Oscar A. Brown

The Thymus-Pituitary Axis and Its Changes during Aging

The pituitary-thymic axis constitutes a bidirectional circuit where the ascending feedback loop is effected by thymic factors of epithelial origin. The aim of the present article is to review the evidence demonstrating that aging brings about a progressive disruption in the integration of this network. In doing so, we briefly review the experimental evidence supporting the view that immune and neuroendocrine aging are interdependent processes. The advantages and limits of the nude mouse as a model of thymus-dependent accelerated aging is also discussed. Next, we review a number of studies which show that the endocrine thymus produces several bioactive molecules, generally called thymic hormones, which in addition to possessing immunoregulatory properties are also active on nervous and endocrine circuits. In particular, the reported activities of thymosin fraction 5 (TF5), thymosin α-1 and thymosin β-4 on β-endorphin, ACTH, glucocorticoids, LHRH and LH secretion in different animal and cell models are reviewed. The known hypophysiotropic actions of other thymic hormones like thymulin, homeostatic thymus hormone (HTH) and thymus factor are summarized. Aging has a significant impact on pituitary responsiveness to thymic hormones. Thus, it has been reported that TF5 and HTH have thyrotropin-inhibiting activity in young but not in old rats. Furthermore, intravenous administration of HTH was also able to reduce plasma GH and increase corticosterone levels in both young and old rats, although these responses were much weaker in the old animals. Further evidence on this topic is discussed. It is proposed that in addition to its central role in the regulation of the immune function, the thymus gland may extend its influence to nonimmunologic components of the body, including the neuroendocrine system. The early onset of thymus involution might therefore act as a triggering event which would initiate the gradual decline in homeostatic potential that characterizes the aging process.

Studies on the prolactin-releasing mechanism of histones H2A and H2B

In previous studies we demonstrated that histone preparations possess multiple effects in vivo on pituitary hormone secretion. We have now studied the specificity and signal transduction pathways involved in the prolactin (PRL)-releasing activity of histones H2A and H2B on perifused and incubated rat pituitary cells. In the perifusion experiments, freshly dispersed pituitary cells were packed into short columns and were continuously perifused with serum-free medium. The substances to be tested (stimuli) were pumped through the perifusion circuit, at the end of which perifusate fractions were collected and PRL measured by specific RIA. In the incubation studies, freshly dispersed pituitary cells were incubated in a metabolic incubator with different stimuli at different doses and for varying times. Perifusion of cells with median eminence extract (1/30), histone H2A (30 microM) or histone H2B (30 microM), generated clear PRL release responses. Cells incubated with histone H2A and H2B showed a dose- and time-dependent stimulatory effect on PRL release which, for H2A, was blocked by peptide MB-35, an 86-120 amino acid synthetic fragment of histone H2A. The polycation, poly-lys was unable to mimic the action of histones. To detect the possible signal transduction pathways involved in the response of lactotrophs to histones, cells were incubated with the calcium ionophore A23187, the calcium chelator EGTA, the intracellular phosphoinositide enhancer LiCl, the intracellular cAMP enhancers caffeine, NaF and forskolin, and the protein kinase C inhibitor, trifluoperazine (TFP). Both EGTA (or EGTA plus A23187 ionophore) and TFP were able to reduce significantly the response of lactotrophs to histones. Our results confirm previous evidence that histones may act as hypophysotropic signals. The data also suggest that calcium- and diacylglycerol-associated pathways participate in these effects.

The Thymus-Neuroendocrine Axis : Physiology, Molecular Biology, and Therapeutic Potential of the Thymic Peptide Thymulin

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 the molecule. After its discovery in the early 1970s, thymulin was characterized as a thymic hormone involved in several aspects of intrathymic and extrathymic T cell differentiation. Subsequently, it was demonstrated that thymulin production and secretion is strongly influenced by the neuroendocrine system. Conversely, a growing core of information, to be reviewed here, points to thymulin as a hypophysotropic peptide. In recent years, interest has arisen in the potential use of thymulin as a therapeutic agent. Thymulin was shown to possess anti‐inflammatory and analgesic properties in the brain. Furthermore, an adenoviral vector harboring a synthetic gene for thymulin, stereotaxically injected in the rat brain, achieved a much longer expression than the adenovirally mediated expression in the brain of other genes, thus suggesting that an anti‐inflammatory activity of thymulin prevents the immune system from destroying virus‐transduced brain cells. Other studies suggest that thymulin gene therapy may also be a suitable therapeutic strategy to prevent some of the endocrine and metabolic alterations that typically appear in thymus‐deficient animal models. The present article briefly reviews the literature on the physiology, molecular biology, and therapeutic potential of thymulin.

