Catecholaminergic signalling through thymic nerve fibres, thymocytes and stromal cells is dependent on both circulating and locally synthesized glucocorticoids.

Glucocorticoids have been shown to modulate the expression of noradrenaline metabolizing enzymes and ß(2)- and α(1B)-adrenoceptors in a tissue- and cell- specific manner. In the thymus, apart from extensive sympathetic innervation, a regulatory network has been identified that encompasses catecholamine-containing non-lymphoid and lymphoid cells. We examined a putative role of adrenal- and thymus-derived glucocorticoids in modulation of rat thymic noradrenaline levels and adrenoceptor expression. Seven days postadrenalectomy, the thymic levels of mRNAs encoding tyrosine hydroxylase, dopamine ß-hydroxylase, monoamine oxidase-A and, consequently, noradrenaline were decreased. Catecholamine content was diminished in autofluorescent nerve fibres (judging by the intensity of fluorescence) and thymocytes (considering HPLC measurements of noradrenaline and the frequency of tyrosine hydroxylase-positive cells), while it remained unaltered in non-lymphoid autofluorescent cells. In addition, adrenalectomy diminished the thymocyte expression of ß(2)- and α(1B)-adrenoceptors at both mRNA and protein levels. Administration of ketoconazole (an inhibitor of glucocorticoid synthesis/action; 25 mg kg(-1) day(-1), s.c.) to glucocorticoid-deprived rats increased the thymic levels of tyrosine hydroxylase, dopamine ß-hydroxylase and, consequently, noradrenaline. The increased intensity of the autofluorescent cell fluorescence in ketoconazole-treated rats indicated an increase in their catecholamine content, and suggested differential glucocorticoid-mediated regulation of catecholamines in thymic lymphoid and non-lymphoid cells. In addition, ketoconazole increased the thymocyte expression of α(1B)-adrenoceptors. Thus, this study indicates that in the thymus, as in some other tissues, glucocorticoids not only act in concert with cateholamines, but they may modulate catecholamine action by tuning thymic catecholamine metabolism and adrenoceptor expression in a cell-specific manner. Additionally, the study indicates a role of thymus-derived glucocorticoids in this modulation.

Cellular and nerve fibre catecholaminergic thymic network: steroid hormone dependent activity.

The thymus plays a critical role in establishing and maintaining the peripheral T-cell pool. It does so by providing a microenvironment within which T-cell precursors differentiate and undergo selection processes to create a functional population of major histocompatibility complex-restricted, self-tolerant T cells. These cells are central to adaptive immunity. Thymic T-cell development is influenced by locally produced soluble factors and cell-to-cell interactions, as well as by sympathetic noradrenergic and endocrine system signalling. Thymic lymphoid and non-lymphoid cells have been shown not only to express beta- and alpha(1)- adrenoceptors (ARs), but also to synthesize catecholamines (CAs). Thus, it is suggested that CAs influence T-cell development via both neurocrine/endocrine and autocrine/paracrine action, and that they serve as immunotransmitters between thymocytes and nerves. CAs acting at multiple sites along the thymocyte developmental route affect T-cell generation not only numerically, but also qualitatively. Thymic CA level and synthesis, as well as AR expression exhibit sex steroid-mediated sexual dimorphism. Moreover, the influence of CAs on T-cell development exhibits glucocorticoid-dependent plasticity. This review summarizes recent findings in this field and our current understanding of complex and multifaceted neuroendocrine-immune communications at thymic level.

Age-associated plasticity of α1-adrenoceptor-mediated tuning of T-cell development.

