Central administration of IL-1 reduces anxiety and induces sickness behaviour in rats.

In the present study, we examined the effects of various doses of recombinant human interleukin-1beta on anxiety-like behaviour, on body temperature, and on behavioural changes typical of sick animals. First, we assessed the behaviour of rats in the elevated plus-maze before and 20 min after intracerebroventricular injection of IL-1 at six doses ranging from 0.001 to 100 ng. After treatment with 0.1 and 100 ng IL-1, animals exhibited different anxiety levels. The dose effect on behavioural performance in the plus-maze appears to be nonlinear (parabolic function), with the highest effects near a 0.1-ng dose and the lowest near doses of 0.0 and 100 ng. In a second set of experiments, we examined the effects of doses of 0.1 and 100 ng IL-1 (which had the most pronounced effects on performance in the plus-maze) on physical parameters over a 24-h period. Using radiotelemetry we measured body temperature, locomotor activity, food intake, and water consumption: a) in animals kept under basal resting conditions, and b) in animals exposed to a novel environment prior to administration of IL-1. Both doses evoked a fever response and reduced locomotor activity, but the increase in body temperature did not correlate with the decrease in locomotor activity and both effects did not occur at the time of behavioural testing. Taken together, our data indicate that central administration of IL-1 has anxiolytic-like properties.

Centrally administered oligodeoxynucleotides in rats: occurrence of non-specific effects.

We studied the effects of various intracerebroventricularly administered oligodeoxynucleotides on body temperature, locomotor activity, food intake and water consumption in rats during a 24 h period with a radio-telemetric system. Both complete phosphorothioate oligodeoxynucleotides and end-inverted oligodeoxynucleotides dose-dependently elevated body temperature, suppressed food and fluid intake and inhibited nighttime activity. Apparently these effects do not depend on the nucleotide sequence because antisense and sense arginine vasopressin and oxytocin oligodeoxynucleotides, as well as a missense oligodeoxynucleotide produced comparable changes in the autonomous and behavioral parameters. In control experiments neither contaminants from the chemical synthesis nor endotoxins produced such effects, whereas native DNA from salmon sperm did. Fever and sickness-like behavior in response to missense phosphorothioate oligodeoxynucleotides were accompanied by elevated concentrations of circulating corticosterone and by a marked increase in interleukin 6 mRNA in brain and spleen, indicating that centrally administered oligodeoxynucleotides stimulate the production of pyrogenic inflammatory mediators in both central nervous system and peripheral tissues. Our results indicate that centrally administered oligodeoxynucleotides produce beside their intended sequence-specific effects also transient and sequence-independent effects due to their nucleic acid structure.

Intracerebroventricular administration of missense oligodeoxynucleotide induces interleukin-6 mRNA expression in brain and spleen of rats.

After intracerebroventricular (i.c.v.) injection of a missense oligodeoxynucleotide (MS-ODN) solution in rats, transcripts of the proinflammatory cytokine interleukin (IL)-6 were induced in hypothalamus, hippocampus, cortex and spleen and increased levels of circulating IL-6 and corticosterone were detected. Moreover, using a biotelemetric method body temperature of rats injected with ODN or Ringer solution was monitored over a period of 24 h after the injection. The i.c.v. injection of ODN induced a fever response which peaked at 6 h post-injection. These observations demonstrate that central administration of ODNs generates an inflammatory response in the central nervous system (CNS) and in the periphery as well.

Acamprosate and alcohol: II. Effects on alcohol withdrawal in the rat.

