i.m. injection of RAd-FTS or RAd-GFP, a control vector. On postnatal day 71, mice were bled and sacrificed, and their pituitaries were immediately dissected, fixed and immunostained for corticotropin. Morphometry was performed by means of an image-analysis system. The following parameters were calculated: volume density (VD: Σ cell area/reference area), cell density (CD: number of cells/reference area), and cell surface (CS: expressed in μm2). Serum thymulin levels were measured by a bioassay, and CRH as well as corticosterone were determined by IRMA and RIA, respectively. Neonatal thymulin gene therapy in the athymic mice restored their serum thymulin levels and increased corticotrope CD, VD and CS in both control and athymic mice. Athymic mice showed only a marginal reduction in corticotrope CD, VD and CS. In these mutants hypothalamic CRH content was slightly increased, whereas adrenal corticosterone tended to be lower. Thymulin administration to adult mice tended to reverse these changes. Our results suggest a possible modulating effect of thymulin on the corticotrope population and the adrenal gland, confirming the existence of a bidirectional thymus-pituitary-adrenal axis.     

Monoclonal antibodies reactive with subsets of mouse and human thymic epithelial cells

We describe monoclonal antibodies (MAB) reactive with subsets of mouse and human thymic epithelial cells. Rat MAb CDR1 reacts with mouse but not human cortical epithelial cells. Immunologic staining of thymic nurse cells in suspension indicates the CDR1 antigen is located on the cell surface. Mouse MAb CDR2 reacts with human but not mouse cortical thymic epithelial cells. Rat MAb MD1 and MD2 detect different determinants expressed by most medullary epithelial cells in mouse thymus but fewer such cells in human thymus. In addition, MD1 detects flattened subcapsular cells rarely in mouse thymus but frequently in human thymus. Two-color stains using an anti-keratin antiserum demonstrate the epithelial nature of the cells reactive with these antibodies. The antigens detected by CDR1 and MD1 first appear during the neonatal period, achieving adult distribution by postnatal days 14 and 4, respectively. The extra-thymic staining of these MAb is described. On the basis of their intra- and extra-thymic reactivities, these MAb differ from those previously reported and may permit dissection of the thymic microenvironment.     

Thymulin and the neuroendocrine system

Thymulin is a thymic hormone exclusively produced by the thymic epithelial cells. It consists of a nonapeptide component coupled to the ion zinc, which confers biological activity to this molecule. After its discovery in the early 1970, thymulin was characterized as a thymic hormone involved in several aspects of intra- and extrathymic T-cell differentiation. Subsequently, it was demonstrated that thymulin production and secretion is strongly influenced by the neuroendocrine system. Conversely, an emerging core of information points to thymulin as a hypophysotropic peptide. Here we review the evidence supporting the hypothesis that thymulin is an important player in the hypophyso-thymic axis.     

Monoclonal antibodies distinguish synthetic peptides that differ in one chemical group

By using human calcitonin (hCT), human calcitonin-gene-related peptide (hCGRP), and a synthetic peptide with a sequence analogous to the 34 C-terminal amino acids of human preprocalcitonin (designated as PQN-34) as haptens in the generation of monoclonal antibodies, we assessed the role of amido and amino groups in paratope-epitope binding. By using peptide inhibition experiments and solid-phase immunoassays, monoclonal anti-hCT antibody CT07 and monoclonal anti-hCGRP antibody CGR01 were found to bind to an antigenic determinant located in the C-terminal segment of the hormones. These epitopes comprise the seven C-terminal amino acids of the hormones, and the presence of the hormone-ending carboxamide group was found to be essential for antibody binding. The corresponding heptapeptides, either bearing a carboxyl group or else linked to a glycine residue at their C-terminal part, failed to react with the antibodies. Moreover, these monoclonal antibodies did not bind to synthetic peptides analogous to the C-terminal region of the hormone precursor molecules that comprised the epitope site flanked by a peptide sequence. In an attempt to assess whether amido groups when present on the side-chain of amino acids may also modulate antibody binding, a monoclonal antibody referred to as QPO1 was produced and was found to recognize an antigenic determinant localized in the N-terminal region of the PQN-34 peptide bearing a glutamine residue as the N-terminal amino acid. The epitope was found to correspond to a topographic assembled site, and binding of QPO1 was found to be substantially dependent on the presence of the free amino and the side-chain amido groups borne by the N-terminal glutamine residue of this peptide PQN-34. In contrast to these findings, an antigenic determinant located in the internal sequence of calcitonin and recognized by monoclonal anti-hCT antibody CT08 was found to be expressed on the mature form of the hormone, as well as on synthetic peptides with sequence mimicking that of preprocalcitonin. These data should guide the choice of synthetic peptide haptens for the production of anti-protein antibodies.