Alpha(1)-adrenoceptors (α(1)-ARs) are involved in neuro-thymic and thymic intercellular communications, and consequently modulation of T-cell development. Ageing is associated with a number of changes in noradrenergic neuro-effector transmission, and possibly intercellular noradrenaline (NA)-mediated communication resulting in altered responses of target cells to NA. Thus, in old animals an altered NA modulation of thymopoiesis via α(1)-ARs may be expected. To test this hypothesis, in old and young adult Wistar rats we examined: 1) thymic NA levels, density of noradrenergic innervation and NA synthesizing cells, as well as α(1)-AR expression, and 2) then the effects of 14-day-long treatment with the α(1)-AR blocker, urapidil, on thymocyte development. Overall, the first part of study suggested augmented NA signalling to thymic cells via α(1)-ARs due to increased NA availability and α(1)-AR thymocyte surface density in old rats. The second part of study supported this assumption. Namely, although in rats of both ages urapidil affected the same thymocyte developmental steps ultimately leading to changes in the relative number of the most mature single positive TCRαß(high) thymocytes, its effects were generally more prominent in old animals. Following urapidil treatment, the percentages of CD4+CD8- cells, including those showing a regulatory CD4+CD25+RT6.1- phenotype, were increased, while CD4-CD8+ cells decreased. In old rats, an augmented thymic escape of immature CD4+CD8+ cells was also registered. In rats of both ages the thymic changes were accompanied by alterations in the proportions of major cell populations in the T-lymphocyte compartment of both peripheral blood and spleen, leading to an increase in the CD4+/CD8+ T-cell ratio. These alterations were also more pronounced in old rats. Moreover, in old rats following urapidil treatment the proportion of TCRαß+cells in the periphery was slightly greater reflecting, most likely, partly enhanced thymic production of regulatory CD161+TCRαß+cells. Thus, the study indirectly suggests an age-associated increase in the basal α(1)-AR-mediated inhibitory influence of NA on thymopoiesis.

Glucocorticoids, master modulators of the thymic catecholaminergic system?

There is evidence that the major mediators of stress, i.e., catecholamines and glucocorticoids, play an important role in modulating thymopoiesis and consequently immune responses. Furthermore, there are data suggesting that glucocorticoids influence catecholamine action. Therefore, to assess the putative relevance of glucocorticoid-catecholamine interplay in the modulation of thymopoiesis we analyzed thymocyte differentiation/maturation in non-adrenalectomized and andrenalectomized rats subjected to treatment with propranolol (0.4 mg.100 g body weight-1.day-1) for 4 days. The effects of beta-adrenoceptor blockade on thymopoiesis in non-adrenalectomized rats differed not only quantitatively but also qualitatively from those in adrenalectomized rats. In adrenalectomized rats, besides a more efficient thymopoiesis [judged by a more pronounced increase in the relative proportion of the most mature single-positive TCRalphabetahigh thymocytes as revealed by two-way ANOVA; for CD4+CD8- F (1,20) = 10.92, P < 0.01; for CD4-CD8+ F (1,20) = 7.47, P < 0.05], a skewed thymocyte maturation towards the CD4-CD8+ phenotype, and consequently a diminished CD4+CD8-/CD4-CD8+ mature TCRalphabetahigh thymocyte ratio (3.41 +/- 0.21 in non-adrenalectomized rats vs 2.90 +/- 0.31 in adrenalectomized rats, P < 0.05) were found. Therefore, we assumed that catecholaminergic modulation of thymopoiesis exhibits a substantial degree of glucocorticoid-dependent plasticity. Given that glucocorticoids, apart from catecholamine synthesis, influence adrenoceptor expression, we also hypothesized that the lack of adrenal glucocorticoids affected not only beta-adrenoceptor- but also alpha-adrenoceptor-mediated modulation of thymopoiesis.

Expression of alpha1-adrenoceptors on thymic cells and their role in fine tuning of thymopoiesis.

The study was undertaken to explore: i) the presence of alpha(1)-adrenoceptors (AR) on thymic lymphoid and non-lymphoid cells and ii) their putative role in T-cell development. The expression of alpha(1)-AR on thymic cells was assessed using both immunohistochemistry and flow cytometric analyses, while their putative role in thymopoiesis was estimated by analyses of thymocyte proliferation and apoptosis, and major thymocyte subset distribution in adult rats subjected to 14-day-long treatment with the alpha(1)-AR blocker urapidil. The presence of alpha(1)-AR was demonstrated on both thymocytes (mainly less mature CD3(-) and CD3(low) cells) and thymic non-lymphoid cells (thymic epithelial cells and CD68-positive cells). Chronic treatment with urapidil increased the thymic weight and thymocyte number. The increase in thymocyte number might, at least partly, be related to an enhanced thymocyte proliferation. In addition, an altered thymocyte subset distribution was observed in these rats. The increase in the percentage of CD4+CD8+ double positive (DP) TCRalphabeta(-) thymocytes was accompanied by the reduction in that of CD4+CD8+ (DP) TCRalphabeta(low) cells, and divergent changes in the percentage of the most mature single positive (SP) TCRalphabeta(high) thymocytes. In urapidil-administered rats the percentage of CD4+CD8- SP TCRalphabeta(high) thymocytes was increased, while that of the CD4-CD8+ TCRalphabeta(high) was reduced, compared with controls. In addition, proportions of CD4+CD25+RT6.1- and CD161+TCRalphabeta+ regulatory cells were increased. Collectively, the results indicate that alpha(1)-AR are involved in complex network of neuro-thymic and intrathymic communications that provide fine tuning of both conventional effector and regulatory T-cell development.

