Basic fibroblast growth factor priming increases the responsiveness of immortalized hypothalamic luteinizing hormone releasing hormone neurones to neurotrophic factors.

The participation of growth factors (GFs) in the regulation of luteinizing hormone releasing hormone (LHRH) neuronal function has recently been proposed, but little is known about the role played by GFs during early LHRH neurone differentiation. In the present study, we have used combined biochemical and morphological approaches to study the ability of a number of GFs normally expressed during brain development, including basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin and insulin-like growth factor I (IGF-I) to induce survival, differentiation, proliferation, and phenotypic expression of immortalized (GT1-1) LHRH neurones in vitro, at early (3-days in vitro, 3-DIV) and late (8-DIV) stages of neuronal differentiation. Comparison of GF-treated vs untreated neurones grown in serum-deprived (SD) medium demonstrated bFGF to be the most potent, and insulin the least active in promoting neuronal differentiation. Thus, at both 3-DIV and 8-DIV, but especially at 8-DIV, bFGF induced the greatest increase in the total length and number of LHRH processes/cell and in growth cone surface area. bFGF was also the most active at 3-DIV, and IGF-I at 8-DIV, in counteracting SD-induced cell death, whereas EGF was the most potent in increasing [3H]thymidine incorporation. All GFs studied decreased the spontaneous release of LHRH from GT1-1 cells when applied at 3-DIV or 8-DIV, except for insulin which was inactive at both time-points and bFGF which was inactive at 8-DIV. Pre-treatment of GT1-1 cells with a suboptimal ('priming') dose of bFGF for 12 h followed by application of the different GFs induced a sharp potentiation of the neurotrophic and proliferative effects of the latter and particularly of those of IGF-I. Moreover, bFGF priming counteracted EGF-induced decrease in LHRH release and significantly stimulated LHRH secretion following IGF-I or insulin application, suggesting that bFGF may sensitize LHRH neurones to differentiating effects of specific GFs during development.

Basic fibroblast growth factor (bFGF) acts on both neurons and glia to mediate the neurotrophic effects of astrocytes on LHRH neurons in culture.

Luteinizing hormone-releasing hormone (LHRH) neurons play a pivotal role in the neuroendocrine control of mammalian reproduction. Astrocytes were shown to be involved in the regulation of LHRH neuronal function, but little is known about the contribution of astroglial-derived factors in the regulation of LHRH neuron development. In order to gain insight into the mechanisms regulating the development of these cells, at morphological and biochemical levels we characterized the neurotrophic effects exerted by young astrocytes (maintained in culture for 8 days in vitro) and old astrocytes (maintained 26 days) on the differentiation, proliferation, and phenotypic expression of immortalized hypothalamic LHRH (GT(1-1)) neurons in vitro. Culturing GT(1-1) cells in the presence of young glia for different time intervals caused a marked acceleration in the acquisition of their neuronal phenotype. At all times examined, GT(1-1) cells cocultured with young glia exhibited a significantly greater extension of processes/cell, larger number of processes/cell and greater surface area of growth cones than GT(1-1) cells grown over nonglial adhesive substrates (polylysine). By contrast, when GT(1-1) neurons were cocultured with old glia, the length of neuronal processes and the growth cone surface area were significantly lower than in control GT(1-1) neurons cultured in the absence of glia. At 3 days in vitro (DIV), GT(1-1) neurons cocultured with young glia exhibited a 50% lower incorporation of [(3)H]thymidine than GT(1-1) neurons cultured without glia. By contrast, in the presence of old glia [(3)H]thymidine incorporation was significantly higher in cells cocultured with glia than in GT(1-1) neurons cultured alone. Localization of the proliferating cells by dual immunohistochemical staining revealed that the incorporation of bromodeoxiuridine (BrdU) was restricted to nuclei of GT(1-1) neurons when these were cocultured with young glia, but associated with both neurons and astrocytes in the presence of old glia. At the functional level, coculture of GT(1-1) neurons with young glia increased the spontaneous release of LHRH as compared to GT(1-1) neurons grown in the absence of glia. By contrast, in the presence of old glia LHRH release in the medium was significantly lower than in controls. Conditioned medium of young glia (ACM-Y) induced significant neurotrophic and functional effects on GT(1-1) cells, but these effects were 50% less potent than the coculture itself. Heat denaturation of ACM-Y totally abolished its neurotrophic and functional properties, indicating that they involved a peptide factor. Suppression of bFGF activity in ACM-Y reduced its neurotrophic activity by approximately 40%, but did not affect its LHRH release-promoting effects. By contrast, neutralization of endogenous bFGF activity in GT(1-1) neurons cocultured with young glia counteracted both neurotrophic and functional effects of young glia. Treatment of old glia with bFGF rescued its neurotrophic and functional effects on GT(1-1) cells. Moreover, the ACM of aged bFGF-treated old glia was the most powerful neurotrophic stimulus for GT(1-1) neurons. These results suggest that: 1) soluble peptidic factors, including bFGF, and mechanism(s) requiring coculture are responsible for the highly potent neurotrophic and functional effects of young glia; 2) the inhibitory effects of old glia on neurite outgrowth and LHRH release are mediated in part by soluble inhibitory molecules and in part by factors requiring coculture with old glia; 3) old glia may revert to a growth-supporting state when treated with bFGF and this functional shift involves a diffusible molecule with potent neurotrophic and functional effects on immortalized LHRH neurons. (c) 2000 Wiley-Liss, Inc.

