Krzysztof Lyson

The effect of interleukin-6 on pituitary hormone release in vivo and in vitro

Intravenously administered interleukin-6 (IL-6), a monokine produced by activated monocytes and folliculostellate cells of the pituitary gland, has been recently reported to elevate plasma ACTH level and to stimulate PRL, GH and LH release from cultured pituitary cells. To determine the site(s) of action of IL-6 in the control of pituitary hormone release, we injected human recombinant IL-6 into the third brain ventricle (3V) of freely moving, conscious male rats. Both 0.05 and 0.25 pmol doses of IL-6 were ineffective to change plasma ACTH in comparison to the values in controls. The maximal IL-6 dose tested of 1.25 pmol increased plasma ACTH within 15 min and the response lasted over 180 min. Plasma TSH levels were significantly lowered by a dose of 0.25 pmol IL-6, but neither the lower dose of 0.05 pmol nor the higher dose of 1.25 pmol altered plasma TSH levels throughout the 180 min of the experiment. Plasma PRL and GH levels were not changed by any IL-6 dose tested. In ovariectomized rats plasma LH and FSH levels were also unaltered by IL-6. The effects of IL-6 on plasma ACTH and TSH were only partially paralleled by increased rectal temperature which suggests that hypothalamic temperature regulating centers were independent of these actions. To evaluate a possible direct effect on the pituitary, IL-6 was incubated in vitro with hemipituitaries under an atmosphere of 95% O2/5% CO2. After 1 h of incubation IL-6 failed to cause any change in the secretion of pituitary hormones throughout a concentration range of 10(-15)-10(-9) M.(ABSTRACT TRUNCATED AT 250 WORDS)

An Interleukin-1-Alpha-Like Neuronal System in the Preoptic-Hypothalamic Region and Its Induction by Bacterial Lipopolysaccharide in Concentrations Which Alter Pituitary Hormone Release

We studied the effect of intravenous injection of lipopolysaccharide (LPS) (30-250 micrograms) on the release of several anterior pituitary hormones as indicated by changes in their concentrations in plasma. Within 30 min after intravenous injection of LPS there was a dose-related stimulation of ACTH release; prolactin (PRL) release was induced only by the highest LPS dose injected (250 micrograms). Even the lowest dose of LPS (30 micrograms) decreased plasma growth hormone (GH) by 60 min. Higher doses lowered plasma GH by 30 min, but thyroid-stimulating hormone release was only significantly inhibited by the highest dose of LPS. The action of LPS seems to be primarily exerted on the central nervous system, since incubation of hemipituitaries with LPS for 3 h in doses ranging from 0.001 to 10 micrograms/ml had no effect on ACTH release. LPS is thought to induce its effects on hormones either by release of cytokines from immune cells which subsequently induce the hormonal changes or possibly by direct action within the hypothalamus. In this report we demonstrate the immunocytochemical localization of a population of interleukin-1 alpha (IL-1 alpha)-like cells in a region extending from the basal forebrain at the level of the diagonal band of Broca, caudally and dorsally to the dorsolateral preoptic region and the hypothalamus at the level of the paraventricular nucleus. Further caudally, IL-1 alpha-like immunoreactive cells were located in the midportion of the amygdala. Two hours after injection of the 125-micrograms dose of LPS, the number of these immunoreactive cells was dramatically increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of Arachidonic Acid Cascade Pathways in lnterleukin-6-Stimulated Corticotropin-Releasing Factor Release in vitro

