Ovarian hormone withdrawal in prepubertal developmental stage does not prevent thymic involution in rats.

The study was undertaken to assess the effects of ovarian hormone withdrawal in prepubertal age on thymopoiesis in 2- (young) and 11-month-old (middle-aged) rats. In ovariectomized (Ox) rats, irrespective of age, thymic weight and cellularity were greater than in age-matched controls, but the values of both parameters exhibited the age-related decline. In addition, although thymopoietic efficiency was increased in both groups of Ox rats when compared with age-matched controls, thymopoiesis exhibited the age-related decline mirrored in the lower numbers of both CD4+ and CD8+ recent thymic emigrants in peripheral blood. This reflected the prethymic changes affecting bone marrow progenitor generation/entry and the thymic alterations encompassing the impaired progenitor progression through early pre-T-cell receptor developmental stages (defined by CD45RC/CD2 expression) and, possibly, a more pronounced decrease in the proliferation of the most mature thymocytes. Apart from the changes at thymocyte level, in Ox rats the age-related alterations in thymic stroma (substantiated in a prominent loss of thymic epithelial cells) were registered. Ovariectomy-induced changes in thymic lymphoid and epithelial component, most probably, influenced each other leading to the increase in thymic expression of interleukin-6 and interleukin-7 mRNAs along with time after ovariectomy. Collectively, the study showed that the withdrawal of ovarian hormones in prepubertal age increases the efficiency of thymopoiesis in young adult rats, but does not prevent decline in thymopoiesis occurring with age.

Role of ovarian hormones in T-cell homeostasis: from the thymus to the periphery.

The study explored the putative role of ovarian hormones in the peripubertal remodelling of peripheral T-cell compartment. Ovariectomy at age of 1 month enhanced the peripubertal rise in CD4+ and CD8+ cell numbers in peripheral blood (PB) and spleen from 2-month-old rats. This reflected maintenance of thymopoietic efficiency at the prepubertal level (judging by numbers of the most mature CD4+ and CD8+ thymocytes and recent thymic emigrants) and alterations in T-cell survival/proliferation in the periphery. Compared with age-matched controls, the frequency of apoptotic cells among CD8+ peripheral blood lymphocytes (PBLs) and CD4+ and CD8+ splenocytes was diminished in ovariectomized (Ox) rats, at least partly, due to lower CD95 surface density. The diminished frequency of the apoptotic T splenocytes could also be associated with the rise in the amount of splenic IL-7 mRNA. Additionally, the latter finding was consistent with the augmented proliferation of CD4+ and CD8+ splenocytes. However, the enhanced proliferation of these cells could also be linked to the rise in IL-2 receptor surface density. This increase was related to the enhanced splenic TNF-α mRNA expression. Additionally, ovariectomy led to the phenotypic alterations in the major PBL and splenic T-cell subsets by diminishing/preventing the peripubertal changes in the frequency of cells at distinct stages of post-thymic differentiation/maturation (recent thymic emigrants, mature naïve and memory cells), and by decreasing the frequency of NKT cells within peripheral CD8+ subsets. In addition to numerical and phenotypic changes in T-cell compartment (due to the lack of ovarian hormone action at both the thymic and peripheral level), Ox rats exhibited a much larger delayed-type hypersensitivity (DTH) response compared with age-matched controls. This suggested the augmented T-cell-mediated immune response in Ox rats compared with aged-matched controls.

Glucocorticoid-catecholamine interplay within the composite thymopoietic regulatory network.

