Brain-borne IL-1 adjusts glucoregulation and provides fuel support to astrocytes and neurons in an autocrine/paracrine manner.

It is still controversial which mediators regulate energy provision to activated neural cells, as insulin does in peripheral tissues. Interleukin-1ß (IL-1ß) may mediate this effect as it can affect glucoregulation, it is overexpressed in the 'healthy' brain during increased neuronal activity, and it supports high-energy demanding processes such as long-term potentiation, memory and learning. Furthermore, the absence of sustained neuroendocrine and behavioral counterregulation suggests that brain glucose-sensing neurons do not perceive IL-1ß-induced hypoglycemia. Here, we show that IL-1ß adjusts glucoregulation by inducing its own production in the brain, and that IL-1ß-induced hypoglycemia is myeloid differentiation primary response 88 protein (MyD88)-dependent and only partially counteracted by Kir6.2-mediated sensing signaling. Furthermore, we found that, opposite to insulin, IL-1ß stimulates brain metabolism. This effect is absent in MyD88-deficient mice, which have neurobehavioral alterations associated to disorders in glucose homeostasis, as during several psychiatric diseases. IL-1ß effects on brain metabolism are most likely maintained by IL-1ß auto-induction and may reflect a compensatory increase in fuel supply to neural cells. We explore this possibility by directly blocking IL-1 receptors in neural cells. The results showed that, in an activity-dependent and paracrine/autocrine manner, endogenous IL-1 produced by neurons and astrocytes facilitates glucose uptake by these cells. This effect is exacerbated following glutamatergic stimulation and can be passively transferred between cell types. We conclude that the capacity of IL-1ß to provide fuel to neural cells underlies its physiological effects on glucoregulation, synaptic plasticity, learning and memory. However, deregulation of IL-1ß production could contribute to the alterations in brain glucose metabolism that are detected in several neurologic and psychiatric diseases.

Behavioural endocrine immune-conditioned response is induced by taste and superantigen pairing.

Administration of bacterial superantigen, such as staphylococcal enterotoxin B (SEB), induces in vivo stimulation of T cell proliferation and cytokine production such as interleukin-2 (IL-2). It has been previously reported that SEB administration induces fever, c-Fos expression in the brain, and hypothalamus-pituitary-adrenal axis activation, demonstrating that the brain is able to sense and respond to SEB. Previously it had been shown that immune functions can be behaviourally conditioned pairing a novel gustatory stimulus together with an immunomodulatory drug or an antigen. We designed an experimental protocol using Dark Agouti rats in which saccharin taste, as conditioned stimulus, was paired with an i.p. injection of SEB (2 mg/kg), as unconditioned stimulus. Six days later, when conditioned animals were re-exposed to the conditioned stimulus they displayed strong conditioned taste avoidance to the saccharin. More importantly, re-exposure to the conditioned stimulus significantly increased IL-2, interferon-gamma and corticosterone plasma levels, in comparison with conditioned animals which had not been re-exposed to saccharin taste. These results demonstrate a behavioural-immune-endocrine conditioned response using a superantigen as unconditioned stimulus. In addition, they illustrate the brain abilities to mimic the unconditioned effects of a superantigen by yet unknown mechanisms.

Interleukin-6: a cytokine to forget.

It is known that proinflammatory cytokines such as interleukin-6 (IL-6) are expressed in the central nervous system (CNS) during disease conditions and affect several brain functions including memory and learning. In contrast to these effects observed during pathological conditions, here we describe a physiological function of IL-6 in the "healthy" brain in synaptic plasticity and memory consolidation. During long-term potentiation (LTP) in vitro and in freely moving rats, IL-6 gene expression in the hippocampus was substantially increased. This increase was long lasting, specific to potentiation, and was prevented by inhibition of N-methyl-D-aspartate receptors with (+/-)-2-amino-5-phosphonopentanoic acid (AP-5). Blockade of endogenous IL-6 by application of a neutralizing anti-IL-6 antibody 90 min after tetanus caused a remarkable prolongation of LTP. Consistently, blockade of endogenous IL-6, 90 min after hippocampus-dependent spatial alternation learning resulted in a significant improvement of long-term memory. In view of the suggested role of LTP in memory formation, these data implicate IL-6 in the mechanisms controlling the kinetics and amount of information storage.

Complex phenotype of mice homozygous for a null mutation in the Sp4 transcription factor gene.