The suppressing effect of acamprosate (calcium-acetyl homotaurinate) on alcohol drinking is well established; however, little is known about its effects upon the alcohol-induced withdrawal syndrome. Male Wistar rats received as a sole drinking fluid a 20% (v/v) alcohol solution for one week. Animals consumed on average 5.3 +/- 0.3 g/kg per day alcohol, which resulted in blood alcohol levels of 38 +/- 14 mg/dl. For the quantification of alcohol withdrawal we used a new radio-telemetric system which enabled us to monitor body temperature, locomotor activity, food and water intake patterns constantly during alcohol withdrawal. Although alcohol intake and the resulting blood alcohol levels were low, clear signs of withdrawal could be observed. Thus, hyperthermia and hyperlocomotion occurred 18 h after the termination of forced alcohol drinking. Food intake was initially enhanced but dropped significantly below basal food intake in control animals one day after the termination of forced alcohol drinking. Acamprosate given twice a day (200 mg/kg, i.p., 8 a.m. and 8 p.m.) reduced hyperlocomotion and food intake significantly in the alcohol withdrawal animals, however, it did not change withdrawal-induced hyperthermia. When acamprosate was given to alcohol-naive animals, it increased locomotor activity and body temperature transiently, in particular during the rats' active night phase. In summary, (i) the radio-telemetric system used in the present study proved to be a very sensitive method for quantifying alcohol-induced withdrawal symptoms; (ii) acamprosate reduced alcohol-induced physical signs of withdrawal, however, this effect could not be observed for all parameters measured, which might be explained by the fact that (iii) acamprosate exerts a slight, transient psychomotor stimulant effects by itself.

Corticosterone controls interleukin-1 beta expression and sickness behavior in the rat.

We studied the effect of corticosterone on interleukin (IL)-1 beta synthesis, body temperature, general activity, food consumption and fluid intake in rats treated with bacterial lipopolysaccharide (LPS). Radiotelemetry was used to assess body temperature and locomotor activity in combination with continuous automated recordings of feeding and drinking. This technique was developed as a novel method to identify and measure sickness behavior in rodents. The animals were (a) sham-operated, (b) adrenalectomized or (c) sham-operated and treated with corticosterone (10 mg/kg, subcutaneously). They were then intraperitoneally injected with vehicle or LPS at a dose (100 micrograms/kg) that in sham-operated rats induced fever and anorexia, reduced spontaneous activity and increased IL1-beta mRNA in spleen and adrenals as determined by Northern blot analysis. Adrenalectomized rats produced larger amounts of splenic IL-1 beta mRNA, reduced their general activity much more and developed a mild adipsia as compared with adrenal-intact animals. Administration of corticosterone 1 h before LPS lowered the splenic IL-1 beta mRNA content compared to LPS-treated adrenal-intact rats that did not receive corticosterone and inhibited fever and anorexia, whereas the glucocorticoid did not attenuate the endotoxin-induced suppression of locomotor activity. Our data suggest that during inflammatory conditions body temperature, sickness behavior and the synthesis of IL-1 beta are controlled by corticosterone. Different components of sickness behavior seem to be independently regulated and are under differential control by glucocorticoids.

Soluble interleukin-6 (IL-6) receptor augments central effects of IL-6 in vivo.

The pleiotropic cytokine interleukin-6 (IL-6) controls both the peripheral and central components of the acute-phase response. These activities are mediated via the IL-6 membrane receptor, but probably also via agonistic soluble IL-6 receptors (sIL-6Rs). In the present study we conducted dose-response experiments with rats that were intracerebroventricularly i.c.v.) injected with recombinant human IL-6 and sIL-6R and determined body temperature, locomotor activity, food intake, and water consumption using radiotelemetry and continuous recordings of feeding and drinking. IL-6 injected i.c.v. at 1, 10, and 100 ng increased body temperature and decreased locomotor activity and food intake, but it did not affect water consumption. When 10 ng sIL-6R, which lacked detectable biological activity, was injected i.c.v. 1 h before 1 ng IL-6, the central effects of IL-6 were enhanced and prolonged, and this was not due to endotoxin contamination of the recombinant proteins. Our data suggest that IL-6 plays an important role in the regulation of body temperature, general activity, and food intake in sick animals. Moreover, we have shown for the first time that it is possible to potentiate the effects of a mediator in vivo by administration of the corresponding receptor, which is a novel pharmacological tool for increasing receptor capacity.

Endotoxin and interleukin 1 decrease the affinity of hippocampal mineralocorticoid (type I) receptor in parallel to activation of the hypothalamic-pituitary-adrenal axis.