This paper highlights the multiple putative thymic and extrathymic points of intersection and interaction between glucocorticoids (GCs) and catecholamines (CAs)--the end-point mediators of the major routes of communication between the brain and the immune system--in the context of intricate thymic T cell-developmental tuning. More specifically, we discuss in detail findings indicating that adrenal GCs can influence thymopoiesis by adjusting directly and/or indirectly (through modulation of pituitary and local ACTH synthesis) not only thymic GC synthesis, in a cell type-specific manner, but also thymic CA bioavailability (via altering CA outflow from sympathetic nerve endings and local CA synthesis), ß and α(1) -adrenoceptor (AR) expression, and/or AR-mediated intracellular signal transduction in thymic cells. In addition, this short review points to GC- and CA-sensitive stages along the multistep T cell-developmental journey and the possible effects of altered GC, and consequently CA signaling, on thymopoietic efficiency.

Role of gonadal hormones in programming developmental changes in thymopoietic efficiency and sexual diergism in thymopoiesis.

There is a growing body of evidence indicating the important role of the neonatal steroid milieu in programming sexually diergic changes in thymopoietic efficiency, which in rodents occur around puberty and lead to a substantial phenotypic and functional remodeling of the peripheral T-cell compartment. This in turn leads to an alteration in the susceptibility to infection and various immunologically mediated pathologies. Our laboratory has explored interdependence in the programming and development of the hypothalamo-pituitary-gonadal axis and thymus using experimental model of neonatal androgenization. We have outlined critical points in the complex process of T-cell development depending on neonatal androgen imprinting and the peripheral outcome of these changes and have pointed to underlying mechanisms. Our research has particularly contributed to an understanding of the putative role of changes in catecholamine-mediated communications in the thymopoietic alterations in adult neonatally androgenized rats.

Age-associated remodeling of neural and nonneural thymic catecholaminergic network affects thymopoietic productivity.

Ageing is associated with a progressive decline in thymic cytoarchitecture followed by a less efficient T cell development and decreased emigration of naïve T cells to the periphery. These thymic changes are linked to increased morbidity and mortality from infectious, malignant and autoimmune diseases in old age. Therefore, it is of paramount importance to understand the thymic homeostatic processes across the life span, as well as to identify factors and elucidate mechanisms driving or contributing to the thymic involution. Catecholamines (CAs) derived from sympathetic nerves and produced locally by thymic cells represent an important component of the thymic microenvironment. In young rats, they provide a subtle tonic suppressive influence on T cell development acting via ß(2)- and α(1)-adrenoceptors (ARs) expressed on thymic nonlymphoid cells and thymocytes. In the face of thymic involution, a progressive increase in the thymic noradrenaline level, reflecting a rise in the density of noradrenergic nerve fibers and CA-synthesizing cells, occurs. In addition, the density of ß(2)- and α(1)-AR-expressing thymic nonlymphoid cells and the α(1)-AR thymocyte surface density also exhibit a pronounced increase with age. The data obtained from studies investigating effects of AR blockade on T cell development indicated that age-related changes in CA-mediated thymic communications, certainly those involving α(1)-ARs, may contribute to diminished thymopoietic efficiency in the elderly. Having in mind thymic plasticity in the course of ageing, and broadening possibilities for pharmacological modulation of CA signaling, we here present and discuss the progress in research related to a role of CAs in thymic homeostasis and age-related decay in the thymic naïve T cell output.

Neonatal androgenization affects the efficiency of ß-adrenoceptor-mediated modulation of thymopoiesis.

We tested the hypothesis that neonatal androgenization affects the efficacy of ß-adrenoceptor (ß-AR)-mediated fine tuning of thymopoiesis in adult female rats by modulating the thymic noradrenaline (NA) level and/or ß-AR expression. In adult rats administered with 1000 µg testosterone enanthate at postnatal day 2 a higher density of catecholamine (CA)-synthesizing thymic cells, including thymocytes, and a rise in their CA content was found. In addition, in these animals increased thymic noradrenergic nerve fiber fluorescence intensity, reflecting their increased CA content, was detected. These changes were followed by an increase in thymic NA concentration. The rise in thymic NA content in thymic nerve fibers and cells was associated with changes in the expression of mRNA for enzymes controlling pivotal steps in NA biosynthesis (tyrosine hydroxylase, dopamine-ß-hydroxylase) and inactivation (monoamine oxidase). In contrast, the thymic level of ß(2)-AR mRNA on a per cell basis and the receptor surface density on thymocytes was reduced in testosterone-treated (TT) rats. As a consequence, 14-day-long treatment with propranolol, a ß-AR blocker, was ineffective in modulating T-cell differentiation/maturation in TT rats. In conclusion, the study indicates the importance of the neonatal sex steroid milieu for shaping the immunomodulatory capacity of the thymic NA/ß-AR signaling system in adult rats.

