Endotoxin induced increases in rat plasma pituitary-adrenocortical hormones are better reflected by alterations in tumor necrosis factorα than interleukin-1β

In order to assess the relative cytokine contribution to endotoxin stimulation of pituitary-adrenocortical hormone secretion, we measured plasma levels of interleukin-1β (IL-Iβ), tumor necrosis factorα (TNFα), adrenocorticotropin (ACTH) and corticosterone following lipopolysaccharide (LPS) challenge in rats. LPS administration induced robust increases in both plasma ACTH and corticosterone levels at 3 h after i.p. injection; while ACTH decreased towards control levels, corticosterone remained at peak concentrations at 6 h after LPS injection. Basal levels of plasma IL-1β were below the sensitivity of the ELISA and basal levels of plasma TNFα were 0.25 ± 0.12 pM. Small but highly variable non-significant increases in plasma IL-1β levels were seen at 3 h and 6 h after injection of LPS. The lack of functional consequences of the small increases in IL-1β levels was demonstrated by unchanged levels of [¹²⁵I]IL-1α binding in liver at 3 h after LPS injection. In contrast, dramatic increases in plasma TNFα concentrations were observed at 3 h and decreased to non-injected control levels at 6 h after LPS injection. There was a significant positive correlation between ACTH and TNFα after LPS injection, while no correlation was seen between ACTH and IL-1β. These data demonstrate differential regulation of IL-1β and TNFα by endotoxin treatment and suggest that TNFα may be a more potent mediator of LPS-induced ACTH secretion in rat.     

A possible role for nitric oxide but not for prostaglanoin E2 in basal and interleukin-1-β-induced prl release in vitro

In previous experiments we have shown that nitric oxide (NO) was able to modulate CRH and ACTH release from cultured rat hypothalamic and anterior pituitary cells, in vitro. Now, we show experimental evidence of an involvement of NO in basal and interleukin-1β-induced prolactin (PRL) release. L-N^G-nitroarginine, an inhibitor of nitric oxide synthetase and hemoglobin, a NO scavenger, impaired basal and interleukin-1-β-induced PRL release, while molsidomine, a NO donor, was able to release PRL and to amplify interleukin-1-β-induced PRL release, confirming a modulatory role for nitric oxide in pituitary hormone secretion. On the other hand, no evidence regarding a possible role of prostaglandin E₂ (PGE₂) in IL-1β-induced PRL release came out from our experiments.     

Interleukin-1β specifically stimulates nitric oxide production in the hypothalamus

In previous experiments we have shown the role of nitric oxide (NO) in basal and interleukin-1β (IL-1β)-induced CRH and ACTH release in vitro. Now, we have studied the possible production of NO from hypothalamic cell cultures, particularly after IL-1β stimulation or L-NOArg inhibition, by high performance liquid Chromatographie (HPLC) assay of L-citrulline production, adding further evidence for a role of NO in IL-1β activity in the hypothalamus.     

Neuropeptide Y modulation of interleukin-1{beta} (IL-1{beta})-induced nitric oxide production in microglia

Given the modulatory role of neuropeptide Y (NPY) in the immune system, we investigated the effect of NPY on the production of NO and IL-1β in microglia. Upon LPS stimulation, NPY treatment inhibited NO production as well as the expression of inducible nitric-oxide synthase (iNOS). Pharmacological studies with a selective Y(1) receptor agonist and selective antagonists for Y(1), Y(2), and Y(5) receptors demonstrated that inhibition of NO production and iNOS expression was mediated exclusively through Y(1) receptor activation. Microglial cells stimulated with LPS and ATP responded with a massive release of IL-1β, as measured by ELISA. NPY inhibited this effect, suggesting that it can strongly impair the release of IL-1β. Furthermore, we observed that IL-1β stimulation induced NO production and that the use of a selective IL-1 receptor antagonist prevented NO production upon LPS stimulation. Moreover, NPY acting through Y(1) receptor inhibited LPS-stimulated release of IL-1β, inhibiting NO synthesis. IL-1β activation of NF-κB was inhibited by NPY treatment, as observed by confocal microscopy and Western blotting analysis of nuclear translocation of NF-κB p65 subunit, leading to the decrease of NO synthesis. Our results showed that upon LPS challenge, microglial cells release IL-1β, promoting the production of NO through a NF-κB-dependent pathway. Also, NPY was able to strongly inhibit NO synthesis through Y(1) receptor activation, which prevents IL-1β release and thus inhibits nuclear translocation of NF-κB. The role of NPY in key inflammatory events may contribute to unravel novel gateways to modulate inflammation associated with brain pathology.     