Gender, neuroendocrine-immune interactions and neuron-glial plasticity. Role of luteinizing hormone-releasing hormone (LHRH).

Signals generated by the hypothalamic-pitutary-gonadal (HPG) axis powerfully modulate immune system function. This article summarizes some aspects of the impact of gender in neuroendocrine immunomodulation. Emphasis is given to the astroglial cell compartment, defined as a key actor in neuroendocrine immune communications. In the brain, the principal hormones of the HPG axis directly interact with astroglial cells. Thus, luteinizing hormone releasing hormone, LHRH, influences hypothalamic astrocyte development and growth, and hypothalamic astrocytes direct LHRH neuron differentiation. Hormonally induced changes in neuron-glial plasticity may dictate major changes in CNS output, and thus actively participate in sex dimorphic immune responses. The impact of gender in neuroimmunomodulation is further underlined by the sex dimorphism in the expression of genes encoding for neuroendocrine hormones and their receptors within the thymus, and by the potent modulation exerted by circulating sex steroids during development and immunization. The central role of glucocorticoids in the interactive communication between neuroendocrine and immune systems, and the impact of gender on hypothalamic-pituitary-adrenocortical (HPA) axis modulation is underscored in transgenic mice expressing a glucocorticoid receptor antisense RNA.

Luteinizing hormone-releasing hormone is a primary signaling molecule in the neuroimmune network.

The brain-pituitary-reproductive axis and the brain thymus-lymphoid axis are linked by an array of internal mechanisms of communication that use similar signals (neurotransmitters, peptides, growth factors, hormones) acting on similar recognition targets. Moreover, such communication networks form the basis and control each step and every level of reproductive physiology. This presentation highlights the extent to which endocrine, neural, glial, or immunologically competent cells may achieve their specific functions using common mechanisms, but employing them to different degrees. In particular, this work will focus on LHRH, the chief hormone orchestrating reproductive events. Within the thymus LHRH plays a unique role of immunomodulator, contributing to the sex-dependent changes in immune responsiveness during the estrous-menstrual cycle as well as pregnancy. From the recent cloning and sequencing of lymphocyte LHRH, the expression of LHRH receptor mRNA in lymphocyte, the transduction mechanisms involved, and the steroidogenic sensitivity of the intralymphocyte LHRH system. It would appear that this peptide may act as an immunological response modifier in the brain-pituitary-lymphoid-gonadal axis. The interplay between neuronal, endocrine, and immune compartments is further emphasized in the study of LHRH-astroglial interactions. Astrocytes are able to manufacture a wide variety of signaling agents and can secrete immunoregulatory molecules that influence immune cells, as well as the glial cells themselves. Astroglia and the immortalized hypothalamic LHRH (GT1-1) neurons communicate with an array of mechanisms, via soluble mediators as well as cell-to-cell contacts. Manipulation of astroglial-derived cytokines and nitric oxide (NO) in GT1-1 neuron-astroglia cocultures, underscores a potential cross-talk between different intra/inter-cellular mediators in the dynamic control of LHRH release. Further studies aimed to disclose at a biochemical and a molecular level such bidirectional, informative network will give us new insights into more general issues concerned with the malfunction of the neuroendocrine-immune axis.