Role of ovarian hormones in age-associated thymic involution revisited.

A commonly held view that ovarian hormones are causally involved in age-associated thymic involution has been recently challenged. In particular, their relevance in the progression of thymic involution has been disputed. To reassess this issue 10-month-old rats with well advanced thymic involutive changes were ovariectomized (Ovx), and after 1 month thymic cellularity, thymocyte development and levels of recent thymic emigrants (RTEs) were examined in peripheral blood and spleen. In addition, the distribution of major conventional and regulatory T-cell subsets was analyzed in the same peripheral lymphocyte compartments. Ovariectomy increased thymic weight and cellularity above the levels in both 10-month-old and age-matched controls indicating that ovarian hormone ablation not only prevented further progression of thymic involution, but also reversed it. The increased thymic cellularity was accompanied by altered thymocyte differentiation/maturation culminating in increased thymic output of naïve T cells as indicated by elevated levels of both CD4+ and CD8+ RTEs in peripheral blood and spleen. The changes in T-cell development produced: (i) a disproportional increase in cellularity across thymocyte subsets, so that relative proportions of cells at all maturational stages preceding the CD4+CD8+ T cell receptor (TCR)alphabeta(low) stage were reduced; the relative numbers of CD4+CD8+ TCRalphabeta(low) cells entering positive selection and their immediate CD4+CD8+ TCRalphabeta(high) descendents were increased, while those of the most mature CD4+CD8- and CD4-CD8+ TCRalphabeta(high) cells remained unaltered; (ii) enhanced cell proliferation across all thymocyte subsets and (iii) reduced apoptosis of cells within the CD4+CD8+ thymocyte subset. The augmented thymic output of naïve T cells in Ovx rats most likely reflected an early disinhibition of thymocyte development followed by increased positive/reduced negative selection, at least partly, due to raised thymocyte surface Thy-1 expression. The greater number of CD4+CD25+Foxp3+ cells in both thymus and peripheral blood suggested augmented thymic production of these cells. In addition, an increased CD4+/CD8+ cell ratio was found in the spleen of Ovx rats. Thus, ovarian hormone ablation led not only to increased diversity of the T-cell repertoire, but also to a new balance among distinct T-cell subsets in the periphery.

Neonatal testosterone imprinting affects thymus development and leads to phenotypic rejuvenation and masculinization of the peripheral blood T-cell compartment in adult female rats.

Exposure of female rodents to testosterone in the critical neonatal period produces defeminization/masculinization of the hypothalamo-pituitary-gonadal (HPG) axis, i.e. neonatal androgenization and postpones axis maturation. To address the hypothesis that HPG axis signaling is involved in the programming of thymic maturation/involution and sexual differentiation we studied the impact of neonatal androgenization on thymic cellularity, development of effector and regulatory T cells, and phenotypic characteristics of peripheral blood T lymphocytes in adult rats. A single injection of testosterone on postnatal day 2 postponed thymic maturation/involution as revealed by organ hypercellularity, increased cellularity of the most mature (CD4+CD8- and CD4-CD8+) TCRalphabeta(high) thymocyte and both recent thymic emigrant (RTE) subsets and caused phenotypic defeminization/masculinization of thymic (decreased CD4+CD8-TCRalphabeta(high)/CD4-CD8+TCRalphabeta(high) cell ratio) and peripheral blood T-cell compartments (decreased CD4+RTE/CD8+RTE and CD4+/CD8+ cell ratio). In addition, neonatal androgenization increased the relative and absolute numbers of both CD4+CD25+Foxp3+ and natural killer (NK) regulatory T cells in peripheral blood. These findings, in conjunction with thymocyte overexpression of Thy-1 that is assumed to reduce negative selection affecting self-reactive cell generation, suggest a new relationship between self-reactive and regulatory T cells. In conclusion, our study provides additional evidence for a role of HPG signals (i.e. sex steroids and gonadotropins) in programming the kinetics of thymic maturation/involution and in establishing immunological sexual dimorphism.