We have demonstrated that centrally administered interleukin-6 (IL-6) stimulates adrenocorticotropin (ACTH) secretion by a direct effect on corticotropin-releasing factor (CRF) release from the hypothalamus. Since metabolites of the arachidonic acid cascade (AAC) have been implicated in mediating actions of cytokines in different tissues and some AAC inhibitors were able to block pyrogenic effects of cytokines and suppress IL-1-induced ACTH secretion, we decided to examine the mechanism of IL-6 action on CRF release in vitro. After a 60-min preincubation in Krebs-Ringer bicarbonate buffer, medial basal hypothalami (MBH) were preincubated for 30 min with dexamethasone (DEX), a phospholipase A2 (PLA2) inhibitor, to block arachidonic acid (AA) formation, or with inhibitors of AA metabolism: a cyclooxygenase inhibitor--indomethacin (IND); a lipoxygenase inhibitor--5,8,11-eicosatriynoic acid (ETI), and an epoxygenase inhibitor--clotrimazole (CLO). Then, the medium was discarded and MBH were incubated with medium or the above compounds and/or IL-6 for 30 min, and CRF release into the incubation medium was measured by radioimmunoassay. As reported previously, 10(-13) M IL-6 increased CRF release, which was significantly suppressed by DEX in a dose-dependent manner. The suppression was already highly significant at a concentration of 10(-11) M DEX and became maximal at 10(-7) M, at which concentration CRF release was no longer stimulated by IL-6. The response to IL-6 was completely blocked at the highest DEX concentration evaluated (10(-5) M). CLO also suppressed IL-6-induced CRF release with a minimal effective dose of 10(-9) M. Suppression was complete at 10(-7) and 10(-5) M.(ABSTRACT TRUNCATED AT 250 WORDS)

Blockade by lnterleukin-1-Alpha of Nitricoxidergic Control of Luteinizing Hormone-Releasing Hormone Release in vivo and in vitro

Nitric oxide (NO) synthase (NOS), the enzyme that converts arginine into citrulline plus NO, the latter a highly active free radical, occurs in a large number of neurons in the brain, including certain neurons in the hypothalamus. Our previous experiments have shown that norepinephrine (NE)-induced prostaglandin E2 (PGE2) release from medial basal hypothalamic explants (MBH) is mediated by NO. Because release of luteinizing hormone (LH)-releasing hormone (LHRH) is also driven by NE and PGE2, we hypothesized that NO controls pulsatile release of LHRH in vivo, which in turn induces pulsatile LH release. Indeed, in vivo and in vitro experiments using an inhibitor of NOS (NG-monomethyl-L-arginine; NMMA) demonstrated that pulsatile LH release is mediated by NO; LHRH release in vitro is also mediated by this free radical. Cytokines that are released from cells of the immune system during infection also inhibit LHRH release. We compared the action of one such cytokine, interleukin-1 alpha (IL-1 alpha), on LHRH release with that of substances which inhibit or induce NO release. Microinjection of IL-1 alpha (0.06 pmol in 2 microliters) into the third cerebral ventricle (3V) of conscious, castrated male rats had an action similar to that of 3V microinjection of NMMA (1 mg in 5 microliters): it blocked pulsatile LH, but not follicle-stimulating hormone (FSH) release. The only difference between the responses to NMMA and IL-1 alpha was that the latency to onset was greater with IL-1 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Alpha-melanocyte-stimulating hormone abolishes IL-1-and IL-6-induced corticotropin-releasing factor release from the hypothalamus in vitro

Alpha-melanocyte-stimulating hormone (alpha-MSH) and adrenocorticotropic hormone (ACTH), peptides derived from the precursor proopiomelanocortin, share amino acid homology at the aminoterminus of ACTH, occur within the pituitary and the brain and are potent antipyretic compounds in cytokine-mediated fever. Because alpha-MSH and ACTH act within the hypothalamus to block leukocytic pyrogen- or cytokine-mediated fever, we hypothesized that these compounds might also be capable of blocking the action of interleukin-1 (IL-1) and interleukin-6 (IL-6) to stimulate corticotropin-releasing factor (CRF) release from the hypothalamus. Mediobasal hypothalami (MBH) were incubated in vitro. After 60 min preincubation in Krebs-Ringer bicarbonate buffer (KRB), MBH explants were incubated for 30 min with KRB alone or KRB containing IL-6 (10(-13) M), IL-1 (10(-16)-10(-10) M) and/or ACTH1-24 (10(-15)-10(-9) M) or alpha-MSH (10(-15)-10(-8) M); CRF release into the incubation medium was measured by RIA. None of the ACTH1-24 or alpha-MSH concentrations changed basal CRF release significantly. As we reported previously, IL-6 (10(-13) M) increased CRF release; this increase was suppressed, in a dose-dependent fashion, by alpha-MSH at concentrations of 10(-13)-10(-11) M, with the maximal inhibitory effect observed at 10(-13) M. ACTH1-24 also exerted a dose-dependent inhibitory effect on IL-6-stimulated CRF release but at even lower concentrations (10(-15)-10(-13) M) with the maximal inhibitory effect observed with the 10(-14) M concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Luteinizing-Hormone-Releasing Hormone, α-Melanocyte-Stimulating Hormone, Naloxone, Dexamethasone and Indomethacin on lnterleukin-2-lnduced Corticotropin-Releasing Factor Release