Luteinizing hormone-releasing hormone (LHRH) receptors in the neuroendocrine-immune network. Biochemical bases and implications for reproductive physiopathology.

It seems apparent that the brain-pituitary-reproductive axis and the brain-thymus-lymphoid axis are linked by an array of internal mechanisms of communication that use similar signals (neurotransmitters, peptides, growth factors, hormones) acting on similar recognition targets. Moreover, such communication networks form the basis and control of each step and every level of reproductive physiology. This work has focused on the LHRH system, a primary central and peripheral clock of both neuroendocrine and immune functions. From the initiation of a sexually organized response, the detection of sexual odors, and the induction of mating behavior, extrahypothalamic and hypothalamic LHRH orchestrates the neuroendocrine modulation of gonadotropin secretion, while its expression within the ovary directly controls specific events such as follicular atresia. The presence of LHRH receptors in oocytes clearly anticipates a potential action of the decapeptide during the process of fertilization and/or implantation. Within the thymus and other peripheral immune organs, LHRH plays a unique role of immunomodulator, contributing to the sex-dependent changes in immune responsiveness during the estrous-menstrual cycle as well as pregnancy. The reciprocity of the neuroendocrine-immune signaling systems is further supported by the ability of sex steroids to modulate thymus-dependent immune functions via direct effects on specific target genes involved in the development of sex dimorphism and sex-dimorphic immune responses, including the downregulation of immune response observed during pregnancy. Such cyclic changes in immune responsiveness could have a physiological implication, such as the decrease or suppression in cell-mediated immunity observed in the postovulatory phase of the cycle and in pregnancy, respectively, and might play a role during the implantation process and the establishment of pregnancy. In this context, the ability of corticosterone to directly inhibit both GR transcript levels as well as a cell-mediated immune response within the thymus, and the modulation of such an inhibitory effect by the sex steroid hormone milieu, may offer an explanation and a molecular mechanism whereby stress may be deleterious for reproduction, also via immunomodulation. On the other hand, hormonally mediated alterations in immunity might also have a pathological implication in sexually related immune diseases. For example, in mouse and humans, lupus erythematosus is more prevalent in females and estrogen accelerates the disease process, while menstruation is known to exacerbate idiopathic thrombocytopenia purpura. Sex steroid hormone milieu might also have a role in controlling the stress response through immunomodulation. Within the placenta, an intricate network of signaling systems controls a delicate interplay between the neuroendocrine hormones, growth factors, and cytokines that are susceptible to play a major local role in the processes of implantation and the establishment and completion of pregnancy. The neuroendocrine and immunomodulatory role of LHRH continues well after parturition because the presence of LHRH-like material within the mammary gland and milk participates in the physiological modulation of hypophyseal, gonadal, and immune functions of the pups. Such a significant role played by the hypothalamic peptide in the modulation of immune responsiveness would indicate LHRH as the signal conveying information to both neuroendocrine and immune cells, with the role of informing and then transducing the messages into appropriate biological responses.(ABSTRACT TRUNCATED)

Neuroendocrineimmunology (NEI) at the turn of the century: towards a molecular understanding of basic mechanisms and implications for reproductive physiopathology.