BACKGROUND: Sp4 is a zinc finger transcription factor which is closely related to Sp1 and Sp3. All three proteins recognize the same DNA elements and can act as transcriptional activators through glutamine-rich activation domains. Unlike Sp1 and Sp3, which are ubiquitous proteins, Sp4 is highly abundant in the central nervous system, but also detectable in many other tissues. RESULTS: We have disrupted the mouse Sp4 gene by a targeted deletion of the exons encoding the N-terminal activation domains. Sp4 knockout mice show a complete absence of Sp4 expression. They develop until birth without obvious abnormalities. After birth, two-thirds die within 4 weeks. Surviving mice are growth retarded. Male Sp4null mice do not breed. The cause for the breeding defect remains obscure since they show complete spermatogenesis. In addition, pheromone receptor genes in the vomeronasal organ appear unaffected. Female Sp4null mice have a smaller thymus, spleen and uterus. In addition, they exhibit a pronounced delay in sexual maturation. CONCLUSIONS: The phenotype of the Sp4null mice differs significantly from those described for Sp1-/- and Sp3-/- mice. Thus, the structural similarities, the common recognition motif and the overlapping expression pattern of these three transcription factors do not reflect similar physiological functions.

Endocrine host responses during early and late phases of tumor development.

It is well established that hormones affect tumor growth. Conversely, inoculation of cells obtained from tumors that had been transplanted for many generations causes changes in the concentration of different hormones before and after tumor detection. We aimed at answering the question of whether hormonal alterations also occur during the development of primary tumors and following transplantation of tumors from early generations. Primary tumors were induced in mice by either the carcinogenic agent 3-methylcholanthrene, which produces fibrosarcomas, or the milk-transmitted mammary tumor virus, which induces adenocarcinomas. The results showed that (i) in both models, an early reduction in plasma insulin and prolactin levels occurred, and in the case of insulin, this reduction was sustained for a prolong period prior to tumor detection, indicating that recognition by the host of emergent tumor cells triggers an endocrine response; (ii) in contrast with multiply transplanted tumors, cells from early transplant generations produced no significant endocrine changes during latency; (iii) irrespective of whether they were primary or transplanted, large tumor burdens caused similar hormonal alterations, consisting of increased corticosterone and growth hormone and decreased insulin, thyroxin, prolactin and sex steroid levels in blood. Our comprehensive longitudinal study demonstrates host endocrine responses during different stages of neoplastic development.

The cytokine-HPA axis feed-back circuit.

Due to their anti-inflammatory effects and their capacity to affect the synthesis of most cytokines and the Th1/Th2 balance, endogenous glucocorticoids contribute to control the basal operation of immune cells. Immune processes result in the production of cytokines, such as IL-1, IL-2, IL-6, IL-11, IL-12, TNF-alpha, and INF-gamma, which can activate the HPA axis. In turn, the resulting increase in glucocorticoid blood levels can affect immune cell activity directly or by controlling the production of cytokines, suggesting the existence of an immunoregulatory cytokine-HPA axis circuit. There is evidence that this circuit plays a relevant role in controlling excessive inflammatory reactions and the non-specific expansion of immune cells with no or low affinity for the antigen that triggers an immune response.

The cytokine-HPA axis circuit contributes to prevent or moderate autoimmune processes.

There is evidence that an immunoregulatory circuit integrated by immune-derived cytokines and the hypothalamus-pituitary-adrenal (HPA) axis operates during certain pathological conditions. For example, it has been shown that IL-1 is the main mediator of the increase in ACTH and corticosterone blood levels detected in models of viral infection and bacterial endotoxins. This endocrine response has protective effects during septic shock. In experimental models of tumors, there are also clear indications that the increase in glucocorticoid levels detected during the growth of a lymphoma is mediated by immune-derived products and contributes to the inhibition of the inflammatory response. The disruption of the cytokine-HPA axis circuit can predispose to autoimmunity. This has been shown in animal models of spontaneous autoimmune thyroiditis, lupus-like disease, and experimental arthritis. More recently, it has been shown that the proper operation of this circuit contributes to preventing or moderating autoimmunity. The recovery of animals from experimental autoimmune encephalomyelitis (EAE) is clearly dependent on an increase in endogenous glucocorticoid levels. It has been recently shown that this endocrine response is, at least in part, triggered by the immune response to the encephalitogenic antigen and mediated by the endogenous IL-1 produced during the disease. These examples support the concept that the cytokine-HPA circuit plays a protective role during certain pathologies and that its disruption can lead to predisposition to or aggravation of autoimmune diseases.

Increased sensitivity of the baroreceptor reflex after bacterial endotoxin.

Lipopolysaccharide (LPS), an endotoxin that elicits the production of several cytokines, induces cardiovascular changes characterized by increased perfusion of immune organs and compensatory sympathetic vasoconstriction in other tissues. We therefore hypothesized that to adapt to altered blood flow distribution following LPS administration, changes in the sensitivity of reflexes that control blood pressure would occur. Our data show that the sensitivity of the baroreceptor reflex increases significantly two and three hours after the intravenous administration of a subpyrogenic dose of the endotoxin. This change in sensitivity that could occur at peripheral or central levels may underlie necessary adjustments of cardiovascular mechanisms during the course of certain immune responses.