Our previous studies have shown that the microinjection of interleukin (IL)-2 into the third ventricle of conscious rats evokes the release of adrenocorticotropin hormone (ACTH) and that its incubation with hemipituitaries in vitro was also effective in releasing ACTH. In the present experiments, we evaluated the effect of IL-2 on the release of corticotropin-releasing factor (CRF) from medial basal hypothalami (MBHs) incubated in vitro and studied the effect of other agents, whose release is altered in stress, on CRF release. IL-2 significantly stimulated CRF release at concentrations of 10(-13) and 10(-14) M, whereas increasing the concentration to 10(-12) to 10(-10) M did not produce significant release of CRF. A high concentration of potassium (55 mM) in the medium also significantly stimulated CRF release and this stimulation was not modified by IL-2. Since high-potassium-induced release of CRF is probably due to opening of voltage-dependent calcium channels, it is likely that IL-2 is releasing CRF by this mechanism. Since the release of luteinizing-hormone-releasing hormone (LHRH) is modified by stress, we evaluated the action of LHRH on CRF release and the release induced by IL-2. Although LHRH failed to alter basal CRF release, except for a slight decrease at 10(-7) M, it completely blocked IL-2-induced CRF release at this concentration. To examine a possible role for opioid peptides in CRF release, the opiate receptor blocker, naloxone (NAL), was tested. At concentrations of 5 x 10(-6) and 10(-5) M, it produced a marked increase in CRF release; however, the simultaneous exposure of MBHs to each of these concentrations of NAL plus IL-2 caused a dose-dependent decrease in IL-2-induced CRF release, suggesting that beta-endorphin or other opioid peptides may play a role in IL-2-induced CRF release. As has been previously shown for IL-1 and IL-6, IL-2-induced CRF release was blocked by alpha-melanocyte-stimulating hormone (alpha-MSH), which at high concentrations also reduced basal CRF release. As in the case of IL-1 and IL-2, dexamethasone (DEX), the highly active synthetic glucocorticoid, although not altering basal CRF release, completely blocked the response to IL-2. The inhibitor of cyclooxygenase, indomethacin (IND), also blocked IL-2-induced CRF release just as it has previously been shown to block IL-1- and IL-6-induced CRF release. The results are consistent with the hypothesis that IL-2 acts on its recently discovered receptors to induce an increase in intracellular calcium. In other experiments, we have shown that this activates nitric oxide (NO) synthase leading to production of NO by a NOergic neuron. NO diffuses to the CRF neuron and activates cyclo-oxygenase leading to generation of prostaglandin E2, which activates adenylate cyclase and increases cyclic AMP release, which then causes extrusion of CRF secretory granules. DEX presumably acts on its receptors on the CRF neuron to inhibit the increase in intracellular calcium and thereby blocks activation of phospholipase A2 necessary for activation of the arachidonic acid cascade. alpha-MSH and LHRH may similarly act on their receptors on these cells to, in some manner, block the pathway. On the other hand, beta-endorphin and/or other opioid peptides inhibit the pathway. Further experiments will be necessary to elucidate the exact points in the pathway at which these compounds are effective.