The interactions between the nervous, endocrine and immune systems require a complex communication network. The central nervous system (CNS) affects the immune system through endocrine, paracrine and neuronal mechanisms. Evidence that this bidirectional communication plays a vital role in the regulation of physiological homeostatic mechanisms while a disfunction of the neuroendocrineimmune balance favors the susceptibility to a number of diseases is derived largely by animal models but also by an increasing number of clinical studies in different fields, including endocrinology, reproductive physiology, pediatrics, oncology, neurology and psychiatry. An increasing number of endocrine hormones, neurotransmitters and neuropeptides are expressed in immune tissues and cells and are actively involved in the physiological regulation of immunity. Conversely, the endocrine and nervous systems harbor receptors for a wide variety of immunologically-derived substances, suggesting potential regulatory feedback loops between the three major integrative bodily systems. Major implications for the reproductive endocrinology field are that psychoneuroendocrine processes may alter fertility via immunomodulation, and that events that occur as part of immune responses influence the neuroendocrine axes, which in turn counter-regulate immune function. In the present article, some features of reproductive-immune interactions will be described, and the neuroendocrineimmune dialogue via the chief reproductive hormone, luteinizing hormone-releasing hormone (LHRH), will be summarized as prototype of intersystem crosstalk. A particular emphasis will be given to the cytokine-LHRH interrelationships both at central (i.e. especially with the astroglial compartment) and peripheral levels. The surprisingly similar communication network systems used by the gonads and the thymus will be summarized, and the sexually-driven dimorphisms dictating female versus male reproductive and immunological capacities reviewed. Evidence that neural, endocrine and immune systems work together as a single unit are emphasized in animal models and human pathologies where interruption of NEI feedback loops results in long lasting pathological consequences for the nervous, endocrine and immune functions.

The immune system response during development and progression of carcinogen-induced rat mammary tumors: prevention of tumor growth and restoration of immune system responsiveness by thymopentin.

A detailed analysis of the immune system response has been performed during the development and progression of dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumors. For this aim, a number of immune parameters (thymocyte and splenocyte proliferative response to T-dependent mitogens, antibody production, lymphocyte subset phenotyping, interleukin 2 receptor expression in resting and activated lymphocytes, thymus morphology and morphometry), were correlated with tumor appearance and growth at different (-7, 0, +15, +30, +60, +90, and +120 days) time intervals after intragastric administration of DMBA, in the absence or the presence of a concomitant treatment with the thymic pentapeptide thymopentin (TP5). A profound and time-dependent immunosuppression characterized the treatment with the carcinogen. Both cell-mediated and humoral immune responses showed a 50% inhibition 2 weeks after DMBA administration, with a peak after 30 days, followed by a plateau until 120 days of observation. The mechanism responsible for reduced ability of thymocytes and splenocytes to respond to both Con-A and PHA was explained by the significant inhibition of one of the key steps of T cell activation, namely the expression of IL-2 receptor in lymphocytes from DMBA-treated animals. The flow cytometric analysis of lymphocyte subpopulations revealed an important reduction in the overall populations of thymocytes and splenocytes. At the thymus gland level, a dramatic reduction of double positive CD4+CD8+ and a decrease of CD4+CD8- and CD4-CD8+ were observed, together with a marked atrophy of the thymic cortex, and impairment of the thymic microenvironment. One hundred and twenty days after DMBA administration, approximately 60 to 70% of the animals developed tumors with a mean tumor surface area of 2.88 +/- 0.86 cm2, and a number of 2.44 +/- 1.0. Treatment with TP5 (100 ng/animal, three times a week, starting a week before DMBA), produced specific effects on different immune compartments and tumoral growth, characterized by a significant reversal of immune depression with a stimulatory effect measured on lymphoproliferative assays, lymphocyte subset distribution, and IL-2 receptor expression. Moreover, thymic atrophy was almost completely prevented in TP5 treated animals. Of major interest, a significant delay in the appearance and growth of tumors was observed in TP5 treated rats. When DMBA-treated animals were followed for the entire observation period (0-120 days) and the immune responsiveness correlated according to tumor progression, stability, or regression, a positive correlation was calculated between the degree of immune system depression and the individual rate of tumor growth; in TP5-treated rats the majority of the tumors were static or regressing tumors.(ABSTRACT TRUNCATED AT 400 WORDS)

Luteinizing hormone-releasing hormone signaling at the lymphocyte involves stimulation of interleukin-2 receptor expression.