Lipopolysaccharides (LPS) activate both the immune and the stress response system. The effects of these bacterial endotoxins involve the release of interleukin 1 (IL1) and other cytokines, which in turn stimulate the hypothalamic-pituitary-adrenal (HPA) axis. We studied the binding properties of the corticosteroid receptor system, which mediates feedback inhibition of the HPA axis, in two brain areas and in the pituitary gland in rats treated with LPS and recombinant murine IL1 beta. The binding properties of the corticosteroid receptors were determined by Scatchard plot analyses of in vitro cytosolic binding of the tritiated mineralocorticoid receptor (MR) radioligand aldosterone and the tritiated glucocorticoid receptor (GR) ligand RU28362. Tissues were collected 48 h after administration of LPS, including a 24-hour period for depletion of endogenous corticosterone. LPS treatment increased the Kd of [3H]aldosterone of the hippocampal MR 4.3-fold and the apparent maximum binding capacity (Bmax) of [3H]aldosterone by 65% during a time interval when the concentration of corticosterone, the endogenous ligand of both hippocampal MR and GR, was elevated in the intact rat. Thereafter, MR binding properties were not different from vehicle-injected controls, at 96 h, when in intact animals the enhanced HPA activity subsided. GRs, determined by binding of [3H]RU28362, were not affected by LPS. IL1 evoked a 2.7-fold increase in the Kd of the hippocampal MR and a 57% increase in Bmax 3 h after injection into the lateral cerebral ventricle. An autoradiographic procedure revealed that the same treatment with IL1 reduced the retention of the tritiated endogenous MR ligand corticosterone by 40-60% in all pyramidal cell layers and in the dentate gyrus of the hippocampus, when a tracer dose of the steroid was administered that gives rise to a concentration around the Kd of the MR. This reduced in vivo retention of corticosterone is predicted in view of the reduced affinity of hippocampal MRs. The data are consistent with the hypothesis that an impaired feedback of the HPA axis via deficient hippocampal MRs contributes to stimulate corticosterone secretion from the adrenals during infection.

Corticosterone modulates interleukin-evoked fever in the rat.

The role of endogenous corticoids in fever responses caused by recombinant murine interleukin (IL)-1 beta and IL-6 was studied in adult male Wistar rats. Adrenalectomy diminished the development of fever after intracerebroventricular (icv) injection of these ILs and lowered body temperature. Intraperitoneal administration of the same doses of ILs did not produce fever in intact animals or hypothermia in adrenalectomized rats, thus suggesting a central site of action of IL-1 beta and IL-6 in these experiments. Chronic replacement with moderate doses of corticosterone restored the fever response in adrenalectomized animals in response to icv administration of IL-1 beta but only partially reversed the fever caused by IL-6. Adrenalectomized animals acutely treated with corticosterone and thereafter with either IL-1 beta or IL-6 developed fever more rapidly than did chronically corticosterone-treated animals. In intact animals corticosterone blocked the fever response to icv injected IL-1 beta. We propose that in the rat corticosterone acts in a bimodal manner on body temperature; it exerts a permissive central effect on the fever response and limits the production of inflammatory mediators in the brain. Conversely, higher corticosterone doses probably reduce the magnitude of the fever response.

The role of the hypothalamic-pituitary-adrenocortical system during inflammatory conditions.

