Gonadotropin-Releasing Hormone in Regulation of Thymic Development in Rats: Profile of Thymic Cytokines.

An increasing body of recent experimental data confirms the impact of neurohormones on fetal development and function of different body systems. The synthesis of many neurohormones starts in fetal tissues before the hypothalamic-pituitary-adrenal and hypothalamic-pituitary-gonadal systems are formed, and their high levels are detected in the bloodstream. Here, we studied the role of gonadotropin-releasing hormone (GnRH) in rat thymus development and tried to reveal possible mechanisms underlying the GnRH effects in early development. Western blotting and reverse transcription-polymerase chain reaction allowed us to identify receptor for GnRH in the fetal thymus with peak expression on embryonic days 17-18 (ED17-18). Blocking the receptors in utero on ED17 by a GnRH antagonist suppressed the concanavalin A-induced proliferative response of T cells in adults. GnRH (10-7 M) increased mRNA expression of interleukin (IL)-4, IL-10, IL-1ß, interferon γ (IFNγ), and tumor necrosis factor α (TNFα) in the thymus of 18-day fetuses after an ex vivo culture for 24 h. The increased mRNA levels of the cytokines in the thymus were accompanied by increased numbers of CD4+ T helpers. Overall, the data obtained confirm the regulatory or morphogenetic effect of GnRH on fetal thymus development mediated by synthesis of thymic cytokines.

Serotonin limits generation of chromaffin cells during adrenal organ development.

Adrenal glands are the major organs releasing catecholamines and regulating our stress response. The mechanisms balancing generation of adrenergic chromaffin cells and protecting against neuroblastoma tumors are still enigmatic. Here we revealed that serotonin (5HT) controls the numbers of chromaffin cells by acting upon their immediate progenitor "bridge" cells via 5-hydroxytryptamine receptor 3A (HTR3A), and the aggressive HTR3Ahigh human neuroblastoma cell lines reduce proliferation in response to HTR3A-specific agonists. In embryos (in vivo), the physiological increase of 5HT caused a prolongation of the cell cycle in "bridge" progenitors leading to a smaller chromaffin population and changing the balance of hormones and behavioral patterns in adulthood. These behavioral effects and smaller adrenals were mirrored in the progeny of pregnant female mice subjected to experimental stress, suggesting a maternal-fetal link that controls developmental adaptations. Finally, these results corresponded to a size-distribution of adrenals found in wild rodents with different coping strategies.

Pattern of MHC class I and immune proteasome expression in Walker 256 tumor during growth and regression in Brattleboro rats with the hereditary defect of arginine-vasopressin synthesis.

Dynamics of the expression of MHC class I, immune proteasomes and proteasome regulators 19S, PA28, total proteasome pool and proteasome chymotrypsin-like activity in Walker 256 tumor after implantation into Brattleboro rats with the hereditary defect of arginine-vasopressin synthesis was studied. The tumor growth and regression in Brattleboro rats were accompanied by changes in the proteasome subunit level unlike the tumor growth in WAG rats with normal expression of arginine-vasopressin gene. In the tumor implanted into Brattleboro rats the immune proteasome level was maximal between days 14 and 17, when the tumor underwent regression. Conversely, the expression of proteasome regulators tended to decrease during this period. Immune proteasomes are known to produce antigen epitopes for MHC class I to be presented to CD8+ T lymphocytes. Enhanced expression of immune proteasomes coincided with the recovery of MHC class I expression, suggesting the efficient presentation of tumor antigens in Brattleboro rats.

Ontogenesis of rat immune system: Proteasome expression in different cell populations of the developing thymus.

Immune proteasomes in thymus are involved in processing of self-antigens, which are presented by MHC class I molecules for rejection of autoreactive thymocytes in adults and probably in perinatal rats. The distribution of immune proteasome subunits LMP7 and LMP2 in thymic cells have been investigated during rat perinatal ontogenesis. Double immunofluorescent labeling revealed LMP7 and LMP2 in thymic epithelial and dendritic cells, as well as in CD68 positive cells - macrophages, monocytes - at all developmental stages. LMP2 and LMP7 were also detected by flow cytometry in almost all thymic CD90 lymphocytes through pre- and postnatal ontogenesis. Our results demonstrate that the immune proteasomes are expressed in all types of thymic antigen presenting cells during perinatal ontogenesis, suggesting the establishment of the negative selection in the thymus at the end of fetal life. The observation of the immune proteasome expression in T lymphocytes suggests their role in thymocyte differentiation besides antigen processing in thymus.