The marked modulation of lymphocyte function exerted by the hypothalamic decapetide LHRH prompted us to study the possible involvement of the neuropeptide in one of the major steps of lymphocyte proliferation, namely the expression of interleukin-2 (IL-2) receptor during in vitro treatment of rat lymphocytes with LHRH agonists (LHRH-A) or antagonists (LHRH-ANTA). The basal proliferative activity of splenocytes and thymocytes from proestrous female rats was significantly stimulated after incubation with LHRH and LHRH-A, but not LHRH fragments [LHRH-(1-3), LHRH-(1-5), and LHRH-(2-6)]. Similarly, in the absence of the mitogenic stimulus, IL-2 receptor expression was significantly stimulated in thymocyte and splenocyte cultures incubated with increasing doses of LHRH or its agonists. The amplification of Concanavalin-A-induced increase in blastogenic transformation of lymphocytes by LHRH was paralleled by a significant stimulation of IL-2 receptor expression. The specificity of such effect was demonstrated by 1) the failure of LHRH fragments [LHRH-(1-6)] to mimick the LHRH stimulatory effect; and 2) the complete reversal produced by simultaneous addition of a potent LHRH-ANTA on IL-2 receptor expression induced by LHRH. Moreover, basal and lectin stimulation of IL-2 receptor-positive cells were significantly inhibited by treatment with the LHRH-ANTA. These data clearly demonstrate that 1) LHRH induction of lymphocyte activation in vitro is accompanied by a specific increase in IL-2 receptor-positive cells; 2) endogenous lymphocyte LHRH may participate in stimulation of IL-2 receptor expression under both basal and stimulated conditions, suggesting that LHRH signaling at the lymphocyte may interact synergistically with intracellular mechanisms responsible for lymphocyte activation.

Blockade of central and peripheral luteinizing hormone-releasing hormone (LHRH) receptors in neonatal rats with a potent LHRH-antagonist inhibits the morphofunctional development of the thymus and maturation of the cell-mediated and humoral immune responses.

The development of the thymus and the hypothalamic-pituitary-gonadal axis are linked by bidirectional hormonally mediated relationships. In the present study, the direct involvement of the neuropeptide LHRH in the maturation of the thymus and development of the cell-mediated and humoral immune responses were assessed after treatment of neonatal (from post-natal day 1-day 5) female rats with a potent LHRH-antagonist (LHRH-anta, p-Glu-D-Phe 2.6,Pro3-LHRH, 50 micrograms/rat), and the effects compared to those resulting from neonatal castration. Whereas in control animals the maturation of mitogenic potential in thymocyte cultures showed a progressive and age-dependent increase, reaching a maximal activity at 30 days of age and then decreasing after puberty onset, in LHRH-anta-treated rats, the thymocyte's proliferative response was completely blocked at 7 days of age and remained very low at each time interval studied, until 3 months of age. A similar effect of the LHRH-anta treatment on splenocyte cultures was measured. Moreover, a reduced percentage of the T-helper lymphocyte subpopulation followed LHRH-anta administration. By contrast, in neonatally castrated rats, blastogenic activity was significantly higher, compared to control cultures, at each stage studied. Treatment with LHRH-anta produced a significant decrease in thymus wt, an alteration of the maturational pattern characterized by a cellular monomorphism, reduced thymocyte volume, reduction of the cortical area, and depauperation of the epithelial microenvironment. Moreover, a morphometric analysis revealed a selective decrease in the large lymphoid cell population of the subcapsular cortex at 7 and 15 days. On the other hand, neonatal castration produced an opposite effect, leading to a marked hypertrophy of the cortical area, and counteracted the post-puberal thymus atrophy. When LHRH-anta-treated adult (3-month-old) rats were challenged with an antigenic stimulus (multiple sc injections of complete Freund adjuvant and BSA) and antibody (anti-BSA antibodies of the immunoglobulin G class) production measured in the serum after 15 days, a marked and significant decrease in immunoglobulin G levels was observed, compared to the values measured in untreated control. The described immune deficiencies in LHRH-anta-treated rats were associated with a clear inhibition of sexual maturation. This study clearly indicates that the blockade of central and peripheral LHRH receptors during a critical period for maturation of both hypothalamus-hypophyseal-gonadal axis and brain-thymus-lymphoid axis dramatically impairs immune system development, suggesting a potential role of the neuropeptide LHRH in the bidirectional programming of both neuroendocrine and immune functions.

Phosphatidylserine counteracts physiological and pharmacological suppression of humoral immune response.