Effects of lipopolysaccharide on neurokinin A content and release in the hypothalamic-pituitary axis

The administration of bacterial lipopolysaccharide (LPS) markedly affects pituitary secretion, and its effects are probably mediated by cytokines produced by immune cells or by the hypothalamo-pituitary axis itself. Since neurokinin A (NKA) plays a role in inflammatory responses and is involved in the control of prolactin secretion, we examined the in vivo effect of LPS on the concentration of NKA in hypothalamus and pituitary (assessed by RIA) and serum prolactin levels in male rats. One hour after the intraperitoneal administration of LPS (250 microg/rat), NKA content was decreased in the posterior pituitary but not in the hypothalamus or anterior pituitary. Three hours after injection, LPS decreased NKA concentration in the hypothalamus and anterior and posterior pituitary. In all the conditions tested, LPS significantly decreased serum prolactin. We also examined the in vitro effects of LPS (10 microg/ml), interleukin-6 (IL-6, 10 ng/ml) and tumor necrosis factor alpha (TNF-alpha, 50 ng/ml) on hypothalamic NKA release. Interleukin-6 increased NKA release without modifying hypothalamic NKA concentration, whereas neither LPS nor TNF-alpha affected them. Our results suggest that IL-6 may be involved in the increase of hypothalamic NKA release induced by LPS. NKA could participate in neuroendocrine responses to endotoxin challenge.     

Interleukin-1 potentiates basal and AVP-stimulated ACTH secretion in vitro--the role of CRH pre-incubation.

The acute-phase cytokine interleukin-1 (IL-1) is known to activate the hypothalamic pituitary adrenal axis, primarily via corticotropin releasing hormone (CRH). The aim of this study was to determine whether IL-1beta could directly stimulate ACTH secretion from perifused equine anterior pituitary cells, and whether CRH pre-incubation affected corticotroph responsiveness. Isolated equine anterior pituitary cells were pre-incubated with media containing 10 nM CRH or vehicle for 20 hours before being loaded onto columns and perifused with 0.02 nM CRH and 100 nM cortisol. Columns were given a 5-minute pulse of arginine vasopressin (AVP, 10 nM), perifused for 4 hours with 0 (control) or 1 nM IL-1beta, then given a further 5-minute pulse of AVP (10nM). ACTH was measured in 5 minute fractions. In the setting of CRH pre-incubation, cells perifused with IL-1beta for 4 hours showed increased basal ACTH secretion compared to control (114 +/- 6 pM vs. 86 +/- 4 pM [means +/- S.E.M.], p < 0.001) and a significantly greater ACTH response to the final AVP pulse (240 +/- 32% vs. 96 +/- 30%, p = 0.009, expressed as % of ACTH response to the initial AVP pulse). The potentiation of AVP-stimulated ACTH release by IL-1 was not observed in cells pre-incubated with vehicle alone. In conclusion, IL-1 increases ACTH release in equine corticotroph cells pre-incubated with CRH and potentiates responsivity to AVP.     

CHRONIC STIMULATION OF THE HYPOTHALAMUS–PITUITARY–ADRENAL AXIS IN RATS BY INTERLEUKIN 1β: CENTRAL AND PERIPHERAL MECHANISMS

The authors have studied mechanisms which could be involved in the sustained activation of the hypothalamus–pituitary–adrenal (HPA) axis during continuous infusion of rats with recombinant human interleukin-1β (IL-1β). First, the effects of 3 days of intracerebroventricular (i.c.v.) infusion of rats with IL-1 on plasma adrenocorticotropin (ACTH) and corticosterone (B) levels were investigated. Thereafter, changes in plasma ACTH and B levels were followed in rats intraperitoneally (i.p.) infused with IL-1β after immunoneutralization of corticotropin-releasing hormone (CRH), hypophysectomy (HPX), macrophage depletion using dichloromethylene diphosphonate (Cl₂MDP)-containing liposomes, adrenalectomy (ADX) and dexamethasone (DEX) administration, respectively. Infusion of IL-1β i.c.v., even in doses as low as 0.1 μg/day, induced significant increases in plasma ACTH and B levels. HPX and ADX rats died within 18 h after starting the IL-1β infusion (0.5 μg/day). Immunoneutralization of CRH significantly decreased and macrophage depletion significantly increased the stimulation of the HPA axis by IL-1 (4.0 μg/day). Administration of high doses of DEX completely abolished the stimulation of the HPA axis by IL-1β (2.0 μg/day). The present study demonstrates that lower doses of IL-1β were able to activate the HPA axis when infused i.c.v. compared with i.p. Regarding stimulation of the HPA axis by chronic i.p. infusion of IL-1β the present study: (1) provides evidence that the CRH system is involved; (2) provides no evidence for a direct stimulatory effect of IL-1β on the release of B by the adrenal gland which is of sufficient magnitude to resist the stress of chronic i.p. IL-1β infusion; (3) shows that endogenous macrophage-derived mediators, induced by i.p. IL-1β infusion, express an overall inhibitory rather than a stimulatory effect on the activity of the HPA axis; (4) demonstrates that exogenous administration of DEX blocks the effect of IL-1β, which fits well in the concept of an immunoregulatory feedback between IL-1β and glucocorticoids.     

