The regulation of CD95 (Fas) ligand expression in primary T cells: induction of promoter activation in CD95LP-Luc transgenic mice.

The interaction between CD95 (Fas) and CD95L (Fas ligand) initiates apoptosis in a variety of cell types. Although the regulation of CD95L expression on activated T cells is an area of intense study, knowledge related to the induction of CD95L promoter activity in primary T cells is lacking. In this report we describe the generation of a novel transgenic mouse strain, CD95LP-Luc, in which murine CD95L promoter sequence controls the expression of a luciferase reporter gene. We use these mice to illustrate several important findings related to transcriptional regulation of CD95L in primary T cells. We demonstrate that maximal CD95L promoter activity occurs only after prolonged T cell stimulation and requires costimulation through CD28. We provide evidence that thymocytes express CD95L/luciferase after strong TCR ligation and that inducible CD95L promoter activation is present, but unequal, in both Th1 and Th2 effector cells. We also illustrate that while agonist peptide presentation by APCs generates robust proliferation during a primary T cell response, the same stimulus induces only modest CD95L promoter activity. These results suggest alternate explanations for the well-characterized delay in CD95-mediated activation-induced cell death following initial ligation of the TCR.

Effects of cholinergic agonists and antagonists on interleukin-2-induced corticotropin-releasing hormone release from the mediobasal hypothalamus.

In previous research we found that interleukin-2 (IL-2)-induced corticotropin-releasing hormone (CRH) release in vitro is mediated by cholinergic activation of nitric oxidergic (NOergic) neurons. The NOergic neurons release nitric oxide that stimulates CRH release. To further characterize the mechanism of IL-2-induced CRH release, the possible role of nicotinic as well as muscarinic receptors in IL-2-stimulated CRH release was evaluated. Medial hypothalamic (MH) explants from adult male rats were preincubated in Krebs-Ringer (KRB) buffer for 45 min followed by incubation for an additional 30 min in fresh KRB or KRB containing various compounds. As previously reported, acetylcholine (ACH) stimulated CRH release in a dose-related fashion. IL-2 (10(-13) M) stimulation of CRH release was unaffected by the lower concentration of ACH (10(-9) M), but surprisingly was inhibited by a 100-fold higher concentration. Atropine (ATR) (10(-7) M) blocked CRH release induced by ACH (10(-7) M) and the release of CRH induced by IL-2. The cholinergic agonist carbachol (CAR) (10(-7) M) also released CRH and this action was blocked by ATR (10(-7) M). CRH release in the presence of CAR was lowered below basal when the concentration of ATR was increased to 10(-6) M. In contrast to ACH, CAR had an additive effect to release CRH when combined with IL-2 (10(-13) M). Nicotine (10(-7) M) also stimulated CRH release and this stimulation was completely blocked by 10(-6) M but not by 10(-7) M of the nicotinic receptor blocker, hexamethonium (HEX). The lower concentration of HEX blocked the stimulatory effect of ACH (10(-7) M) and IL-2 on CRH release. Combined blockade with ATR plus HEX completely blocked the action of ACH and even reduced the CRH concentration to below basal values. Furthermore, combined blockade completely blocked the release of CRH induced by IL-2. We conclude that nicotinic as well as muscarinic receptors play an important role in CRH release, and that they both act to mediate IL-2-stimulated CRH release.

Enhanced propagation of epileptiform activity through the kindled dentate gyrus.

Extracellular recordings were performed in combined hippocampal-entorhinal cortex (HC-EC) slices obtained from control and commissural kindled rats to investigate the propagation of epileptiform activity from the entorhinal cortex (EC) to the hippocampus (HC) after chronic epilepsy. Lowering extracellular Mg2+ concentration in control slices induced epileptiform activity consisting of spontaneous epileptiform bursts in area CA3 and of electrographic seizures in the EC. In contrast, the CA3 region of HC-EC slices obtained from kindled rats displayed significantly longer lasting epileptiform bursts and electrographic seizures. The electrographic seizures that were absent in controls propagated from the EC because disconnecting the HC from the EC stopped their occurrence in the CA3, whereas epileptiform bursts persisted with an unaltered pattern and frequency. Thus the area CA3 is affected by kindling and contributes to the spread of epileptiform activity within the EC-HC complex. We developed a method to induce focal epileptiform activity in the EC by locally perfusing the gamma-aminobutyric acid-A (GABA) antagonist bicuculline (50 mM) in 10 mM KCl containing artificial cerebrospinal fluid. This method enabled us to investigate the propagation of epileptiform discharges from the disinhibited EC to the DG without affecting the DG with the epileptogenic medium. We show here that kindling facilitates the propagation of epileptiform activity through the DG. These data are consistent with the normal function of the DG as a filter limiting the spread of epileptiform activity within the HC-EC complex. This gating mechanism breaks down after chronic epilepsy induced by kindling.

Effects of luteinizing-hormone-releasing hormone, alpha-melanocyte-stimulating hormone, naloxone, dexamethasone and indomethacin on interleukin-2-induced 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.

