Endocrine control of thymic serum factor production in young-adult and old mice

The influence of different endocrinological manipulations on the blood concentration of serum thymic factor (FTS) was studied in young-adult and old mice. Among the experimentally induced endocrinopathies in youth, hypothyroidism and diabetes caused strong reductions of FTS levels, which were restored to normal by the appropriate hormonal substitutive therapy. Removal of adrenals or gonads has no significant effect on FTS level. Old mice, which show undetectable levels of FTS and low levels of thyroxine, can regain the capacity to produce FTS, provided they are treated with thyroxine. The variations of FTS blood levels in the course of endocrinological manipulations were due to a direct or indirect effect exerted on the recipient thymus. Hormonal treatment of thymectomized mice did not induce any FTS-like activity in their sera, nor did hormones interfere in vitro with the bioassay used to test for FTS. These data suggest that the neuroendocrine balance modulates the synthesis and/or the release of FTS from the thymus during the whole life of the organism and that the decline of FTS production with advancing age is largely dependent on age-associated endocrinological imbalances.     

Respective influence of extrinsic and intrinsic factors on the age-related decrease of thymic secretion.

The serum level of a circulating thymic factor (FTS) described in our laboratory diminishes in mice after adult thymectomy and with age. However, thymuses from old mice, when grafted into young adult thymectomized recipients lacking circulating FTS, can still partially restore the circulating FTS level of the recipients, whereas newborn thymuses are less efficient in restoring the serum level of FTS in old recipients than in young adult thymectomized recipients. Taken together, our results suggests that in addition to an intrinsic deficiency of the thymic secretion, "environmental" factors play a role in the disappearance of circulating FTS with age, the more so since we observed in old mouse sera factors inhibiting the in vitro biologic activity of FTS, factors that are absent from young mouse sera.     

Some endocrine aspects of the thymus gland.

In studies of the mouse thymus, lymphocyte mitoses are seen to be most frequent in the thymus cortex. There is evidence from thymic grafts that a hypothetical factor, thymopoietin, may stimulate mitosis of thymic lymphocytes. It is a factor which is postulated to act in conjunction with the PAS-positive mesenchymal reticular cells and epithelial reticular cells of the cortex. The thymus medulla is necessary for the integrity of thymic grafts, and may also elaborate a secretion for maintaining the cellular functions of the gland. Thymectomy has been used as a gauge for judging normal thymic function and results, in the mouse, in lymphopenia, degeneration of spleen and lymph nodes, delayed rejection of skin allografts, reduced ability of spleen cells to mount the graft versus host reaction, and reduced primary immune response to certain antigens. Correction of these deficiencies offers a means of evaluating various thymic extracts and grafts. Lymphocytosis-stimulating hormone (LSH) is known to maintain the peripheral lymphoid organs and cause lymphocytosis in the thymectomized animal. Diffusion chamber studies of thymic grafts also show restored lymphoid tissue by a cell-free factor (CIF). These two factors may be the same and probably represent the basis of the highly purified lymphocyte-stimulating proteins, LSHr and LSHh, which restore the L/P ratio in thymectomized animals and may stimulate lymphopoiesis in spleen and lymph nodes. LSHr, unlike LSHh, increases the total lymphocyte count. LSHr has been found to increase the humoral antibody response in neonatal mice both by the PFC technique and by direct hemolysis of sheep erythrocytes. Homeostatic thymic hormone (HTH) is a thymic extract of small molecular weight and contains nucleic acid. In the thymectomized guinea pig it has been found to maintain normal levels of lymphocytes in the blood, spleen and lymph nodes, to restore antibody titers to typhoid H antigen and to restore the toxic allergic reaction. Thymic humoral factor (THF) is of smaller molecular weight (less than 1,000) and probably is not a protein. It also enhances lymphoid proliferation in neonatally thymectomized mice. There is evidence that THF participates in humoral antibody formation because it stimulates PFC formation from neonatally thymectomized mice after inoculation with sheep erythrocytes. Its effects on cell-mediated immunity are seen from findings that injection of THF restores the ability of thymectomized mice to reject skin allografts. THF enables spleen cells from thymectomized or neonatal animals to mount the graft versus host reaction, and causes maturation of bone marrow cells and spleen or lymph node cells so that they can participate in the graft versus host reaction. It has been reported to stimulate lymphocytes to kill isogeneic tumor cells in vitro. Thymosin is protein extracted from the thymus. It has been found to alleviate leukopenia slightly and provide some improvement in lymphoid histology in thymectomized mice...     