Vasopressin pressor receptor-mediated activation of HPA axis by acute ethanol stress in rats

The plasma arginine vasopressin (AVP), ACTH, and corticosterone levels and the hypothalamic corticotropin-releasing hormone (CRH) content were measured after oral administration of 1 ml of 75% ethanol to rats, a model known to induce acute gastric erosions and stress. Elevated plasma AVP, ACTH, and corticosterone levels were detected 1 h after ethanol administration. Treatment with the vasopressin pressor (V(1)) receptor antagonist [d(CH(2))(5)Tyr(Me)-AVP] before ethanol administration significantly reduced the ACTH and corticosterone level increases. A higher hypothalamic CRH content was measured at 30 or 60 min after ethanol administration. V(1) receptor antagonist injection, 5 min before ethanol administration, inhibited the rise in hypothalamic CRH content. The protein synthesis blocker cycloheximide prevented the hypothalamic CRH content elevation after stress. The AVP-, CRH-, and AVP + CRH-induced in vitro ACTH release in normal anterior pituitary tissue cultures was also prevented by pretreatment with the V(1) receptor antagonist. The results support the hypothesis that stress-induced AVP may not only act directly on the ACTH producing anterior pituitary cells but also indirectly at the hypothalamic level via the synthesis and release of CRH.     

Role of glucose in interleukin-1β production by lipopolysaccharide-activated human monocytes

When monocytes are activated with endotoxin (lipopolysaccharide [LPS]), they make and release several mediators, including interleukin-1β (IL-1β). This study was undertaken to investigate the role of glucose in IL-1β production by these cells. IL-1β was produced in a dose-dependent manner to glucose concentration in the culture medium. The uptake of (³H)2-deoxyglucose in monocytes was stimulated by LPS 1,554% after 10 minutes, 6,095% after 2 hours, then gradually declined after 4 hours of incubation. The inhibition of the uptake of (³H)2-deoxyglucose by either 10 μM cytochaiasin B or phloretin, added at the time of monocyte activation, was accompanied by significant reduction in ATP/ADP ratio and the inhibition of the production of IL-1β by activated monocytes. The synthesis of total protein did not change in monocytes activated in the absence of glucose in the culture medium, nor in the presence of either 10 μM cytochalasin B or phloretin. The export of IL-1β from LPS-activated monocytes was not inhibited by either 10 μM cytochalasin B or phloretin, nor in the absence of glucose in the culture medium. These data suggest that (1) glucose is required for LPS-induced IL-1β production by monocytes; (2) glucose is the major source of ATP for IL-1β production; (3) glucose transporter (GLUT 1) does not control the export of IL-1β. © 1993 Wiley-Liss, Inc.     

Growth hormone-releasing peptide-2 stimulates secretion and synthesis of adrenocorticotropic hormone in mouse pituitary

Growth hormone (GH)-releasing peptides (GHRPs) are synthetic peptides which induce strong GH release in both animals and humans. Among them, GHRP-2 is known to stimulate GH release by acting at both hypothalamic and pituitary sites, but also induces adrenocorticotropic hormone (ACTH) release in healthy subjects. GHRP-2 may stimulate ACTH release directly via GHRP receptor type 1a in ACTH-producing tumors. GHRP-2 increases ACTH secretion in rat in vivo, but not ACTH release from rat primary pituitary cells. In the present study, in order to elucidate the mechanism underlying ACTH secretion by GHRPs, mouse pituitary cells were stimulated by GHRP-2. GHRP receptor mRNA was expressed in the mouse pituitary, and GHRP-2 directly stimulated secretion and synthesis of ACTH in the mouse anterior pituitary cells. GHRP-2 increased intracellular cyclic AMP production. H89, a potent protein kinase A (PKA) inhibitor, and bisindolylmaleimide I, a selective protein kinase C (PKC) inhibitor, inhibited the GHRP-2-induced ACTH release, and that H89, but not bisindolylmaleimide I, inhibited the GHRP-2-induced proopiomelanocortin mRNA levels. Together, the GHRP-2-induced ACTH release was regulated via both PKA and PKC pathways in the mouse pituitary cells, while ACTH was synthesized by GHRP-2 only via the PKA pathway.     