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)

Alpha-melanocyte-stimulating hormone inhibits corticotropin-releasing factor release by blocking protein kinase C.

Cytokine-induced release of corticotropin-releasing factor (CRF) from hypothalamic explants in vitro can be inhibited by femtomolar concentrations of alpha-melanocyte-stimulating hormone (alpha-MSH). Because the mechanism of the anticytokine action of alpha-MSH remains unknown, we examined if the peptide inhibits CRF release by interference with various steps in the activation of CRF release. Previous studies have shown that CRF release is induced by activation of phospholipase A2 (PLA2). Therefore, we examined the effect of alpha-MSH on the action of melittin (MEL), a PLA2 activator. After 60 min preincubation in Krebs-Ringer bicarbonate buffer, medial basal hypothalami were incubated for 30 min with Krebs-Ringer bicarbonate buffer or MEL with or without alpha-MSH (10(-11) to 10(-16) M). CRF release into the incubation medium was measured by RIA. As reported previously none of the alpha-MSH concentrations used changed basal CRF release nor did any concentration of alpha-MSH significantly alter CRF release induced by MEL (10 micrograms/ml). Thus, alpha-MSH alters cytokine-induced CRF release at a step unrelated to the activation of PLA2. Because activation of PLA2 requires an increase in intracellular calcium ion (Ca2+) concentrations, we evaluated the effect of alpha-MSH on the release of CRF induced by a high concentration of potassium (56 mM). This concentration of potassium induced a 3.5-fold increase in CRF release that was not affected by alpha-MSH. Protein kinase C (PKC) stimulates CRF release. Consequently, we examined the effect of alpha-MSH on CRF release induced by phorbol myristate acetate (PMA), which in the presence of Ca2+ stimulates PKC.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of anti-inflammatory alpha-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.

Cyclosporin A inhibits interleukin-2-induced release of corticotropin-releasing hormone.

Cyclosporin A (CSA), a potent immunosuppressive drug, has recently been shown to bind with high affinity to the immunophilin, cyclophilin. Calcineurin, the calcium-dependent protein phosphatase, binds the cyclophilin/CSA complex, rendering it inactive and blocking dephosphorylation of phosphoproteins. Very high concentrations of cyclophilin have been reported in the brain with a localization identical to that of calcineurin. We have reported that interleukin-2 (IL-2) releases corticotropin-releasing hormone (CRH) by generation of nitric oxide (NO). Nitric oxide synthase (NOS), the enzyme in nitric oxidergic neurons that converts arginine into citrulline plus NO, is inactive in the phosphorylated state. We hypothesized that cyclosporin might therefore inhibit IL-2-induced acute CRH release by blocking the dephosphorylation of NOS by calcineurin. Consequently, we examined the effect of CSA on the release of CRH from mediobasal hypothalami (MBH) in vitro in 'basal' conditions and in the presence of IL-2, which we had previously shown to stimulate CRH release acutely in this preparation. Incubation of MBH for 30 min with IL-2 (10(-13) M), the concentration that was most effective in previous experiments, evoked a significant release of CRH. CSA at 10(-6) or 10(-8) M did not alter basal release of CRH; however, addition of either concentration completely blocked the IL-2-induced release of CRH. This acute action of CSA within the brain is probably mediated by blockade of the dephosphorylation of NOS by calcineurin.

Blockade by interleukin-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)

Role of nitric oxide in interleukin 2-induced corticotropin-releasing factor release from incubated hypothalami.

Stimulation of corticotropin-releasing factor (CRF) release from the hypothalamus by interleukin 2 (IL-2) was recently demonstrated. Cytokines induce nitric oxide synthase (NOS), an enzyme that converts L-arginine into L-citrulline and nitric oxide (NO). NO is believed to be responsible for the cytotoxic action of these agents. The constitutive form of NOS occurs in neurons in the central nervous system and NO appears to play a neurotransmitter role in cerebellar and hippocampal function. We explored the probability that IL-2 and synaptic transmitters might release CRF via NO. The effects of L-arginine, the substrate for NOS, and NG-monomethyl-L-arginine (NMMA), a competitive inhibitor of NOS, on IL-2-induced CRF release were studied using mediobasal hypothalami (MBHs) incubated in vitro in Krebs-Ringer bicarbonate buffer. L-Arginine did not alter basal and IL-2-induced CRF release after 30 min of incubation but significantly elevated both basal and IL-2-induced CRF release when MBHs were incubated 30 min longer, presumably because the endogenous substrate had been depleted after the initial 30-min incubation period. In 30-min incubations, both carbachol, an acetylcholineomimetic drug, and norepinephrine stimulated CRF release. There was an additive effect of incubation of the MBHs in the presence of carbachol (10(-7) M) and IL-2 (10(-13) M). On the other hand, coincubation of MBHs with norepinephrine (10(-6) M) and IL-2 (10(-13) M) did not produce any additive effect. Addition of NMMA, an inhibitor of NOS, at 1 or 3 x 10(-4) M completely suppressed IL-2-induced release of CRF as well as that caused by IL-2 plus carbachol. In contrast, the release of CRF induced by norepinephrine was not blocked by 3 x 10(-4) M NMMA. The data indicate that IL-2 can activate constitutive NOS leading to increased NO release, which activates CRF release. It appears that NO is also involved in the release of CRF induced by carbachol but not by norepinephrine.