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.     

Thymic modulation of IL-2 and IL-4 synthesis by peripheral T cells

In this paper we provide several lines of evidence to support the hypothesis that the thymus can exert regulatory influences on the functional capabilities of mature recirculating T cells. Our studies demonstrate that while the IL-2-producing potential of T cells that repopulate the secondary lymphoid organs of lethally irradiated and stem cell-reconstituted mice is significantly reduced compared to that of T cells harvested from normal mice, the amount of IL-4 produced by the T cells of these experimental animals is equivalent to, or greater than, the amount produced by T cells from control animals. In addition, we determined that the amount of biologically active IL-2 and IL-4 secreted by T cells harvested from lethally irradiated animals who reconstitute their hematopoietic and immune systems under the influence of nonirradiated thymic epithelial grafts is essentially identical to the amount produced by T cells harvested from nonirradiated control animals. Collectively, these findings suggest that: (1) the alterations observed in the lymphokine-producing potential of T cells harvested from lethally irradiated and stem cell-reconstituted mice is not due to a direct effect of ionizing radiation on the T lymphocytes themselves, and (2) the exposure of the epithelial cells of the thymus to ionizing radiation during marrow-ablative regimens abrogates or modifies a component of thymic function which can influence the lymphokine-secreting potential of recirculating T cells. Further evidence for thymic involvement in the regulation of lymphokine production by peripheral T cells comes from our finding of a post-thymectomy time-dependent reduction in the capacity of T cells from animals to produce IL-2. Coincident with this reduction, T cells harvested from peripheral lymphoid organs of thymectomized animals demonstrated an augmentation in their IL-4-producing capabilities. The finding that treatment of thymectomized animals with the androgen steroid hormone dehydroepiandrosterone reestablished a normal IL-2-producing potential by their T cells makes it unlikely that the reduced capacity to produce IL-2 was secondary to a loss in fresh thymic emigrants.     

The proliferative potential of CFUs from the bone marrow of thymectomized mice]

Proliferative potential of CFUs in bone marrow of young and adult mice (1.5-25 months) and thymus influence on this property were studied. It has been shown on the model of adult thymectomized mice that during "steady state" hematopoiesis, proliferative potential of bone marrow CFUs does not depend on the animals age and on thymic factors.     

Effect of chorionic gonadotropin on the structure and endocrine function of the thymus gland in mice

The thymic secretory function changes in the pubertal female mice under the influence of chorionic gonadotrophin (CG). The latter is activated after administration of hormone in doses, corresponding to its levels in animals in different terms of pregnancy and is depressed with doses, greatly exceeding those levels. The lack of the effect in thymectomized mice testifies to the realization of the modulating CG action at the thymus level. Changes of mass and cellularity of the thymus and spleen reflect dose-dependent alteration of the thymocyte migration rate.     

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.     

In vitro modulation of mouse natural killer (NK) cell activity by the serum thymic factor (FTS)

Previous studies indicated that the serum thymic factor (FTS) could modulate in vivo the level of splenic natural killer (NK) cell activity in mice. The present report shows that such an effect is also observed after a short term in vitro incubation of the effector cells with FTS. The regulatory effects of FTS result in an increase or a decrease of the splenic NK cell cytotoxicity depending upon the age and the mouse strain. Furthermore, FTS is able to enhance the NK cell activity of thymus and bone marrow cells which are known to be weakly reactive in NK cytotoxicity. Depletion experiments demonstrated that the FTS-induced increase of NK cell activity was not mediated by Thy 1+ cells nor macrophages, thus suggesting a direct action of FTS on the effector cells. Comparative studies using other thymic hormones revealed similar patterns of reactivity. These results favor the hypothesis of a close relationship between the thymus and NK cells.     

Influence of thymectomy on the lymphoid regeneration of hematopoietic bone marrow after sublethal irradiation]

In C57BL mice, bone marrow lymphoid regeneration after a sublethal irradiation is modified by a graft of normal marrow cells. This effect is suppressed in thymectomized mice since a lymphoid peak is observed after a 350 R irradiation; its composition is heterogeneous: small lymphocytes, lymphoblasts and peculier cells named "X cells". The same phenomenon is observed in mice where all the thymocytes and thymus derived and peripheral lymphocytes are destroyed. These results exclude that bone marrow lymphoid regeneration after irradiation is due to a migration of lymphoid cells of thymic origin to the marrow. They could be explained by the effect of a humoral thymic factor on marrow lymphopoiesis.     