Binding of Anti-Inflammatoryα-Melanocyte-Stimulating-Hormone Peptides and Proinflammatory Cytokines to Receptors on Melanoma Cells

alpha-Melanocyte-stimulating hormone (alpha-MSH1-13), a peptide derived from proopiomelanocortin, has remarkable anti-inflammatory and antipyretic activities. This peptide and a tripeptide that forms the COOH-terminal portion of the molecule (alpha-MSH11-13; Lys Pro Val) inhibit inflammation when given centrally or peripherally. Because of the similarity in their actions, the tripeptide has been presumed to be the amino acid message sequence underlying the effects of alpha-MSH1-13. To test the possibility that the two peptides occupy the same receptors, competitive binding experiments were performed with B16 mouse melanoma cells that are known to have alpha-MSH1-13 receptors. In these experiments, alpha-MSH11-13 did not inhibit binding of a radiolabelled alpha-MSH1-13 analog. This finding suggests that alpha-MSH1-13 and alpha-MSH11-13 exert their anti-inflammatory/antipyretic/anticytokine effects via stimulation of separate receptors. Because alpha-MSH inhibits the effects of several cytokines including inflammation caused by interleukin (IL)-6 and IL-8, the capacity of these cytokines to compete for alpha-MSH binding sites was tested. There was no evidence that these proinflammatory cytokines bind to alpha-MSH receptors on murine melanoma cells. Although further tests with host cells involved in inflammation are required, the latter result is the first evidence that the mechanism of anticytokine action of alpha-MSH does not depend upon peptide/cytokine competition for binding sites.

Effect of Thymosin α1 on Hypothalamic Hormone Release

Thymosin α1 (Tα1) is a well-characterized immunopotentiating polypeptide originally isolated from calf thymus. We have recently shown in vivo, probable hypothalamic effects of Tα1 to decrease the release of the pituitary hormones, TSH, PRL and ACTH from the pituitary gland. Therefore, in the present study we evaluated the effect of the peptide on the release of hypothalamic regulatory hormones: thyrotropin-releasing hormone (TRH) and corticotropin-releasing hormone (CRH), as well as somatostatin (SRIH), from medial basal hypothalamic (MBH) fragments incubated in vitro. After a preliminary time-course study indicated that a 30-min incubation period was optimal, it was used for all the other experiments. At the end of the incubation the tissue was still able to respond to a depolarizing K+ concentration for 15 min by a 4-fold increase of TRH concentration compared to control basal release during the preceding 30 min. Tα1 was shown to inhibit the release of TRH and CRH from MBH fragments incubated in vitro with a minimal effective dose (MED) of 10-9M. SRIH and CRH release was also inhibited but the MED for these peptides was 10∼9M. The relative responsiveness to the action of Tα1 was TRH greater than CRH, which was greater than SRIH. This correlated with our previous in vivo results for pituitary hormone release, except in the case of SRIH since we previously did not detect any significant effect of the peptide on growth hormone release. Finally, we evaluated the possible involvement of other neurotransmitters in the effect of Tα1 on TRH release. A serotonin receptor blocker, metergoline, which blocked the inhibitory effect of serotonin on TRH release, also prevented the inhibitory effect of Tα1 on the release of TRH. These results suggest the possible involvement of a serotonergic mechanism in the inhibitory effect of Tα1 on TRH release.

Endocrine Aspects of Neuroimmunomodulation: Methods and Overview

This article presents methods used to evaluate the actions of cytokines at the hypothalamic and pituitary level and then summarizes the current status of our knowledge of their actions on the hypothalamic–pituitary unit. This field has developed explosively in the last few years, following the discovery of the structure of these small proteins produced by the immune system.

The effect of nerve growth factor on DNA synthesis, cyclic AMP and cyclic GMP accumulation by mouse spleen lymphocytes

Nerve growth factor (NGF), a trophic neuropeptide, is known to stimulate development, and to be important in the maintenance and survival of sympathetic and sensory neurons. Considering the presence of specific receptors on the surface of spleen cells, the effect of 2.5s nerve growth factor on 3H-thymidine uptake, cAMP and cGMP accumulation in mouse spleen lymphocytes has been studied. It was found that NGF added in vitro at the concentrations between 4 x 10(-7) and 4 x 10(-8) M significantly inhibited the incorporation of 3H-thymidine into lymphocytes DNA and increased cAMP levels in a dose-dependent manner but had no effect on cGMP levels. The maximal stimulation of cAMP synthesis occurred between 5 and 30 min after the NGF addition to the culture medium. When NGF was administered in vivo a significant dose-dependent inhibition of the lymphocytes proliferation was observed. These results indicate that an early increase of cAMP concentration is responsible for the antiproliferative action of NGF on mouse spleen lymphocytes and suggest that NGF could play an important role in the regulation of immune system function.