Interleukin-1beta and tumor necrosis factor-alpha mediation of endotoxin action on growth hormone

In humans and sheep, endotoxin (LPS) administration results in increased growth hormone (GH) concentrations. To determine the role of cytokines in the effect of LPS on GH, sheep were challenged with IL-1beta or TNF-alpha. GH data were compared with results with LH, where the major effects of LPS are known to act via the hypothalamus. Intracerebroventricular (icv) administration of IL-1beta or TNF-alpha did not alter plasma concentrations of GH. Endotoxin was then administered intravenously (iv) in combination with icv injection of IL-1 receptor antagonist (IL-1RA), TNF antagonist (sTNF-R1), or saline. Administration of LPS increased GH (P < 0.0001), although coadministration of IL-1ra or sTNF-R1 icv did not alter GH response to LPS. In contrast, plasma concentrations of LH were profoundly inhibited by icv administration of either cytokine (P < 0.03), but the LH response to LPS was not altered by cytokine antagonists. Intravenous administration of either IL-1beta or TNF-alpha increased plasma concentrations of GH (P < 0.0001). Administration of IL-1RA and sTNF-R1 iv prevented LPS-induced increases in GH. Although LH was suppressed by high iv doses of IL-1beta (P = 0.0063), the antagonists did not alter the LH response to LPS. To determine whether LPS might directly activate GH release, confocal microscopy revealed colocalization of CD14, the LPS receptor, with GH and, to a lesser extent, LH and some prolactin (PRL)-containing cells, but not ACTH or TSH. These data are consistent with the effects of LPS on GH secretion originating through peripheral cytokine presentation to the pituitary, as well as a potential to act directly on selective populations of pituitary cells via CD14.     

Effect of dexamethasone on the release of pituitary hormones in monolayer cultures of rat pituitary cells

The effect of dexamethasone on the release of ACTH, GH, PRL, LH and TSH was studied in monolayer cultures of rat pituitary cells in 4-hour incubation. With or without the addition of rat hypothalamic extract, the release of GH was significantly inhibited by dexamethasone at concentrations higher than 10(-9) M, although less remarkably than that of ACTH. Intracellular ACTH and GH were unchanged. PRL, LH and TSH were not affected. These results indicate that dexamethasone, when exerted for 4 hours, suppressed the release of GH as well as ACTH, at least in part, at the pituitary level.     

Unmasking of LPA1 receptor-mediated migration response to lysophosphatidic acid by interleukin-1β-induced attenuation of Rho signaling pathways in rat astrocytes

Action mechanism of lipopolysaccharide (LPS), interleukin-1β (IL-1β), and lysophosphatidic acid (LPA) to regulate motility, an important process of astrogliosis, was investigated in rat astrocytes. While LPA exerted no significant effect on the cell migration, the prior treatment of the cells with LPS or IL-1β resulted in the appearance of migration activity in response to LPA. The LPS induction of the migration response to LPA was associated with the production of IL-1β precursor protein and inhibited by the IL-1 receptor antagonist. The IL-1β treatment also allowed LPA to activate Rac1. The LPA-induced Rac1 activation and migration were inhibited by pertussis toxin, a small interfering RNA specific to LPA(1) receptors, and LPA(1) receptor antagonists, including Ki16425. However, the IL-1β treatment had no appreciable effect on LPA(1) receptor mRNA expression and LPA-induced activation of ERK, Akt, and proliferation. The induction of the migration response to LPA by IL-1β was inhibited by a constitutively active RhoA. Moreover, LPA significantly activated RhoA through the LPA(1) receptor in the control cells but not in the IL-1β-treated cells. These results suggest that IL-1β inhibits the LPA(1) receptor-mediated Rho signaling through the IL-1 receptor, thereby disclosing the LPA(1) receptor-mediated G(i) protein/Rac/migration pathway.     

Site of action of 5-hydroxytryptophan and 5-hydroxytryptamine on the hypothalamo-pituitary-adrenal system in vitro.