Thymic transplantation across an MHC class I barrier in swine.

Thymic tissue transplantation has been performed previously in adult mice to induce donor-specific tolerance across allogeneic and xenogeneic barriers. We have now attempted to extend this technique to a large animal preclinical model and describe here our initial studies of allogeneic thymic transplantation in miniature swine. Two miniature swine were thymectomized before thymic tissue transplantation, and two remained euthymic. Donor thymic tissue was harvested from SLA class I-mismatched juvenile pigs and placed into recipient sternocephalicus muscle, kidney capsule, and omentum. A 12-day course of cyclosporin A was started on the day of transplantation. Allogeneic thymic engraftment could only be achieved in euthymic and not in thymectomized miniature swine using this treatment regimen. Both nonthymectomized animals showed good graft development, with evidence of thymopoiesis, as indicated by positive CD1 and host-type SLA class I immunoperoxidase staining of immature graft-infiltrating cells. Both animals also demonstrated donor-specific T cell hyporesponsiveness, as measured by MLR and cell-mediated lympholysis. The thymic grafts continued to develop despite the appearance of high levels of anti-donor specific cytotoxic IgG Abs. Thus, thymic tissue transplanted across an SLA class I barrier can engraft and support host thymopoiesis in euthymic miniature swine. The presence of the host thymus was required for engraftment. These data support the potential of thymic transplantation as part of a regimen to induce donor-specific tolerance to xenogeneic organ grafts.     

Cellular and molecular interactions of thymus with endocrine organs and nervous system.

T-cell ontogenesis has been disclosed to depend on the interactions of thymus with endocrine glands and nervous system as follows: i/ Thymic deprivation not only impaired the immunological development but also brought about the dysgenesis of pituitary anterior lobe. Conversely, hypophysectomy resulted in thymus atrophy with the disturbed immune responses. ii/ Binding of pituitary acidophilic cell hormones to their receptors on thymus epithelial cells (TECs) augmented the release of thymic hormonal peptides (THPs) in vitro. iii/ Elevation of blood glucocorticoid level after stress caused atrophy of thymus cortex through double positive thymocyte apoptosis. Morpho-molecular alterations of cytoplasm preceded nuclear damage in the apoptotic thymocytes. iv/ Administration of thymosin to the streptozotocin-induced diabetic mice repressed mononuclear cell infiltration to the pancreatic islets. v/ Autonomic nerve fibers innervate thymic parenchyma. Binding of acetylcholines (Achs) to Ach receptors on TECs enhanced protein synthetic activity which seemed to connect with THP production. vi/ Thymectomy not only depressed the immune responses but also accelerated the reduction of leaming and memory ability with aging. The operation appears to disturb the brain adrenoceptor functions and to suppress the regulatory roles of hypothalamus to other nervous tissues. vii/ Several kinds of THPs, separated from the culture supernatant of TEC line by high performance liquid chromatography, showed a favorable effect on the thymocytes at different stage of differentiation and maturation. viii/ Thymosin, thymulin and THPs were capable of proliferating and differentiating thymocytes in vitro. However, the administration of each thymic product to the thymus-deprived animals could not restore from their "wasting disease". Since TECs are composed of a heterogeneous population, it would be one of essential ways for isolating "true thymus hormone" (TTH) to use the material which consists of functionally homogeneous subset of TECs. ix/ An additional grafting of pituitary gland to the thymus-grafted nude mice improved the disturbed T-cell ontogeny. Accordingly, the administration of "TTH" and pituitary acidophilic cell hormones might be more hopeful procedure for rescuing the thymus-deprived animals from "wasting disease".     

Radioprotective effects of serum thymic factor in mice.