Infections and injury are often accompanied by the production of large quantities of proinflammatory mediators such as cytokines and eicosanoids. These substances have been shown to efficiently activate the hypothalamic-pituitary-adrenocortical (HPA) system. The glucocorticoid hormones secreted from the adrenal cortex seem to be crucial for survival because they have an inhibitory influence on inflammatory processes, which, if uncontrolled, may become toxic for the host. Furthermore, these steroid hormones are known to support thermogenesis by inducing or repressing key enzymes of carbohydrate, lipid, and protein metabolism, and thus may also facilitate energy mobilization during fever, which usually accompanies infectious diseases. Finally, a number of studies suggest that glucocorticoids attenuate debilitating symptoms of inflammatory mediators, such as sleepiness, loss of appetite, and suppression of reproductive functions. One can assume that glucocorticoids exert similar behavioral effects during inflammatory conditions, which are seen in infectious diseases. Corticotropin-releasing hormone (CRH), the major hypothalamic component of the HPA system, is a putative mediator of the central effects of cytokines and autacoids because it inhibits growth, reproduction, and food intake. In contrast, CRH decreases sleep duration. Vasopressin, another hypothalamic peptide of the HPA system, counteracts fever and sickness behavior and is thought to support recovery from inflammatory diseases. Apparently, a well-balanced, concerted action of proinflammatory mediators, glucocorticoids, and hypothalamic peptide hormones provides not only an efficient principle for combating microorganisms and support of tissue repair but also for self-protection of the host during the stress of inflammation. Therefore, an impairment of the HPA system under inflammatory conditions often has severe pathological consequences, for example, in patients suffering from Addision's disease and arthritis.

Cytokines and the brain corticosteroid receptor balance: relevance to pathophysiology of neuroendocrine-immune communication.

Interleukin-1 (IL-1) and interleukin-6 (IL-6), and their cognate receptors, are expressed in hippocampal neurons, which are targets for corticosteroid hormones. Corticosteroids bind to intracellular receptors, that is, mineralocorticoid (MRs) and glucocorticoid receptors (GRs). MRs respond to low concentrations of the steroid, while higher concentrations are needed for additional activation of GRs. MR occupation appears relevant in hippocampal neurons for stability of ongoing transmission, for basal activity and sensitivity of the stress response system, and for behavioural reactivity and response selection. Additional transient GR activation suppresses excitability, facilitates recovery from the stress response, and promotes information storage. Thus, the balance of MR- and GR-mediated effects appears critical for the long-term control exerted by corticosteroids over specific aspects of neuronal activity, stress responsiveness, and behavioural adaptation. Administration of IL-1 produces a long-lasting increase in corticosterone. IL-1 also influences MR function in hippocampus and causes a shift in the MR/GR balance, which may underlie prolonged activation of the HPA axis during an immune response.

Cellular localization of interleukin 6 mRNA and interleukin 6 receptor mRNA in rat brain.

The distribution of interleukin 6 (IL-6) mRNA and IL-6 receptor (IL-6R) mRNA in the brain of adult male rats was studied at the light microscope level by in situ hybridization histochemistry using 35S-labelled oligonucleotides. The transcripts of both genes were localized in the pyramidal neurons and in the granular neurons of the hippocampus, in neurons of the habenular nucleus as well as in the dorsomedial and ventromedial hypothalamus, in the piriform cortex, in scattered neurons of the cortex and in granular cells of the cerebellum. The medial preoptic nucleus and the anterior tip of the lateral ventricle contained mRNA encoding IL-6 and its receptor. Moreover, white matter areas, such as the internal capsule, which consist of only fibres and glial cells, were found to have autoradiographic signals above background. The mRNAs for IL-6 and IL-6R in hippocampus and cerebellum are not different, as shown by Northern blot analyses of RNA isolated from these tissues. We postulate that the cytokine IL-6 is expressed constitutively in discrete regions of the CNS and that it is involved in the mechanisms coordinating metabolic, behavioural and neuroendocrine changes not only during illness but also under normal physiological conditions. Our results suggest that IL-6 mRNA and IL-6R mRNA are colocalized, thus supporting a role of the cytokine in autocrine and paracrine communication.

Interleukin-1 beta, but not interleukin-6, impairs spatial navigation learning.

The effects of the cytokines interleukin-1 and -6 (IL1 beta, IL6; 100 ng) on spatial learning were examined in the Morris water maze. Intracerebroventricular injection of IL1 or IL6 before the training on day 1 did not influence the acquisition of spatial navigation. However, IL1 administered at 60 min, but not immediately before the training, resulted in impaired performance of spatial navigation the following day. In contrast, IL6 administered at both times had no effect. In a second experiment the same doses of IL1 and IL6 increased the body temperature of rats in a time-related fashion. The temperature effect of IL1 developed after a delay of 120 min, while the IL-6-effect was immediate. Comparable behavioral changes might accompany infections or inflammatory diseases and therapeutic cytokine administration.