5-hydroxytryptamine at a concentration of 0.04 microgram/ml was able to block the ACTH release caused by hypothalamic tissue (CRF) while it was ineffective when a hypothalamic extract containing CRF was used. 5-hydroxytryptophan added to rat adrenal tissue caused a dose-dependent increase in corticosterone production. In a dose of 0.04 microgram/ml, 0.4 microgram/ml and 4.0 microgram/ml, 5-hydroxytryptophan was able to inhibit the ACTH release caused by hypothalamic tissue in vitro. However 0.04 microgram/ml was ineffective on the increase in ACTH secretion elicited by hypothalamic extract CRF. The data suggest that the inhibitory action of 5-hydroxytryptophan and 5-hydroxytryptamine is exerted at the hypothalamic level by inhibiting the release and/or synthesis of the corticotrophic releasing factor.     

The effect of serotonin agonist 1-(trifluoromethylphenyl)-piperazine on corticotropin releasing factor and arginine vasopressin in rat hypothalamic nuclei

Effects of 1-(m-trifluoromethylphenyl)-piperazine, a serotonin agonist, were examined on rat plasma levels of adrenocorticotropin (ACTH) and arginine vasopressin (AVP), and on hypothalamic contents of corticotropin releasing factor (CRF) and AVP, to investigate the role of brain serotonin in ACTH regulation. Both plasma ACTH and AVP levels increased markedly 30 min after injection of the compound and were still elevated at 80 min. CRF and AVP contents in the median eminence decreased 30 min after injection but returned to the basal levels by 80 min. The AVP content in the supraoptic nucleus was elevated 80 min after injection. The CRF and aVP content did not significantly change in the paraventricular, suprachiasmatic and arcuate nuclei. Serotonin or 1-(m-trifluoromethylphenyl)-piperazine did not stimulate the release of ACTH in pituitary cell cultures. These results suggest that both CRF and AVP were secreted into the portal vessels by 1-(m-trifluoromethylphenyl)-piperazine to release ACTH from the anterior pituitary and that both the ACTH and AVP release were stimulated via the brain serotonergic mechanism.     

The effect of somatostatin and of prolyl-leucyl-glycinamide (MIF) on ACTH release in dispersed pituitary cells.

The hypothalamic releasing and release-inhibiting peptides have multiple effects on more than one pituitary hormone. In this study the action of the two hypothalamic inhibiting factors, somatostatin (GH-IH) and MSH release-inhibiting factor, prolyl-leucyl-glycinamide (MIF), on ACTH release were studied. Increasing concentrations of GH-IH and MIF were added to 1 ml of a suspension of dispersed anterior pituitary cells from male rats. Both GH-IH and MIF (10^?5 to 10^?11 M) were without effect on basal ACTH secretion of normal and of adrenalectomized rats. However, both peptides, within certain concentration ranges, inhibited the ACTH release stimulated by rat hypothalamic extracts or by arginine vasopressin. The most effective concentrations were 35 nM MIF or 6 nM GH-IH. Beyond these concentrations no further suppression was observed. Our results indicate that somatostatin and MIF can inhibit ACTH release, but only in a state of steroid deprivation and within a limited dose range.     

TRAF6 mediates IL-1β/LPS-induced suppression of TGF-β signaling through its interaction with the type III TGF-β receptor

Transforming growth factor-β1 (TGF-β1) is an important anti-inflammatory cytokine that modulates and resolves inflammatory responses. Recent studies have demonstrated that inflammation enhances neoplastic risk and potentiates tumor progression. In the evolution of cancer, pro-inflammatory cytokines such as IL-1β must overcome the anti-inflammatory effects of TGF-β to boost pro-inflammatory responses in epithelial cells. Here we show that IL-1β or Lipopolysaccharide (LPS) suppresses TGF-β-induced anti-inflammatory signaling in a NF-κB-independent manner. TRAF6, a key molecule in IL-1β signaling, mediates this suppressive effect through interaction with the type III TGF-β receptor (TβRIII), which is TGF-β-dependent and requires type I TGF-β receptor (TβRI) kinase activity. TβRI phosphorylates TβRIII at residue S829, which promotes the TRAF6/TβRIII interaction and consequent sequestration of TβRIII from the TβRII/TβRI complex. Our data indicate that IL-1β enhances the pro-inflammatory response by suppressing TGF-β signaling through TRAF6-mediated sequestration of TβRIII, which may be an important contributor to the early stages of tumor progression.