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.     

Production of anti-thymulin (FTS) monoclonal antibodies by immunization against human thymic epithelial cells

A monoclonal antibody specific for thymulin (FTS), a thymic hormone initially isolated from serum, was obtained by cell fusion using spleen cells from BALB/c mice immunized with cultured human thymic epithelial cells. Hybridomas were selected according to their capacity to produce antibodies binding specifically to thymic epithelial cells in culture (as assessed by indirect immunofluorescence) and their ability to absorb in vitro the biological activity of synthetic and natural hormone preparations and to induce in vivo the disappearance of endogenous circulating thymulin. In this way monoclonal antibodies were obtained that recognized a subpopulation of nonlymphoid cells on frozen sections of mouse and human thymuses. The epithelial nature of these cells was assessed using an antikeratin antiserum. The binding of the antibodies to thymic cells was completely abolished by its absorption with the synthetic hormone or normal (but not of thymectomized) mouse serum. The thymic specificity of the antibody was further confirmed by the complete absence of binding to sections of all the various lymphoid and epithelial organs examined (from both humans and mice). Double labeling experiments using the monoclonal antibody described above and a monoclonal antibody prepared by immunization with the synthetic peptide showed that the two antibodies bound to the same cell. These results provide further evidence for the exclusive presence of the thymic hormone thymulin in thymic epithelial cells.     

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.     

Increase of circulating levels of thymulin in hyperprolactinemia and acromegaly

The production of thymulin by the thymic epithelium is under complex control involving the endocrine system. Experimental models have suggested that prolactin (PRL) and growth hormone (GH) participate in this regulation but this has not been documented in humans. Using a bioassay we measured circulating thymulin levels in patients with hyperprolactinemia (n = 21), acromegaly (n = 15), or both (n = 6). Thymulin was elevated in these three groups of patients compared with normal subjects or with patients with pituitary disease but no excess in PRL or GH. Contrasting with observations in control groups, thymulin did not decrease as a function of age in patients. No correlation between thymulin and PRL or GH levels was observed while thymulin and insulin-like growth factor 1 levels were correlated. A new radioimmunoassay used in some patients for thymulin determination yielded similar results. Overall these data demonstrate that PRL and GH are involved in the hormonal control of thymulin production by the thymic epithelium in the human.     

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.     

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.     

Influence of growth hormone on thymic endocrine activity in humans

The thymus produces humoral factors that induce the proliferation and differentiation of T cells which are responsible for cell-mediated immunity. Experimental data have suggested that this thymic hormone production is modulated by the neuroendocrine network and, in particular, by growth hormone (GH) and thyroid hormones. To study the role played by GH in thymic endocrine activity in humans, the circulating level of one of the best known thymic peptides, i.e. thymulin (Zn-FTS), has been determined, after a washout period of 2 weeks without GH treatment, in GH-deficient children before and after a single injection of GH. The basal thymulin level is consistently lower in GH-deficient children than in healthy age-matched controls. A single injection of GH induces a significant increment of the thymulin level for at least 48 h. Since thymulin activity may also depend on zinc bioavailability, on thyroid hormone turnover and on the eventual presence of thymulin-inhibitory substances, all these aspects have been checked. No supporting evidence regarding the existence of these kinds of interferences in GH-deficient children has been substantiated. A positive correlation has been found between the serum level of insulin-like growth factor I, but not of GH, and thymulin activity. These data suggest that GH may directly or indirectly modulate the thymic endocrine function in humans. Whether and to what extent such a modulation is relevant to the functioning of the immune system remains to be ascertained.     

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.     

Impaired peripheral zinc metabolism in patients with senile dementia of probable Alzheimer’s type as shown by low plasma concentrations of thymulin

Plasma concentrations of a zinc carrier peptide, namely thymulin, were measured according to a bioassay in young donors, healthy elderly, and patients with senile dementia of Alzheimer’s type (SDAT). Thymulin is a hormone released by thymic epithelial cells and its biological activity on cells of immune system is dependent on the presence of one molecule of zinc bound to the peptide. Plasma from different subjects were fractionated by gel filtration to yield protein-bound thymulin and free thymulin. The biological activity of the peptide was then assessed in the two different fractions. The activity of protein-bound thymulin was higher in young donors than in elderly of SDAT patients, being the lowest in SDAT. Addition of zinc ions to plasma fractions increased the thymulin activity of samples from elderly and SDAT patients to levels observed in young donors. Thymulin activity in free thymulin fractions was lower in the elderly than in the young and was practically undetectable in SDAT patients. The addition of zinc ions normalized the activity of thymulin in these fractions from both the elderly and SDAT patients. These findings confirm the presence of an altered zinc status in the elderly and suggest that an impaired zinc metabolism may be present in SDAT patients.     

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.     

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.     

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.     

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.     