Thymulin stimulates prolactin and thyrotropin release in an age-related manner

Thymulin is a Zn-bound nonapeptide produced by the thymic epithelial cells (TEC) whose secretion is modulated by prolactin (PRL) and thyroid hormones, among other hormones. We assessed the ability of thymulin to influence the release of PRL and thyroid stimulating hormone (TSH) from dispersed anterior pituitary (AP) cells from young, middle-aged and senescent Sprague-Dawley female rats. Perifused and incubated AP cells were used in different sets of experiments and PRL and TSH release was measured by radioimmunoassay. Perifusion of young and senescent AP cells with thymulin doses ranging from 10(-8) to 10(-5) M gave a logarithmic dose response pattern for both hormones. Supernatants from TEC lines also showed PRL and TSH secretagogue activity. Hormone release was always lower in the senescent cells. AP cells incubated with 10(-8) to 10(-3) M thymulin showed a time- and dose-dependent response for both hormones, the latter being bell-shaped with a maximum at 10(-7) M thymulin. Preincubation of thymulin with an anti-thymulin serum completely quenched the secretagogue activity of the thymic hormone. Coincubation of thymulin with TRH revealed a synergistic release of PRL and TSH in AP cells from all age groups. The calcium chelator EGTA but not the calcium ionophore A23187, blocked the hormone-releasing activity of thymulin in AP cells. The cAMP enhancers, caffeine, NaF and forskolin, significantly increased the thymulin-stimulated release of PRL and TSH, while trifluoperazine, a protein kinase C inhibitor, had no effect. The inositol phosphate enhancer LiCl, potentiated the action of thymulin on PRL and TSH. The present results suggest that the TRH-like activity documented here for thymulin is a receptor-mediated effect which appears to involve calcium, cAMP and inositol phosphates. Senescence but not middle age, appears to impair AP cell responsiveness to thymulin.

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.

Sexual dimorphism in the age changes of the pituitary lactotrophs in rats.

It is known that aging is associated with alterations in hypothalamic and pituitary functions. In the present study, we have undertaken a quantitative immunohistochemical assessment of the lactotroph cell population as well as prolactin (PRL) secretion, in male and female rats of different ages. Pituitaries from young (3 months), old (20 months) and senescent (29 months) male and female Sprague-Dawley rats were processed for the immunohistochemical detection of lactotrophs. Serum PRL was measured by a homologous RIA. Additionally, the in vitro PRL secretory activity was estimated by perifusion of pituitary cells from senescent animals. Analysis of morphometric parameters revealed age-related changes of PRL cell population in animals of both sexes. The cell density (CD), surface density (SD) and volume density (VD) decreased with age in both male and female rats. However, CD as well as SD appeared to have increased in females when compared to males, either in young or old animals, while VD was higher only in old females. The pituitaries of senescent females displayed chromophobic microadenomas on a background of diffuse PRL cell hyperplasia. Prolactin serum levels showed a marked increase with age in females, but only a modest elevation in males. In senescent females, PRL production per cell was reduced. We conclude that in rats, there exists a clear sexual dimorphism in the age-related changes of pituitary PRL cells.

In Vitro Studies on the Thymus-Pituitary Axis in Young and Old Ratsa

In recent years a growing body of evidence suggests that the immune system is functionally linked to the nervous and endocrine systems, thus constituting an integrated homeostatic network.’*2 Within this network, the neuroendocrine system monitors and controls the physical and chemical variables of the internal milieu. Through antigenic recognition, the immune system perceives an internal image of the macromolecular and cellular components of the body and reacts to alterations in this image. It can therefore be said that the immune system participates in the “biologic” homeostasis of the organism. During early life the thymus gland appears to be essential for proper maturation of the neuroendocrine system as suggested by multiple endocrine alterations caused by neonatal thymectomy or congenital absence of the thymus in m i ~ e ~ . ~ (Goya, Sosa, and Dardenne, unpublished results). In fact, it is now well established that the endocrine thymus produces immunoregulatory substances, some of which are also active on nervous and endocrine circuit^.^ Also, in vivo evidence suggests that in addition to reduced activity of the endocrine thymus, old rodents show significant desensitization of the neuroendocrine system to thymic signals. Thus, thymosin fraction five (TFS) and homeostatic thymus hormone (HTH), two partially purified thymic preparations of bovine origin, are reported to possess thyrotropin (TSH)-inhibiting activity in young but not in old rats.” When administered intravenously to young rats, HTH markedly reduced growth hormone secretion, an effect that was much weaker in old animals.8 Intravenous administration of HTH also activated the adrenal axis in young and, to a lesser extent, in old rats9 Considering that thymus removal induces generalized endocrine alterations, the multi-hormone changes elicited by HTH in vivo suggested that this preparation might

Peripheral and mesencephalic transfer of a synthetic gene for the thymic peptide thymulin.

