Gordana Leposavić

Differential effects of gonadectomy on the thymocyte phenotypic profile in male and female rats

As an organ responsible for generation of T-cell repertoire the thymus occupies a central position in establishment of mature immune response. To assess the potential role of the gonadal steroids in development and maintenance of immunological sexual dimorphism, the effects of gonadectomy pre- and postpuberty on the thymocyte profile of male and female rats were examined. Rats aged 30 days or 75 days were gonadectomized; 30 days later the thymic cellularity was estimated and the expression of the cell surface antigens (CD4 and CD8) and the T-cell receptor (TCR) alpha beta was analyzed by flow cytometry. Regardless of age at surgery, the thymus weight and total thymocyte yield were greater in sham-operated males than females; this sexual dimorphism in thymic cellularity persisted after gonadectomy. Sexual dimorphism in the composition of thymocyte subsets was also evident in sham-operated rats, with males expressing a higher percentage of CD4-8- cells, and remained after gonadectomy of adult rats. In male rats, gonadectomy at day 75 increased the percentage of CD4+8- single-positive and TCR alpha beta + cells. In contrast, in females, ovariectomy decreased the percentages of CD4+8- single-positive, CD4-CD8- double-negative, and TCR alpha beta + cells and increased the percentage of CD4+CD8+ double-positive cells. In the immature rats gonadectomy increased the percentages of CD4+CD8- single-positive and TCR alpha beta + thymocytes and decreased the percentages of double-positive and double-negative cells in males, while in the female it increased the percentage of CD4+8- single-positive thymocytes. Gonadectomy at that age abolished the sexual dimorphism in the expression of accessory molecules (i.e., CD4/CD8), but facilitated gender-specific expression of TCR alpha beta. In conclusion, the results suggest that the gonadal steroids are more important for the development than for the maintenance of the sexual dimorphism in the thymocyte composition.

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.

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 α- and β-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.

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.

In vivo modulation of the distribution of thymocyte subsets by female sex steroid hormones.

This study examined the effects of the principal ovarian steroids, 17 beta-estradiol (E) and progesterone (P), on the thymic structure and on the intrathymic development of T-cells. Adult female rats were ovariectomized (OVX) and treated for 14 days with physiological doses of either E or P; controls received an equivalent volume of vehicle. Ovariectomy produced a marked increase (vs. sham-operated controls) in thymus weight, which was associated with an increase in the volume and cellularity of both the medulla and cortex. Treatment of OVX rats with E reduced the thymic weight to value, which was significantly lower than that of sham-operated controls decreasing the volume of cortex below level in sham-OVX rats, and reversing the effect of ovariectomy on the volume of medulla. P only prevented the increases in thymus weight and cortical volume induced by OVX. However, unlike E, it had no discernable effect on the medullary volume. E treatment reduced the cellularity of the cortex and medulla to values, which were lower than those of sham-OVX rats, while P only reversed the effects of OVX on the cellularity of both the compartments. Ovariectomy also had a profound effect on the thymocyte profile, increasing the proportion of CD4+8+TCR alpha beta- cells and producing a corresponding decrease in the relative proportions of all TCR alpha beta high cell subsets. The decrease in the latter was opposed by treatment with E or P. However, the sensitivity of the less mature cells (except CD4-8-TCR alpha beta-, the percentage of which was reduced by both hormones) to the two hormones differed. E reduced the relative proportion of CD4-8+TCR alpha beta-, CD4-8+TCR alpha beta low and CD4+8+TCR alpha beta- cells, while P increased the percentage of CD4-8+TCR alpha beta low cells. The results suggest that E and P affect both the lymphoid and nonlymphoid compartments of the thymus, and that while P increases the volume of the nonlymphoid component of the medulla, E has the opposite effect. The finding that ovariectomy decreased while E and P increased the relative proportion of the most mature thymocytes, which include CD4-8-TCR alpha beta high cells that are believed to harbour potentially autoreactive cell clones, is particularly interesting and may relate to the high propensity of autoimmune diseases in females.

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.

Age-dependent morphometrical changes in the thymus of male propranolol-treated rats.

