In vitro and in vivo effects of mercuric chloride on thymic endocrine activity, NK and NKT cell cytotoxicity, cytokine profiles (IL-2, IFN-gamma, IL-6): role of the nitric oxide-L-arginine pathway.

Mercury (Hg2+) affects cell-mediated immunity, including thymulin production. Thymulin, a zinc-dependent thymic hormone synthesized by thymic epithelial cells (TECs), is involved in NK cell cytotoxicity and Th1 cytokine production (IL-2 and IFN-gamma), which in turn affect both NKT and classic NK spleen cell cytotoxicity. High doses of Hg2+ induce an inflammatory status, increased production of IL-6 and consequent Th1/Th2 imbalance as well as cell-mediated immune depression. The mechanisms by which Hg+ affects the cell-mediated immune response are still unclear. The nitric oxide (NO) pathway may be implicated. The aim of this work was to further explore its noxious role in innate and adaptive immunity and to study the possible role played by the NO pathway. Young Balb/c mice treated in vivo for 1 month with 1.0 mg HgCl2/kg b.w. showed low thymulin activity, depressed NO production (as measured by nitrite and nitrate plasma levels), impaired classic NK spleen cell cytotoxicity, decreased Th1 (IL-2 and IFN-gamma) cytokine profiles, and increased IL-6 production. In vitro, 10(-6) M of HgCl2 inhibited active thymulin kinetics, TEC proliferation, NKT cell cytotoxicity and Th1 cytokine production, whereas IL-6 increased. L-arginine restored thymulin activity, TEC proliferation, NKT cytotoxicity, cytokine profiles and nitrite and nitrate plasma levels both in vivo and in vitro. Since L-arginine is the substrate for NO production, it may compensate for the cell-mediated immune defect induced by HgCl2, via the arginine-NO-pathway. L-arginine is also able to reduce glomerular kidney IgG antibodies deposits induced by higher dose of HgCl2 administration.

Reversibility of thymulin production impairment by L-arginine supplementation in mice exposed to inorganic mercury.

Immunotoxicological effects fo mercury on peripheral immune system are known. We had previously in vitro found that mercuric chloride inhibits thymulin production in mouse thymus cultures at concentrations as low as 10(-8) M. In this study, thymus efficiency, assessed as production of active and total thymulin, was evaluated in vivo using young mice that were injected sc every 3 days for 4 weeks with saline containing mercuric chloride at different concentrations (0 -controls-, 0.001 or 1.0 mg HgCl2/kg body weight). The results show that both the doses are able to cause a significant reduction in active and total thymulin production. Since arginine enhances immune efficiency some of the animals also received a diet supplemented with arginine in order to evaluate a possible role of arginine during mercury intoxication. The data show that arginine has a protective effect on thymic endocrine efficiency. Mice, treated with the lowest dose of mercury and receiving and arginine supplemented diet, produced active and total thymulin like mercury untreated mice. Arginine is an aminoacid which may be found in various amounts in different foods, some foods are particularly rich in arginine i.e. peanuts, stock fish. We suggest that the daily arginine intake may account for individual susceptibility to the mercury-induced immunological effects which are found in mercury occupationally exposed workers.

Restoration of the thymus in aging mice by in vivo zinc supplementation.

A multiparametric study of the thymus was performed in normal aging mice (12-15 months old) submitted to a mild oral zinc supplementation during 3-6 months as compared to age-matched control mice. First, this study demonstrated that in rodents, zinc levels are significantly reduced with aging and can be restored to values close to those observed in young animals after 6 months of zinc supplementation. Second, our data showed that oral zinc administration stimulates thymus growth and partially restores the microenvironmental as well as lymphoid compartments of the organ. Regarding thymic endocrine function, a significant increase in thymulin levels and a concomitant decrease in plasma thymulin inhibitors were observed, suggesting that the age-related decline of thymic function might at least partially be due to extrinsic factors, such as zinc deficiency. The total number of thymic lymphocytes was consistently increased, without significant changes in CD4/CD8 defined thymocyte subsets. Finally, structural changes of the thymus epithelium were also detected, including the disappearance of epithelial cysts frequently observed in old animals, reappearance of a normal pattern of the thymic epithelial cell network, and a decrease in the extracellular matrix network. Taken together, these data suggest that aging-related physiological zinc deficiency induces some relevant changes in thymus structure and function which can be partially corrected by a mild oral zinc supplementation.

Modulation of thymic endocrine function by thyroid and steroid hormones.