Peripubertal orchidectomy transitorily affects age-associated thymic involution in rats

The role of gonadal hormones in induction and, particularly, maintenance/progression of rat thymic involution, which normally starts around puberty, was reassessed by examining the effects of peripubertal orchidectomy on thymic weight and morphometric parameters at different times up to the age of 10 months. Up to 6 months post-castration both thymic weight and cellularity in orchidectomized (Cx) rats were greater than in age-matched control rats, sham Cx (Sx). The increase in thymic cellularity reflected an increase in thymocyte proliferation rate (the proportion of proliferating cells was 18.6 ± 0.7 percent in 2-month-old Cx (N = 5) vs 13.4 ± 0.3 percent (N = 5) in age-matched Sx rats) followed by reduced sensitivity to apoptotic signals (apoptotic thymocytes were 9.8 ± 0.9 percent in 2-month-old Cx (N = 5) vs 15.5 ± 0.3 percent (N = 5) age-matched Sx rats). However, 9 months post-orchidectomy, neither thymic weight and cellularity nor any of the morphometric parameters analyzed differed between Cx and control rats. The reduction of thymic cellularity in Cx rats to control values may be related to increased sensitivity of their thymocytes to apoptotic signals in culture (72.6 ± 1.2 percent in 10-month-old vs 9.8 ± 0.9 percent in 2-month-old Cx rats) followed by reduced responsiveness to proliferative stimuli (14.1 ± 0.2 percent in 10-month-old vs 18.6 ± 0.7 percent in 2-month-old Cx rats). Thus, the study indicates that the effects of peripubertal orchidectomy on thymic weight and cellularity, as well as on the main morphometric indices, are long-lasting but not permanent, i.e., that removal of the testes can only postpone but not prevent age-related organ atrophy and consequently functional deterioration of the immune system.

Beta-adrenoceptor blockade ameliorates the clinical course of experimental allergic encephalomyelitis and diminishes its aggravation in adrenalectomized rats.

As glucocorticoids influence both catecholamine synthesis and adrenoceptor expression by immune cells, the current study was undertaken to distinguish their direct effects on the development of experimental allergic encephalomyelitis from those induced by alteration of catecholamine signaling. We examined the influence of 16-day-long beta-adrenoceptor blockade with propranolol (0.40 mg/100 g body weight/day, s.c.) beginning 3 days before immunization on the development of experimental allergic encephalomyelitis in adrenalectomized (7 days before immunization) and in non-operated male Dark Agouti rats. Adrenalectomy aggravated the clinical course of experimental allergic encephalomyelitis. In contrast, propranolol attenuated both the clinical signs of the disease and decreased the number of lesions in the spinal cord. Furthermore, propranolol prevented adrenalectomy-induced aggravation of the disease course without affecting mortality. We also found that the percentage of CD4(+)CD25(+) T lymphocytes (recently activated or regulatory cells) was increased in peripheral blood of experimental allergic encephalomyelitis rats over that in the corresponding non-immunized and bovine serum albumin immunized rats. However, the percentage of these cells was reduced in adrenalectomized and/or propranolol-treated experimental allergic encephalomyelitis rats compared to control experimental allergic encephalomyelitis rats. Our findings, coupled with the clinical course of the disease and the underlying pathomorphological changes, clearly suggest that differential mechanisms were responsible for the changes in the percentage of CD4(+)CD25(+) T lymphocytes in propranolol-treated adrenalectomized rats and only propranolol-treated rats with experimental allergic encephalomyelitis. Our results, when viewed globally, indicate that: i) beta-adrenoceptor-dependent mechanisms are involved in the immunopathogenesis of experimental allergic encephalomyelitis, ii) experimental allergic encephalomyelitis has a more severe course in adrenalectomized rats and iii) beta-adrenoceptor-mediated mechanisms operate in adrenalectomy-induced aggravation of the disease.

Neonatal castration affects intrathymic kinetics of T-cell differentiation and the spleen T-cell level.