Regulation of interleukin 6 gene expression in rat.

The effects of ip endotoxin administration on interleukin 6 (IL6) transcripts in brain and in peripheral tissues of rats were studied together with the effects of this treatment on IL6 and corticosterone concentrations in blood serum. Northern blot analyses showed a rapid increase of IL6 transcripts in spleen, pituitary gland, and adrenals that was paralleled by pronounced elevations in serum IL6 and corticosterone levels. Adrenalectomy further enhanced the induction of IL6 messenger RNA (mRNA) in spleen and pituitary gland and augmented the increase in serum IL6 bioactivity after lipopolysaccharide (LPS) injection. Corticosterone pretreatment (10 mg/kg) completely blocked the increase of IL6 in serum and IL6 mRNA in spleen, adrenals, and hypophysis. In several brain areas, low amounts of IL6 mRNA were detected under basal, noninflammatory conditions, but in response to LPS there was no change in the IL6 mRNA in hippocampus, hypothalamus, and cerebellum. Neither adrenalectomy nor peripheral injections of sublethal LPS doses of up to 10 mg/kg were capable of increasing IL6 mRNA in the hippocampus. The data do not support the hypothesis that central IL6 biosynthesis via transcription of the gene contributes to the endotoxin-mediated activation of the hypothalamic-pituitary-adrenal system. The results, however, clearly demonstrate that LPS-induced IL6 gene expression is subject to glucocorticoid suppression in peripheral tissues.

Corticosteroid receptor plasticity in the central nervous system of various rat models.

The action of corticosteroids in the central nervous system (CNS) is mediated by two distinct corticosteroid receptors: the mineralocorticoid and glucocorticoid receptors (MR and GR respectively). Using an established in vitro binding assay system, MR and GR binding parameters were determined in the hippocampal, hypothalamic and pituitary cytosol of various rat models. In the (pharmaco-)genetically selected rat lines, the apomorphine susceptible (APO-SUS) rats showed a significant increase in the hippocampal and pituitary MR binding capacities (but not affinity) compared to those in the apomorphine-unsusceptible (APO-UNSUS) rats. In immunologically-altered Lewis (LEW/N) rats and in spontaneously hypertensive rats (SHR), increased hippocampal MR capacity (but not affinity) and hypothalamic MR capacity were observed compared to their respective control, Wistar (WIST) and Wistar Kyoto (WKY) rats. In addition, compared to WKY rats, SHR rats also showed a much greater pituitary but lesser hypothalamic GR binding capacity. In rats subjected to alteration in environmental conditions, the long-term effects of a short inescapable stress resulted in a significant increase in both hippocampal MR and GR while the pituitary and hypothalamic MR and GR do not differ in the stress and control groups. In rats subjected to a defeat test, a decrease in hippocampal MR and GR was observed 3 weeks (but not 1 week) later.

Peripheral and central regulation of IL-6 gene expression in endotoxin-treated rats.

The distribution of IL-6 mRNA in the rat brain was studied by in situ hybridization using 35S-labelled oligonucleotides. The mRNA encoding IL-6 was found in hippocampus, hypothalamus, cerebellum as well as in other brain areas. Microscopic analyses revealed both neuronal and glial cell localization of the mRNA. Subsequently, Northern blot analyses were performed with RNA isolated from spleen, adrenals, pituitary gland, hypothalamus, hippocampus and cerebellum of rats injected intraperitoneally with a non toxic dose of LPS. A rapid induction of the IL-6 mRNA was observed in the peripheral organs, whereas no change in IL-6 transcripts could be measured in the brain. It is concluded that the local synthesis and release of IL-6 in pituitary and adrenal gland might be involved in the activation of the HPA axis following an endotoxin challenge.