Phosphatidylserine (PS) is a necessary cofactor for protein kinase C (PKC) activation, and changes in the synthesis of PS have been shown to participate in the mechanism(s) involved in the transmembrane signaling of interleukin 1 (IL-1). In view of the age-associated defects in T-cell functions, in the present study we have addressed the question of whether an in vivo treatment with PS might interfere with such processes. Furthermore, the effect of an in vitro treatment with PS in human peripheral blood monocytes (PBMC) or splenocytes activated with a lectin mitogen, on the expression of IL-2 receptor, was assessed. While the process of ageing was accompanied by a marked decline of humoral response monitored by anti-BSA antibodies (of the IgG class) production, following immunization with BSA in complete Freund adjuvant, chronic treatment with PS (50 mg/kg, in drinking water), reversed this effect, raising specific antibody titers to levels practically indistinguishable from those measured in young animals. Pharmacological depression of humoral immune response induced by a treatment of adult animals with dexamethasone was similarly reversed by a chronic treatment with PS. While only a pharmacological concentration (10(-5) M) of PS significantly increased IL-2 receptor expression in activated human PBMC, simultaneous treatment of PBMC with inactive doses of PS and the pharmacological activator of PKC (phorbol myristate acetate, PMA, 10(-8) M) resulted in a synergistic stimulation of Tac+ cells. Furthermore, in cultures of rat splenocytes PS (10(-6) M) significantly stimulated the expression of IL-2 receptor, and concomitant addition of PS (10(-7) M) to Con A-stimulated splenocytes produced a significant potentiation of IL-2 receptor induction. The present results indicate that in vivo treatment of ageing animals with the specific phospholipid PS is able to reverse the physiological decline of the humoral immune response induced by the ageing process. Moreover, treatment of young rats with PS reversed the pharmacological associated depression of specific antibody production. The in vitro effects of the phospholipid on human PBMC and rat splenocytes might suggest that PS is implicated in T-cell activation through its action on IL-2 receptor.

Luteinizing hormone-releasing hormone-binding sites in the rat thymus: characteristics and biological function.

The present study was designed to explore the effects of LHRH and its agonists on immune system function. As a first step, to identify a putative site of action, the very potent and stable LHRH agonist (LHRH-A), [D-Ser(TBU6)] des-Gly10-LHRH ethylamide (buserelin), was used as an iodinated ligand to characterize LHRH receptors in a membrane preparation of rat thymus, a key organ of the immune system. The effects of LHRH and LHRH-A were then investigated on the proliferative capacity of rat thymocytes exposed in vitro to a mitogen and on ornithine decarboxylase specific activity. In addition, to determine whether LHRH-A treatment in vivo might directly influence thymic function, we treated hypophysectomized (hypox) rats with a moderately high dose of LHRH-A for a period of 2 weeks, and thymocyte mitogenic capacity, thymus weight, and the histological and functional appearance of the thymus were then assessed. Specific binding of LHRH-A to rat thymic membrane preparations is a saturable process, depending on both time and temperature of incubation, but differs markedly from binding to the rat pituitary or ovarian LHRH receptor in its low binding affinity. Binding is optimal in the absence of chelating agents (EDTA) or divalent metal ions, and increases linearly with increasing protein concentration. Binding is specific for LHRH, LHRH-A, and antagonists. Both the C-terminal amide and N-terminal regions of the LHRH molecule were required for binding, and amino acid substitutions at position 6 markedly enhanced and at position 8 markedly reduced binding potencies in rat thymic tissue. A number of peptides, proteins, and other agents had no effect on the specific binding of LHRH-A to thymic membrane preparations. The binding affinity (Ka) of the membrane receptor of the rat thymus for the LHRH superagonist buserelin was 8.4 x 10(8) M-1, while a higher binding affinity (Ka = 2.8 x 10(9) M-1) was calculated for the ovarian LHRH-binding site. Preincubation of rat thymocytes with LHRH-A for 20 h induced a significant dose-dependent increase in the proliferative response to the mitogen Concanavalin-A, monitored by [3H]thymidine incorporation. Using native LHRH, it was also possible to elicit stimulatory effects on the same parameter, although much higher concentrations were required than with LHRH-A. Furthermore, simultaneous addition of a LHRH antagonist, abolished the LHRH effect on thymocytes. Ornithine decarboxylase specific activity under lectin stimulation was also significantly increased by LHRH-A in cultures of rat thymocytes.(ABSTRACT TRUNCATED AT 400 WORDS)