To test putative interdependence in the ontogenesis of the hypothalamic-pituitary-gonadal and thymic-lymphatic axes, thymocyte differentiation and maturation was examined in neonatally castrated (Cx) adult rats. In the hypercellular thymi of Cx rats, the proportion of the least mature CD4(-)CD8(-)TCRalphabeta(-) triple negative (TN) thymocytes was reduced, while the proportions of all downstream double positive (DP) subsets (TCRalphabeta(-), TCRalphabeta(low) and TCRalphabeta(high)) were increased when compared with neonatally sham-castrated (Sx) adult rats. This suggested an accelerated thymocyte transition from the TN to DP TCRalphabeta(low) developmental stage accompanied by an increased positive/ reduced negative thymocyte selection. The increased thymocyte surface density of Thy-1, which is implicated in thymocyte hyposensitivity to negative selection, in Cx rats further supports the previous assumption. The finding that the proportions of both single positive (SP) TCRalphabeta(high) thymocyte subsets were reduced, while their numbers were increased (CD4(+)CD8(-)) or unaltered (CD4(-)CD8(+)), coupled with results demonstrating an increased level of CD4(-)CD8(+) cells without changes in that of CD4(+)8(-) cells in the spleen indicate: (i) accelerated differentiation and maturation of the positively selected DP TCRalphabeta(high) thymocytes towards CD4(-)8(+) TCRalphabeta(high) cells followed by increased emigration of the mature cells and (ii) decelerated differentiation and maturation towards CD4(+)8(-)TCRalphabeta(high) cells in Cx rats. Furthermore, the unaltered proportion of intrathymically developing CD4(+)CD25(+)Foxp3(+) regulatory cells in Cx rats, in light of putative hyposensitivity of thymocytes to negative selection suggesting reduced elimination of autoreactive cells, may provide a firm basis for understanding the reasons behind increased susceptibility of Cx rats to autoimmune disease induction.

Long-term beta-adrenergic receptor blockade increases levels of the most mature thymocyte subsets in aged rats.

Age-related increase in the density of thymic noradrenergic fibres and noradrenaline (NA) concentration is proposed to be associated with thymic involution and altered thymopoiesis. To test this hypothesis thymocyte differentiation/maturation and thymic structure were studied in 18-month-old male Wistar rats subjected to 14-day-long propranolol (P) blockade of beta-adrenoceptors (beta-ARs). The treatment primarily resulted in changes in the T-cell receptor (TCR)-dependent stages of thymopoiesis, which led to an increase in both the relative and absolute numbers of the most mature single positive (SP) CD4(+)CD8(-) (including cells with the CD4(+)CD25(+) regulatory phenotype) and CD4(-)CD8(+) TCRalphabeta(high) thymocytes. Accordingly, in the thymi of these rats an increase in both numerical density and absolute number of medullary thymocytes encompassing mainly the most mature SP cells was found. These findings, together with an increase in the thymocyte surface expression of the regulatory molecule Thy-1 (CD90) (implicated in negative regulation of TCRalphabeta-dependent thymocyte selection thresholds) in the same rats, may suggest increased positive/reduced negative thymocyte selection. Collectively, the results indicate that a decline in thymic efficiency in generating both conventional and regulatory T cells, and consequently in immune function, in aged rats may be, at least partly, attenuated by long-term blockade of beta-ARs with P.

Characterization of thymocyte phenotypic alterations induced by long-lasting beta-adrenoceptor blockade in vivo and its effects on thymocyte proliferation and apoptosis.

Adult male Wistar rats were subjected to propranolol (P, 0.40 mg/100 g/day) or saline (S) administration (controls) over 14 days. The expression of major differentiation molecules on thymocytes and Thy-1 (CD90) molecules, which are shown to adjust thymocyte sensitivity to TCRalphabeta signaling, was studied. In addition, the sensitivity of thymocytes to induction of apoptosis and concanavalin A (Con A) signaling was estimated. The thymocytes from P-treated (PT) rats exhibited an increased sensitivity to induction of apoptosis, as well as to Con A stimulation. Furthermore, P treatment produced changes in the distribution of thymocyte subsets suggesting that more cells passed positive selection and further differentiated into mature CD4+ or CD8+ single positive (SP) TCRalphabeta(high) cells. These changes may, at least partly, be related to the markedly increased density of Thy-1 surface expression on TCRalphabeta(low) thymocytes from these rats. The increased frequency of cells expressing the CD4+25+ phenotype, which has been shown to be characteristic for regulatory cells in the thymus, may also indicate alterations in thymocyte selection following P treatment. Inasmuch as positive and negative selections play an important role in continuously reshaping the T-cell repertoire and maintaining tolerance, the hereby presented study suggests that pharmacological manipulations with beta-AR signaling, or chemically evoked alterations in catecholamine release, may interfere with the regulation of thymocyte selection, and consequently with the immune response.