Localization of interleukin 6 mRNA and interleukin 6 receptor mRNA in rat brain.

The cytokine interleukin 6 (IL6) has several effects on the central nervous system in addition to the well established regulation of the acute phase inflammatory response. Therefore, the distribution of IL6- and IL6 receptor mRNA in the rat brain has been investigated by in situ hybridization using [35S]-labeled oligonucleotides. The messages of both genes were found in the CA1-CA4 regions as well as in the dentate gyrus of the hippocampus, in the habenulae, the dorsomedial and the ventromedial hypothalamus, in the internal capsule, the optic tract and in the piriform cortex. These data indicate both neuronal and glial localization of IL6 and IL6 receptor and their involvement in an autocrine or paracrine action of the cytokine in centrally regulated functions including neuroendocrine control.

The early induction of the actin-sequestering peptide thymosin beta 4 in thymocytes depends on the proliferative stimulus.

The expression of the actin-sequestering peptide, thymosin beta 4, was analyzed in proliferating rat thymocytes, activated by diverse stimuli, during the early G1 phase and the S phase. In the presence of concanavalin A a 6.3-fold increase of thymosin beta 4 occurred already after 1 h of stimulation without elevation of the corresponding mRNA level. In contrast, during the S phase the increase of thymosin beta 4 (2.5-fold) was accompanied by a higher mRNA level, but did not exceed the growth related increase of total protein. Stimulation with a crosslinked antibody against rat T cell antigen receptor or stimulation with phorbol 12-myristate 13-acetate (PMA) and Ca(2+)-ionophore A23187, separately or in combination, did not lead to the marked increase of the thymosin beta 4 concentration in the early G1 phase but resulted in elevated thymosin beta 4 peptide and mRNA levels during the S phase. It therefore appears that protein kinase C activation and a rise in cytoplasmic Ca(2+)-concentration are not exclusively responsible for the stimulation of thymosin beta 4 specific translation in thymocytes. This assumption was reinforced by the observation that inhibition of the protein kinase C activity by 1-(5-isoquinolinylsulfony)-2-methylpiperazine (H-7) did not affect the cellular thymosin beta 4 content 1 h and 48 h after concanavalin A (Con A) stimulation.

Nucleotide and nucleic acid metabolism in rat thymocytes during cell cycle progression.

A complete cell cycle of mature, concanavalin A (Con A) stimulated rat thymocytes was documented by analyzing the cell number as well as the content and synthesis of DNA and RNA. Cell cycle progression is accompanied by an elevation of class I, II and III RNA polymerase activities (about 10-fold) in the S phase maximum, 48 h after stimulation. Moreover, maximal cellular contents of DNA, ATP, ADP and AMP were observed at this culture period, whereas the RNA level peaked at 60 h. The synthesis of purine and pyrimidine nucleotides de novo was detected by use of [14C]HCO3-. Maximal incorporation rates of [14C]HCO3- into nucleotides (de novo synthesis) and of [3H]adenine into adenylates ('salvage pathway') occur during the S phase. However, the de novo synthesis rates were markedly lower than those of the 'salvage pathway'. The highest cellular level of the nucleotide precursor 5-phosphoribosyl-1-pyrophosphate (8.4-fold increase) also coincided with the S phase.

Cell-cycle-regulated expression of thymosin beta 4 in thymocytes.

Thymosin beta 4 belongs to a family of ubiquitous peptides present at a high cellular content but still with an unknown intracellular function. The expression of this peptide was studied in concanavalin-A-stimulated, proliferating rat thymocytes during cell cycle progression. An early, transient 10-fold increase of the peptide occurred 1 h after stimulation without elevation of the corresponding mRNA level. This increase coincided with that of thymosin beta 4 biosynthesis. The sharp decline of the thymosin beta 4 content was not due to a secretion of the peptide into the medium. During S phase and mitosis, the biosynthetic rates as well as mRNA content, but not the cellular thymosin beta 4 concentration, increased again. After 96 h of culture the values returned to those of quiescent cells.