Age-associated remodeling of thymopoiesis: role for gonadal hormones and catecholamines.

The present review summarizes recent data on age-related thymic changes termed thymic involution, and highlights the putative role of perturbances in extrathymical and, possibly, intrathymical production of gonadal steroids and catecholamines in this process. Thymic involution has been envisaged as an extremely complex process involving multifactorial mechanisms along the bone marrow-thymic axis that accounts for the major manifestations of immunosenescence. These mechanisms include basic cell aging processes (for example, cell replication and programmed cell death) and processes unique to the immune system (such as generation of the T cell receptor repertoire and control of potentially autoreactive cells). Given that the onset of age-associated thymic involution coincides with the rise in gonadal steroid levels at puberty, a causal link between these events has been suggested. It has been shown that: (1) peripubertal gonadectomy causes substantial decrease in the level of noradrenaline in adult male and female thymus and (2) catecholamines, acting via alpha- and beta-adrenoceptor, produce suppressive effects on the thymic cellularity and production of both effector and regulatory T cells. Furthermore, the possibility that gonadal steroids contribute to thymic involution is discussed in this paper. In light of recent data indicating that effects of gonadal hormone deprivation on the thymic cellularity and function are long lasting but transitory, a putative role for the intrathymic sex steroid/catecholamine production in assuring the organ involution, under conditions of their limited supply by extrathymic sources, is also considered.

Catecholamines as immunomodulators: a role for adrenoceptor-mediated mechanisms in fine tuning of T-cell development.

In its simplest form, effective T cell-mediated immunity emanates from the expansion of specific T cells activated in response to antigen. In establishing and maintaining the peripheral T-cell pool, the thymus plays a critical role. It does so by providing a microenvironment within which T-cell precursors proliferate, differentiate and undergo selection processes to create a fully functional population of major histocompatibility complex restricted, self-tolerant T cells. The control of the thymic function involves intrathymic, as well as sympathetic nervous and endocrine system signalling. In addition to postganglionic noradrenergic fibres, both thymic lymphoid and non-lymphoid cells, including epithelial cells and macrophages, have been demonstrated to express tyrosine hydroxylase (TH), and suggested to form a local non-neural catecholaminergic cell network. A higher level of noradrenaline has been found in male than in female rat thymi, and a role of gonadal hormones in providing this dimorphism has been demonstrated. In addition, thymic lymphoid and non-lymphoid cells, including those expressing TH, have been found to bear beta- and alpha1-adrenoceptors (ARs) and a role of gonadal hormones in regulation of, at least, beta-AR density and signalling has been suggested. These findings have also entailed conclusion that catecholamines (CAs) influence T-cell development, not only via neurocrine/endocrine, but also via autocrine/paracrine action. Generally, CAs have been shown to exert an inhibitory influence on thymopoiesis. Role of alpha1- and beta-AR-mediated mechanisms in maintaining thymic homeostasis and in fine tuning of both conventional and regulatory T-cell development is discussed in the manuscript.

Sexual dimorphism in the catecholamine-containing thymus microenvironment: a role for gonadal hormones.