Serum thymic factor (FTS) reduced mortality of mice after total-body irradiation with 7.56 Gy X rays. The radioprotective effect was achieved by daily repeated subcutaneous injections of 3-100 micrograms FTS, while doses higher than 300 micrograms/day/mouse were neither radioprotective nor toxic. Similarly, degeneration of the spleen was moderated by 3-100 micrograms FTS but not by 500 micrograms FTS in sublethally (3.78 Gy) irradiated mice. Histological examination showed that hematopoiesis was enhanced in the spleen by daily injections of 10 micrograms FTS. Spleen cells from the FTS-treated mice incorporated more [3H]thymidine in culture with or without concanavalin A. The treatment with FTS increased the production of colony-stimulating factor in the spleen as well as in peritoneal macrophage-like cells, and caused a significant increase in the number of granulocyte-macrophage colony-forming cells both in the spleen and in the femoral bone marrow. Furthermore, FTS prevented a decrease in circulating neutrophils in the sublethally irradiated mice. Prominent overshoot recovery of myelopoiesis, which occurred occasionally in sublethally irradiated mice, did not occur in the FTS-treated mice. The decrease in blood erythrocytes was also significantly reduced. These observations imply that this thymic hormone has potential as a radioprotector.     

Thymus hormones do not induce proliferative ability or cytolytic function in PNA+ cortical thymocytes

A variety of thymus hormone preparations, as well as drugs known to perturb cell differentiation, were tested for their ability to induce nonfunctional cortical thymocytes to become functional precursor cells. Murine cortical thymocytes, defined as the high peanut agglutinin (PNA) binding or as the low H-2K, major [86%] thymocyte subpopulation, were isolated by fluorescence-activated cell sorting. Their function was assessed in a high cloning efficiency, growth factor saturated, concanavalin A-stimulated limit-dilution culture system, determining the number of precursors of extended clones (PTL-p), or determining with a lectin-mediated tumor-lysis readout the number of precursors of cytolytic clones (CTL-p). The hormone preparations tested were crude or partially purified culture supernatants from thymus "epithelial" monolayers (TES), soluble extracts of thymic nonlymphoid tissue (STF), semipure thymus humoral factor (THF), and the pure peptides thymopoietin 32-36 (TP5) and "facteur thymique sérique" (FTS). These preparations were either added directly to the limit dilution cultures, or were first preincubated with the cells, which were then subjected to limit-dilution culture. In no case did the hormone preparations cause any increase in the level of PTL-p or CTL-p in the PNA+ or low H-2K thymocyte population, even though a conversion of only a few percent to functional cells could have been detected. Two possible explanations are considered. One is that the main function of these materials is to control post-thymic peripheral T cells, rather than to induce intrathymic differentiation. Another is that the typical cortical thymocyte is beyond the stage at which thymocytes can be induced by hormones, a view that is strengthened by the failure of either 5-azacytidine or the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate to activate these cells. In this latter explanation the true intrathymic target of hormone action may be an earlier, and very minor, thymus subpopulation.     

Hypothalamic control of thymic function.

Removal of the pituitary gland results in atrophy of the thymus. As the former is under the control of hypothalamus, destruction of anterior portion of the hypothalamus (AHTL) would be expected to negatively influence the thymic function. Contrary to our expectation, however, the thymus became hypertrophic and serum level of growth hormone (GH) markedly increased, when the anterior portion of the hypothalamus was destroyed in rats at 1 month of age and older. The results suggested that AHTL removed the cells secreting GHRIH (growth hormone release inhibitory hormone), but not GHRH (growth hormone releasing hormone), leading to increased pituitary secretion of GH. This high serum level of GH appeared to be responsible for the thymic hyperplasia occurring after AHTL. In other words, the development and aging of the thymus appear to be dependent on the serum level of GH which is under the balance of positive (GHRH) and negative (GHRIH) signals from the hypothalamus. In rats and mice, the serum level of GH is very high just after birth, quickly declines in young adults and does not change greatly thereafter. Thus, it is likely that the initial positive signal is high just after birth and decreasing thereafter with a concomitant increase of negative signal, leading to the onset ofthymic atrophy at around puberty, in association with sex steroid release.     

Role of neuroendocrine hormones in murine T cell development. Growth hormone exerts thymopoietic effects in vivo.