New approach to study of T cellular immunity development: parallel investigation of lymphoid organ formation and changes in immune proteasome amount in rat early ontogenesis.

The expression pattern and distribution of proteasome immune subunits LMP7 and LMP2 in the developing rat spleen and liver as well as the periarterial lymphoid sheath formation were investigated. LMP7 and LMP2 were detected by immunoblotting in the spleen on the 21st embryonic day and during the first postnatal days in equal amounts. Their levels increased by the 8th and 18th postnatal days. Double immunofluorescent labeling the spleen cells revealed LMP7 and LMP2 in T and B lymphocytes localized in the red pulp in embryogenesis. Few T lymphocytes were discovered in periarterial zones on the 8th postnatal day. T lymphocytes filled these zones and formed lymphoid sheaths by the 18-19th day. In the liver, LMP7 and LMP2 were revealed by the 17-19th postnatal day. Immunofluorescent analysis showed their presence in hepatocytes at this period. The data suggest that T cell-mediated immune response in relation to hepatocytes is possible beginning from 18th to 19th postnatal day.

Noradrenergic regulation of galanin expression in the supraoptic nucleus in the rat hypothalamus. An ex vivo study.

Galanin is coexpressed with vasopressin and oxytocin in magnocellular neurons of the rat neuroendocrine hypothalamus. Various physiological stimuli, such as osmotic stimulation or lactation, that affect vasopressin and oxytocin expression and release also modulate galanin expression. Magnocellular neurons are highly innervated by noradrenergic inputs from the brainstem. The noradrenergic system plays a critical excitatory role in the activation of vasopressin-expressing and oxytocin-expressing neurons. Here, we have evaluated the possible regulation of Gal expression by noradrenaline in the magnocellular neurons of supraoptic nucleus in an ex vivo acute model of rat hypothalamic slices. The slices containing the supraoptic nucleus were incubated with 10(-4) M noradrenaline for 1 or 4 hr. The levels of galanin and galanin mRNA were estimated by semiquantitative immunohistochemistry and in situ hybridization, respectively. Our results show that the amount of galanin-immunopositive material in the cell bodies of the magnocellular neurons increased significantly after incubation with noradrenaline compared with control slices at the same time point and that this effect was more pronounced after 4 hr than after 1 hr. In situ hybridization showed that radiolabeling of the supraoptic nucleus with a radioactive galanin probe increased slightly after 1 hr of incubation and increased considerably after 4 hr of incubation with noradrenaline. Our study shows that galanin may be a target in the regulation of the hypothalamic magnocellular-neurohypophysial system by noradrenaline.

Hypothalamic control of mitogen-induced proliferative responses and luteinizing hormone-releasing hormone levels in thymus and peripheral blood of rat fetuses.

The role of endogenous luteinizing hormone-releasing hormone (LHRH) in the development of concanavalin A (ConA)-induced proliferative responses was studied in rat fetuses. Preliminary treatment of fetuses in utero with either the LHRH receptor antagonist or anti-LHRH antibodies resulted in the suppression of ConA-induced proliferative responses of thymocytes. LHRH and LHRH-immunopositive cells, morphologically similar to thymocytes, were detected in intact fetal thymus. A significant content of LHRH was also found in the peripheral blood of fetuses. The LHRH content in thymus and plasma was similar in males and females. Surgical ablation of the hypothalamus resulted in 2-fold decreases in thymus and plasma levels of LHRH in 21-day-old fetuses compared to sham-operated fetuses. It was concluded that LHRH regulates mitogen-induced proliferative responses of thymocytes during prenatal ontogenesis in the rat. The main source of plasma LHRH at that period is the hypothalamus. Moreover, LHRH is synthesized in the fetal thymus. Thus, LHRH is suggested to have not only a central effect but also to be involved in autocrine or paracrine regulation of proliferative immune responses.