The study was undertaken to explore whether there were: i) apart from neural and circulatory, some other sources of catecholamines (CAs) in rat thymus and ii) gender-specific differences in thymic CA levels, and if so to elucidate the role of sex steroids in this phenomenon. Tyrosine hydroxylase (TH) immunoreactivity was found in thymocytes and thymic epithelial cells (some of which showed morphological features of nurse cells). The density of CA-synthesizing cells was greater in male than in female rats. Noradrenaline (NA), but not dopamine (DA), was detected in thymocytes. NA and DA levels in thymi, and the NA level in thymocytes, were higher in male rats. To explore the putative role of sex steroids in this dichotomy in the thymi of adult rats gonadectomized (Gx) or sham-Gx at the age of 30 days the density of TH+ cells and CA levels were measured. Gonadectomy abolished sexual dimorphism in the density of thymic TH+ cells (diminishing their density in male rats) and thymic CA levels (the NA levels were reduced in rats of both sexes and also the DA level in male rats). Therefore, it can be assumed that testicular and ovarian hormones control thymic NA and DA levels via different mechanisms. Moreover, in Gx rats, despite the decrease in the overall thymic NA level, an increase in the thymocyte NA level was found indicating that gonadal hormones exert differential effects on the NA level in distinct thymic cellular compartments.

Early postnatal castration affects thymic and thymocyte noradrenaline levels and beta-adrenoceptor-mediated influence on the thymopoiesis in adult rats.

The interactions among the nervous, endocrine and immune system were studied by examining: i) thymic and thymocyte catecholamine levels in adult rats castrated (Cx) at postnatal day 3 and ii) effects of 14-day-long propranolol (P) treatment on main thymocyte differentiational molecule expression in adult non-Cx and Cx rat. The results demonstrated that castration in early postnatal period lowers levels of both neurally- and thymocyte-derived noradrenaline in adult rats, and thereby diminishes beta-adrenoceptor-mediated fine tuning of the T-cell differentiation/maturation. In non-Cx rats P affected TCRalphabeta-dependent stages of thymocyte differentiation/maturation decreasing frequency of CD4+8+ double positive (DP) TCRalphabeta(low) cells entering selection processes and increasing relative number of positively selected DP TCRalphabeta(high) (most likely due to an increased thymocyte surface density of Thy-1 that is involved in negative control of TCRalphabeta-mediated signaling/selection thresholds) and the most mature CD4+8- TCRalphabeta(high) cells (including CD4+25+ regulatory cells). However, in Cx rats P failed to produce any significant changes in thymocyte subset composition.

Age-associated changes in CD90 expression on thymocytes and in TCR-dependent stages of thymocyte maturation in male rats.

To elucidate the effects of ageing on T-cell-maturation, in 3- and 18-month-old rats, we analysed the expression of: (i) CD4/CD8/TCRalphabeta and (ii) Thy-1, which is supposed to be a regulator of TCRalphabeta signalling, and thereby the thymocyte selection thresholds. Since an essential role for TCRalphabeta signalling in the development of CD4+25+T(reg)-cells was suggested, the frequency of these cells was also quantified. We demonstrated that, as for mice, early thymocyte differentiational steps within the CD4-8- double negative (DN) developmental stage are age-sensitive. Furthermore, we revealed that TCRalphabeta-dependent stages of T-cell development are affected by ageing, most likely due to an impaired expression of Thy-1 on TCRalphabeta(low) thymocytes entering selection processes. The diminished frequency of the post-selection CD4+8+ double positive (DP) cells in aged rats, together with an overrepresentation of mature single positive (SP) cells, most probably suggests more efficient differentiational transition from the DP TCRalphabeta(high) to the SP TCRalphabeta(high) developmental stage, which is followed by an increase in pre-migration proliferation of the mature SP cells. Moreover, the study indicated impaired intrathymic generation of CD4+25+T(reg)-cells in aged rats, thus providing a possible explanation for the increased frequency of autoimmune diseases in ageing.