Growth hormone (GH) and other neuroendocrine mediators have been implicated previously in T cell development. We therefore examined thymic development in DW/J dwarf mice. DW/J mice lack acidophilic anterior pituitary cells and consequently are totally deficient in the production of GH, as well as other neuroendocrine hormones. DW/J dwarf mice had greatly hypoplastic thymi that continued to decrease in size as the mice aged. Characterization of the different T cell subpopulations within the thymi of dwarf mice indicated a deficiency in CD4+/CD8+ double-positive thymocytes. This deficiency of progenitor cells became more complete as the mice aged culminating in the total disappearance of this subpopulation in some dwarf mice > 3 mo of age. Analysis of the lymph nodes indicated that a population of double-positive (CD4/CD8) T cells appeared in some mice concurrent with the disappearance of double-positive cells in the thymus suggesting that these thymocytes may have migrated to the periphery. However, peripheral T cells from dwarf mice did exhibit Ag-specific responses indicating that these mice have functional T cells. Treatment of the mice with recombinant human GH, which binds both murine growth hormone receptors and murine prolactin receptors, or ovine GH, which binds murine growth hormone receptors but not murine prolactin receptors, resulted in an increase in thymic size and the reappearance of the CD4+/CD8+ double-positive cells within the thymus. Additionally, after GH treatment, the double-positive cells disappeared from the lymph nodes. The thymi of mice treated with GH failed to attain normal size but did develop a normal distribution of T cell progenitors. Thus, GH exerts significant thymopoietic effects in vivo. Neuroendocrine hormones may be important for normal T cell differentiation to occur within the murine thymus.     

Circulating thymic hormone levels in zinc deficiency

The effect of zinc deficiency (Zn^?) on the circulating thymic hormone (FTS) levels in A/J mice was studied. After 3 weeks of feeding the mice a Zn^? diet, FTS levels were markedly reduced and after 17 weeks, FTS was undetectable. By contrast, the zinc-supplemented (Zn⁺) group seemed to maintain FTS levels better than the normal diet group with aging. On the other hand, spleen spontaneous rosette-forming cells (sRFC) were studied for their azathioprine (AZ) sensitivity in A/J mice on different diets. The Zn^? mice had fewer sRFC than did the normally fed or Zn⁺ mice. The role of zinc in controlling levels of FTS and thus thymic function is discussed.     

Sera from both normal and thymectomized animals produce elevations of cyclic AMP in thymocytes.

Cyclic AMP content in mouse thymocytes has been measured after incubation either with sera or serum fractions from normal or thymectomized (Tx) mice and pigs or with a synthetic circulating pig thymic factor. Sera from both Tx and normal pigs and mice induced an increase in cyclic AMP in mouse thymocytes, whereas the synthetic pig thymic factor did not. It is concluded that the increase in cyclic AMP in mouse thymocytes should be used with caution for the evaluation of circulating thymic hormone levels.     

Alterations in thymocyte surface markers after in vivo treatment by serum thymic factor.

Three T cell markers (heterologous sheep rosette, autologous rosette, and theta antigen) have been studied in thymocytes after in vivo injection of a single dose of serum thymic factor (Facteur Thymique Sérique: FTS). Within 24 h after such treatment, sheep erythrocyte-binding cells increased 5 to 8 times, while autologous rosette-forming cells decreased by a factor of 3. Using cytotoxicity assays (trypan blue exclusion test and chromium release) it was observed that thymocytes from FTS-treated mice presented a higher sensitivity to antitheta serum and complement than control mice. These results are compatible with the hypothesis that exposure of immature T cells to thymic hormone may induce T cell maturation, as assessed by decreased number of immature autologous rosette forming cells (RFC) and increased number of mature T cells (sheep RFC and theta-positive cells). Facteur Thymique Sérique appears as an interesting probe to define the various intra-thymic cellular compartments of differentiation.     

Lymphocyte-mediated cytotoxicity: effects of ageing, adult thymectomy and thymic factor.

Adult thymectomy, as well as ageing, depressed splenic lymphocyte-mediated cytotoxicity (LMC) in the mouse. Ageing depressed significantly LMC as early as 19 weeks of age, independently of the number of cells used for immunization. Thymectomy affected LMC only when supoptimal numbers of immunizing allogeneic cells were used. This effect peaked at 6 to 12 weeks after thymectomy. No difference between thymectomized and normal mice was observed when LMC was tested 16 to 20 weeks after thymectomy, at an age when normal control mice themselves already showed a lowered LMC due to ageing. The effect of in vivo treatment with a circulating thymic factor (TF), which was shown to disappear with ageing as well as after adult thymectomy, has been tested in adult thymectomized mice and normal young and ageing mice. TF treatment prevented LMC depression in adult thymectomized mice, whereas it depressed paradoxically splenic LMC in normal young and old mice. The possible mechanisms of the effects of adult thymectomy, ageing, and thymic factor on the different T cell subsets involved in allogeneic killer cell generation are discussed.