Nitric oxide mediates norepinephrine-induced prostaglandin E2 release from the hypothalamus.

Nitric oxide (NO), formed by conversion of arginine to citrulline and NO by NO synthase, mediates relaxation of vascular smooth muscle. NO synthase has been demonstrated by immunocytochemical methods in neurons in various parts of the central nervous system including the hypothalamus. The latter finding suggested to us that NO might play a role in controlling the release of hypothalamic peptides. We have previously shown that norepinephrine mediates the release of luteinizing hormone-releasing hormone (LHRH) from LHRH terminals in the median eminence into the hypophyseal portal veins, which transport LHRH to the anterior pituitary gland to trigger release of luteinizing hormone from gonadotrophs. LHRH release from these terminals requires increased release of prostaglandin E2 (PGE2). PGE2 activates adenylate cyclase to produce cAMP, and then cAMP induces the exocytosis of LHRH secretory granules. In view of the evidence above and because of the developing evidence for the importance of NO in the central nervous system, it occurred to us that NO might be involved in this process. Consequently, we evaluated the role of NO in the release of PGE2 from medial basal hypothalamic fragments. As previously reported, norepinephrine (10 microM) increased PGE2 release from the hypothalamic fragments. The inhibitor of NO synthase NG-monomethyl-L-arginine (NMMA, 300 microM) blocked the stimulation of PGE2 release induced by norepinephrine but had no effect on the basal release of PGE2. Sodium nitroprusside (100 microM), which liberates NO, also elevated PGE2 release from the hypothalamic fragments. This elevation was not affected by NMMA, presumably because NMMA blocks enzymatic generation of NO but does not alter NO liberated by nitroprusside. When the NO liberated by nitroprusside was inactivated by hemoglobin (2 micrograms/ml), the effect of nitroprusside on PGE2 release was completely inhibited. Neither NMMA nor hemoglobin altered the basal release of PGE2, which indicates that NO is not responsible for basal PGE2 release. Addition of L-arginine (10 microM to 1 mM), the substrate for NO synthase, had no effect on basal PGE2 production. These results indicate that NO synthase is not activated in unstimulated hypothalamic fragments in vitro. The results suggest that norepinephrine activates NO synthase leading to the production of NO, which subsequently activates cyclooxygenase and results in the production of PGE2. PGE2 then activates adenylate cyclase leading to generation of increased cAMP, which induces exocytosis of secretory granules of LHRH and other neuropeptides released by PGE2. The indication that NO is essential to norepinephrine-induced release of PGE2 from hypothalamic fragments provides insight into the mechanism of LHRH release and the results open the possibility that the importance of NO to neuronal functions may be widespread in the nervous system.

Effect of thymosin alpha 1 on hypothalamic hormone release.

Thymosin alpha 1 (T alpha 1) is a well-characterized immunopotentiating polypeptide originally isolated from calf thymus. We have recently shown in vivo, probable hypothalamic effects of T alpha 1 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. T alpha 1 was shown to inhibit the release of TRH and CRH from MBH fragments incubated in vitro with a minimal effective dose (MED) of 10(-11) M. SRIH and CRH release was also inhibited but the MED for these peptides was 10(-9) M. The relative responsiveness to the action of T alpha 1 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 T alpha 1 on TRH release.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of arachidonic acid cascade pathways in interleukin-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)

Induction of adrenocorticotropic hormone release by interleukin-6 in vivo and in vitro.

Recent reports show that cytokines such as interleukin-1 (IL-1), tumor necrosis factor (TNF) and intravenously administered interleukin-6 (IL-6) stimulate adrenocorticotropic hormone (ACTH) release. Both IL-1 and TNF are known to be potent inducers of IL-6, a monokine produced by activated monocytes and folliculo-stellate cells of the pituitary gland and released from the hypothalamus. To determine the site(s) of action of IL-6 in the control of ACTH release, we injected human recombinant IL-6 into the third brain ventricle (3V) of freely moving, conscious male rats and measured ACTH by RIA. Both 0.05 pmole and 0.25 pmole 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 pmole increased plasma ACTH within 15 min and the response lasted over 180 min. The effects of IL-6 on plasma ACTH 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 hr of incubation IL-6 failed to cause any change in the secretion of ACTH throughout a concentration range of 10(-15) to 10(-9) M. Increased ACTH secretion into the incubation medium was found only with 10(-13) M IL-6 after a 2-hr incubation. The results support a possible role for IL-6 at both hypothalamic and/or pituitary levels to stimulate ACTH release.

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)

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.