Synthesis and biological activity of eight thymulin analogues

Eight analogues of thymulin, a thymic nonapeptide involved in several aspects of T-cell differentiation, were synthesized by the conventional method in solution. Four were modified in residue 7 (Ala, D-Ala, D-Leu or Sar instead of Gly) and two in residue 8 (D-Ser or Thr instead of Ser); in the others, the Gly6-Gly7 sequence was replaced either by a single glycyl residue or by a triglycyl sequence. The biological activity of the analogues was determined in the rosette assay: five exhibited a prolonged activity in vivo with respect to thymulin. All the analogues inhibited the binding of tritiated thymulin to thymulin receptors on three human lymphoblastoid T-cell lines (HSB2, 1301 and CEM) with the same order of magnitude as non-labelled thymulin.     

Thymulin gene therapy prevents the reduction in circulating gonadotropins induced by thymulin deficiency in mice

Integrity of the thymus during perinatal life is necessary for a proper maturation of the pituitary-gonadal axis in mice and other mammalian species. Thus congenitally athymic (nude) female mice show significantly reduced levels of circulating gonadotropins, a fact that seems to be causally related to a number of reproductive derangements described in these mutants. Interestingly, a number of in vitro studies suggest that the thymic peptide thymulin may be involved in thymus-pituitary communication. To determine the consequences of low serum thymulin in otherwise normal animals, we induced short (8 days)- and long (33 days)-term thymulin deficiency in C57BL/6 mice by neonatally injecting (intraperitoneally) an anti-thymulin serum and assessed their circulating gonadotropin levels at puberty and thereafter. Control mice received an irrelevant antiserum. Gonadotropins were measured by radioimmunoassay and thymulin by bioassay. Both long- and short-term serum thymulin immunoneutralization resulted in a significant reduction in the serum levels of gonadotropins at 33 and 45 days of age. Subsequently, we injected (intramuscularly) an adenoviral vector harboring a synthetic DNA sequence (5'-ATGCAAGCCAAATCTCAAGGTGGATCCAACTAGTAG-3') encoding a biologically active analog of thymulin, methionine-FTS, in newborn nude mice (which are thymulin deficient) and measured circulating gonadotropin levels when the animals reached 52 days of age. It was observed that neonatal thymulin gene therapy in the athymic mice restored their serum thymulin levels and prevented the reduction in circulating gonadotropin levels that typically emerges in these mutants after puberty. Our results indicate that thymulin plays a relevant physiological role in the thymus-pituitary-gonadal axis.     

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     

Immunomodulatory potential of thymulin-Zn(2+) in the alveolar epithelium: amelioration of endotoxin-induced cytokine release and partial amplification of a cytoprotective IL-10-sensitive pathway

The immunomodulatory potential of thymulin in the perinatal epithelium is not well characterized. In an in vitro model of fetal alveolar type II epithelial cells, we investigated the exhibition of an anti-inflammatory activity of this peptide hormone. Thymulin selectively ameliorated, in a dose-dependent manner, the endotoxin-induced release of IL-1 beta (IC(50) = 657 ng. ml(-1)), but showed no inhibitory effect on IL-6 and TNF-alpha. Zinc, an anti-inflammatory antioxidant, which is required for the biological activity of thymulin, reduced the secretion of IL-1 beta (IC(50) = 62 microM), TNF-alpha (IC(50) = 1000 microM), and, to a lesser extent, IL-6. This cation (100 microM) amplified the effect of thymulin on IL-1 beta and TNF-alpha (IC(50) < 0.1 ng. ml(-1)), but not on IL-6. Analysis of whether thymulin is up-regulating a counterpart anti-inflammatory signaling loop revealed the involvement of an IL-10-sensitive pathway. These results indicate that thymulin acts as a novel dual immunoregulator by enhancing an anti-inflammatory cytoprotective response and depressing an inflammatory signal, an effect synergistically amplified, in part, by cationic zinc.     

Thymic hormones and prostaglandins. II. Synergistic effect on mouse spontaneous rosette forming cells

Prostaglandins (PGs) have been assumed to play a role in the biological activity of thymic hormones (TH). Indeed, it has been shown that type E-PGs are able to mimic the action of several TH. Moreover, indomethacin interferes in the rosette assay, which still represents the most commonly used bioassay for the evaluation of TH and, in particular thymulin levels, in biological fluids. Previously, our attempt to modulate PG production by different TH showed that none of the TH tested affect PGE2, 6-keto-PGF1 alpha, PGF2 alpha and TXB2 production by spleen cells from control and thymectomized (Tx) mice, while indomethacin was able to inhibit the spontaneous PG production. Here, we investigated a possible role for each endogenously produced PG in the experimental conditions of the rosette assay, in order to define: 1) whether or not there was a specificity of action of a given PG; and 2) to analyze the pattern of action between thymulin and the endogenously produced PGs. We demonstrated that PGE2 and 6-keto-PGF1 alpha are the PGs which are physiologically involved in the rosette assay, according to their levels of endogenous production, and that they are able to synergize with thymulin. This synergy was demonstrated in two ways: 1) by adding anti-PGE2 and anti-6-keto PGF1 alpha-antibodies, which prevent part of the thymulin effect, or 2) by simultaneous addition of PG and thymulin, at concentrations far lower than those which correspond to their thymulin-like effect. Moreover, PGE2 addition, at concentration close to that found to be endogenously produced, partially reversed the indomethacin-induced effect in the rosette assay. In conclusion, if PGs do not act as mediators of thymulin, they are able to synergize in one of its biological action.     

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.