In order to elucidate a putative role of neurally derived noradrenaline in the thymus development, and in maintenance of adult thymus structure, sexually immature male rats (21-day-old at the beginning of treatment) and young adult animals (75-day-old on the beginning of treatment) were treated with the non-selective beta-adrenoceptor antagonist propranolol (0.40 mg/100 g BW/day, s.c.) for 15 consecutive days, and their thymuses were analyzed stereologically. The effects of beta-adrenoceptor blockade were much more pronounced in sexually immature than in adult rats. In immature propranolol-treated rats the thymus size and volumes of both the main compartments (cortex and medulla) were significantly decreased reflecting, at least partly, a reduction in the overall number of thymocytes. Furthermore, in both the cortical subcompartments (outer and deep cortex) the mean diameter of thymocytes was increased. However, in adult rats exposed to propranolol treatment, only the volume of interlobular connective tissue was enlarged, whereas in the outer part of the cortex the mean thymocyte diameter was increased. These results indicate that the lack of sympathetic input (via beta-adrenoceptors) during the prepubertal period of development diminishes the normal thymus growth and/or accelerates the thymic involution that starts at puberty, immediately after its maximum size is reached, while it is less significant for the maintenance of the thymus size and structure in adults. Additionally, they suggest that distinct cell types, as well as thymocyte subsets, are sensitive to lack of beta-adrenoceptor-mediated influences in sexually immature and adult rats.

Thymopoiesis following chronic blockade of β-adrenoceptors

Abstract The present study was undertaken in order to further clarify putative role of the adrenergic innervation in the regulation of the intrathymic T‐cell maturation. For this purpose adult male DA rats were subjected to either 4‐day‐ or 16‐day‐long propranolol treatment (0.40 mg propranolol/100 g/day, s.c.) and the expression of CD4/8/TCRαβ on thymocytes, as well as thymocyte proliferative and apoptotic index, was assessed in these animals by flow cytometric analysis. Propranolol treatment, in spite of duration, increased both the thymocyte proliferative and apoptotic index (vs. respective vehicle‐treated controls). In 4‐day‐treated animals the thymus cellularity and thymus weight remained unaltered, while in 16‐day‐treated rats the values of both of these parameters were reduced (since increase in the thymocyte apoptotic index overcame that in the proliferative index). The treatments of both durations affected the thymocyte phenotypic profile in a similar pattern, but the changes were more pronounced in rats exposed to the treatment of longer duration. The relative proportion of the least mature CD4−8− double negative (DN) TCRαβ− cells was increased, those of thymocytes at distinct differentiational stages on the transitional route to the CD4+8+ double positive (DP) TCRαβlow stage decreased (all subsets of TCRαβ− in both groups of rats, and those with low expression of TCRαβ in rats subjected to 16‐day‐long treatment) or unaltered (all subsets of TCRαβlow cells in 4‐day‐treated rats). Furthermore, the percentage of CD4+8+ DP TCRαβlow cells was significantly elevated, as well as those of the most mature CD4+8− TCRαβhigh and CD4−8+TCRαβhigh cells (the increase in the percentage of former was much more conspicuous than that of the latter), while the relative proportion of their direct detectable precursors (CD4+8+ DP TCRαβhigh) was reduced. Thus, the present study: i) further supports notion of pharmacological manipulation of adrenergic action as an efficient means in modulation of the T‐cell development, and hence T‐cell‐dependent immune response, and ii) provides more specific insight into T‐cell maturation sequence point/s particularly sensitive to β‐adrenoceptor ligand action.

Differential effects of chronic propranolol treatment on the phenotypic profile of thymocytes from immature and adult rats

To elucidate a putative role of beta-adrenoceptors in the modulation of intrathymic T-cell maturation, the expression of major differentiational antigens (CD4/CD8 and TCR alphabeta) on the thymocytes from both immature (aged 21 day at the beginning of the treatment) and adult (aged 75 days at the beginning of treatment) male rats subjected to a 15-day-long propranolol treatment (0.40 mg/100 g/day, s.c.) was analyzed by two- and one-color flow cytometry, respectively. Rats of matched age injected with saline served as controls. The propranolol treatment in immature but not adult rats caused a significant reduction in both the relative thymus weight and total thymocyte yield. In addition, a significant increase in the percentage of CD4+ 8+ double-positive cells, with a proportional decrease in the relative proportion of CD4+ 8- single positive cells, was found in immature rats. In contrast, a slight but significant decrease in the percentage of CD4+ 8+ cells with a parallel increase in the relative proportion of CD4+ 8- cells was found in adult rats. In both groups of rats, the percentage of TCR alphabeta(total) thymocytes was increased: in immature rats this was due to an increase in the percentage of TCR alphabeta(low) thymocytes, while in the adult rats it reflected a rise in the relative proportion of TCR alphabeta(high) cells. In conclusion, the study revealed that propranolol treatment in both immature and adult rats alters the relative proportion of CD4+ 8+ and CD4+ 8- thymocytes, but in opposite fashion, and the data suggest that this treatment affects distinct fractions within the population of CD4+ 8+ thymocytes with respect to expression of TCR alphabeta. The results also indicate that, regardless of rat sexual maturity, the development of thymocytes towards CD4- 8+ T-cells is relatively insensitive to long-lasting beta-adrenoceptor blockade.

Early postnatal castration affects thymic and thymocyte noradrenaline levels and β-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.