Thymulin is a thymic peptide with antiinflammatory activity in the brain. We constructed a recombinant adenoviral vector, RAd-FTS, expressing a synthetic DNA sequence encoding met-FTS, a biologically active analog of thymulin and used it for peripheral and central gene transfer in rats. Thymulin concentration in serum and brain tissue was determined by bioassay. Reporter gene expression in the substantia nigra (SN) was quantitated by enzymohistochemistry or fluorescence microscopy using an appropriate image analysis software. A single intramuscular injection (10(8) plaque forming units (pfu)/animal) of RAd-FTS in thymectomized rats (nondetectable serum thymulin) induced supraphysiologic serum thymulin levels for at least 110 days (123+/-22 fg/ml versus 598+/-144 fg/ml in intact and vector-injected rats, respectively). Stereotaxic intranigral injection of RAd-FTS induced steady expression levels of met-FTS for at least 90 days, whereas expression of adenovirally transferred reporter genes coding for green fluorescent protein fused to HSV thymidine kinase (GFP-TK)(fus) or E.coli beta-galactosidase (beta-gal), declined drastically within a month (% transgene expression in the SN on post-injection day 30 relative to day 2 was: 18, <1 and 125%, for beta-gal, (GFP-TK)(fus) and met-FTS, respectively). We conclude that RAd-FTS constitutes a suitable biotechnological tool for the assessment of peripheral and central thymulin gene therapy in animal models of nigral dopaminergic neurodegeneration induced by pro-inflammatory agents.

Thymus and Aging: Potential of Gene Therapy for Restoration of Endocrine Thymic Function in Thymus-Deficient Animal Models

Objective and Background: The aim of the present article is to discuss the potential of gene therapy for thymic hormones as a novel therapeutic strategy to treat dyshomeostatic states associated with athymia, as Di George syndrome, or hypothymic conditions like those associated with AIDS, chronic stress or aging. First we review the advantages of the athymic (nude) mouse as an animal model to implement experimental thymic hormone gene therapy strategies to restore endocrine thymic function. The aging rat, known to be markedly hypothymic, is also considered as an alternative model. Methods and Expected Results: The possibility of constructing adenoviral vectors harboring a synthetic gene for the thymic hormone thymulin is discussed. The adenoviral vector so constructed would then be injected intramuscularly in nude mice or senile rats. Transduced myocytes should then begin to act as an ectopic source of thymulin thus restoring circulating thymulin levels to normal youthful levels. Conclusion: We conclude that the implementation of thymulin gene therapy should provide novel biotechnological tools that will boost basic studies on the molecular biology of thymulin and would also allow an assessment of the potential of gene therapy to restore circulating thymulin levels in thymodeficient animal models.

HISTONES AS EXTRACELLULAR MESSENGERS: EFFECTS ON GROWTH HORMONE SECRETION

Histones display hormone‐like properties when present in extracellular fluids. The authors report that histones H2A and H2B possess growth hormone (GH)‐releasing activityin vitroand describe the specificity and signal transduction pathways involved in these effects. Perfused and incubated rat pituitary cells were used in different sets of experiments and GH release was measured by radio‐immunoassay (RIA). Perfusion of cells with 30μmhistone H2A or H2B, generated significant GH secretory responses. Cells incubated with histone H2A showed a dose‐ and time‐dependent stimulatory effect on GH release which was blocked by peptide MB35, a synthetic fragment of histone H2A. Incubation of pituitary cells with the GH secretagogue GHRP‐6, and histones revealed an additive release of GH, whereas GHRH and histones revealed a synergistic effect. The basic peptide poly‐Lys did not mimetize the action of histones. Both EGTA and the protein kinase C inhibitor trifluoperazine, but not the calcium ionophre A23187, were able to reduce significantly the GH response of somatotrophs to histones. Pituitary cell incubation with 30μmforskolin alone or in the presence of H2A or H2B, stimulated GH release in the same magnitude. The results confirm previous evidence that histones may act as hypophysotropic signals and suggest, although do not prove, that this activity is receptor dependent. Calcium‐ and diacylglycerol‐associated pathways participate in these effects.