The thymic epithelium is responsible for the secretion of thymic hormones that intervene in some steps of intra- and extra-thymic T cell differentiation. In the present paper, we studied the in vivo and in vitro influences of thyroid and steroid hormones on the secretion of thymulin, one of the chemically-defined thymic hormones. Triodothyronine injected in young mice or applied into supernatants of cultured thymic epithelial cells resulted in an increase of thymulin synthesis and secretion, respectively evaluated by the analyses of thymulin containing cells and thymulin levels. The influence of steroids was investigated in vivo by steroid depletion (adrenalectomy and/or castration) and in vitro by the addition of various steroids and/or their specific antagonists to cultured thymic epithelial cells. Surgical ablation of adrenals and/or gonads induced a transient depletion of circulating thymulin and, by feedback mechanism, an increase in the numbers of thymulin containing cells. From the in vitro data we could conclude that the various steroid hormones (that exhibited a stimulatory effect) can act directly on the thymic epithelium, probably via specific receptors. The bulk of data above described represents a strong evidence for the physiological involvement of the neuroendocrine network, on the hormonal function of the thymic epithelium.

Thymic dysfunction in histiocytosis-X.

In some patients with histiocytosis-X there is a deficiency of suppressor T cells, which is corrected in vitro by incubation with either crude calf thymic extract or thymostimulin. This finding suggests that a thymus deficiency could be involved in this disease. Low levels of serum thymic factor (FTS) are found in patients with histiocytosis-X. Their plasma contain factors capable of inhibiting biological activity of FTS in vitro. Elucidation of the mechanism by which FTS is inhibited would be helpful in understanding the immunological defect in histiocytosis-X. The presence of evidence of thymic dysfunction and the fact that patients respond to thymic hormone therapy suggest that histiocytosis-X could be due to a primary immunodeficiency syndrome. Thymulin, which stimulates the generation of suppressor T cells, could be of benefit in therapy.

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.

Feedback regulation of the secretion of a thymic hormone (thymulin) by human thymic epithelial cells in culture.

The production of the thymic hormone, thymulin (FTS), was studied in primary cultures of human thymic epithelium by immunofluorescence using monoclonal anti-thymulin antibodies. The number of thymulin-containing cells and the thymulin level in the culture supernatant increased gradually during the culture. Addition of synthetic thymulin to the culture medium reduced significantly the increase of thymulin-containing cells. Conversely, addition of monoclonal anti-thymulin antibody from the beginning of the culture exacerbated the spontaneous increase of thymulin-containing cells and abrogated the effects of thymulin. Combined with similar data previously reported in vivo, these results demonstrate that thymulin is actively produced by cultured thymic epithelial cells and that its synthesis can be down-regulated by the hormone itself.

Thymic hormone deficiency in normal ageing and Down's syndrome: is there a primary failure of the thymus?

Normal individuals aged over 50 and most young Down's syndrome (DS) subjects had markedly reduced concentrations of circulating thymic hormone (facteur thymique sérique, FTS). Plasma from these two groups contained factors capable of inhibiting biological activity of FTS in vitro. Addition of zinc sulphate to plasma samples from DS subjects or the older individuals induced concentrations of FTS comparable to those observed in young healthy people and completely prevented FTS-inhibitory activity. These findings suggest that biologically active circulating thymic hormone is bound to zinc. The decline in thymic hormone activity in older individuals and DS subjects may be the result of changes in the mechanism of zinc-dependent activation of FTS molecules, which are probably associated with marginal zinc deficiency rather than with a primary failure of the thymus. Addition of zinc salt to plasma samples unmasks the presence of inactive FTS molecules.

Thymic dysfunction in the mutant diabetic (db/db) mouse.

Thymic function has been explored in genetically diabetic homozygous C57BL/KsJ (db/db) mice by evaluating their serum thymic factor (FTS) levels with a rosette assay. As previously reported for other autoimmune mice (NZB or MRL/I mice), the age-dependent decline of FTS levels was significantly accelerated in diabetic mice when compared to heterozygous littermates. Furthermore, FTS inhibitory molecules were detected in db/db mouse sera (as early as 10 wk of age) as evaluated by their ability to absorb in vitro the activity of synthetic FTS in the rosette assay, and in vivo for their capacity to induce the disappearance of endogenous FTS when injected into normal mice. These inhibitors were shown to be immunoglobulins. Histologically, the thymus presented an accelerated involution starting with a cortical lymphocytic depletion and an increased number of Hassall's corpuscles. Ultrastructural studies showed alterations in thymic epithelial cells, mainly represented by an increasing number of cytoplasmic vacuoles. By means of indirect immunofluorescence with anti-FTS monoclonal antibodies, it was shown that the number of FTS+ cells was reduced in db/db mouse thymuses: at the age of 22 wk, diabetic mice had 10 times fewer FTS+ cells than heterozygotes of the same age. Taken together, these results indicate important abnormalities in the thymus of diabetic mice. It is possible that the associated lymphocyte dysfunction plays a role in the pathogenesis of the autoimmune disease presented by db/db mice.