Not all peripheral immune stimuli that activate the HPA axis induce proinflammatory cytokine gene expression in the hypothalamus.

Administration of low doses of lipopolysaccharide (LPS) that do not disrupt the blood-brain barrier (BBB) results in the expression of interleukin-1 beta (IL-1 beta), IL-6, and tumor necrosis factor-alpha (TNF alpha) in the hypothalamus in parallel to stimulation of the hypothalamus-pituitary-adrenal (HPA) axis. This endocrine response is triggered by peripheral cytokines, and we recently obtained evidence that brain-borne IL-1 contributes to its maintenance. LPS preferentially stimulates cells of the macrophage lineage and B lymphocytes. The possibility that primarily stimulation of other types of peripheral immune cells also results in the expression of proinflammatory cytokines in the brain and in the activation of the HPA axis was investigated. Our results showed that, in contrast to LPS, administration of the superantigen staphylococcal enterotoxin B (SEB), which stimulates T cells by binding to appropriate V beta domains of the T-cell receptor, did not result in induction of IL-1 beta, IL-6, and TNF alpha expression in the hypothalamus. Furthermore, although IL-2 transcripts in the spleen were highly increased, expression of this gene was not detected in the brain. However, as with LPS, SEB administration also results in elevated levels of glucocorticoids in blood. Therefore, our data suggest that increased expression of proinflammatory cytokines in the brain is not a necessary step in the stimulation of the HPA axis by SEB.

Sympathetic innervation affects superantigen-induced decrease in CD4V beta 8 cells in the spleen.

The stimulation by superantigens of T cells expressing an appropriate V beta chain results in a strong proliferative response that is followed by a state of energy specific for the antigen used. This model was used to continue our studies on immunoregulatory host neuroendocrine responses. We have recently found that four days after administration of the superantigen staphylococcal enterotoxin B (SEB) into mice, that is, at an early stage of the anergic phase, the decrease in the percentage of splenic CD4V beta 8 was accompanied by a decrease in the splenic concentration of the sympathetic neurotransmitter noradrenaline (NA) as compared to vehicle-injected mice. No comparable changes were detected in the kidney. At this point, blood levels of NA, adrenaline, and corticosterone were comparable in SEB- and vehicle-injected mice. We have also found that the decrease in splenic CD4V beta 8 cells was not observed in animals that had been chemically sympathectomized prior to the administration of the superantigen. These results indicate that the sympathetic response induced by SEB may have immunoregulatory implications.

A reduction in blood insulin levels as a host endocrine response during tumor development.

It has been previously reported that endogenous insulin levels decrease during tumor growth. We have now studied whether this host endocrine response is independent of the way in which the tumor is induced. For this purpose, animals transplanted with tumor cells induced by 3-methylcholanthrene (MCA) or 7,12-dimethylbenz(a) anthracene (DMBA), or with EL-4 lymphoma cells, and animals that develop autochthonous tumors induced by MCA or the murine mammary tumor virus (MMTV) were used. These procedures result in the induction of tumors of different histologic types: fibrosarcoma, mammary adenocarcinoma and lymphoma. The results obtained showed that a reduction in insulin levels preceded the overt appearance of tumors in all models of syngeneic or autochthonous tumors studied but not when DMBA-induced tumor cells were administered into allogeneic recipients. Reduced levels of insulin before tumor detection appeared to affect the onset of MCA-induced tumors. Indeed, those mice with a late tumor onset were those that had a more pronounced decrease in insulin blood levels during the induction phase of autochthonous MCA-induced tumors. Soluble factors associated with tumor growth seem to mediate the reduction in insulin blood levels in mice transplanted with EL-4 tumor cells. The results obtained indicate that the reduction in insulin levels detected is a consequence of the recognition of tumor cells by the host, and seems to be independent of the histologic type of the neoplastic cells that develop. Pharmacological interventions at the levels of mechanisms that control insulin output should clarify the relevance of decreased levels of this hormone for tumor development.

Norepinephrine stimulates lymphoid cell mobilization from the perfused rat spleen via beta-adrenergic receptors.

The possibility that norepinephrine (NE) influences lymphoid cell outflow independently of its vasoconstrictor action was investigated in the perfused rat spleen. Using agents that affect the vasoconstrictor tonus of the spleen, we observed an inverse correlation between flow resistance and splenic cell output. The curve obtained served as a reference for evaluating effects of different treatments on the number of cells that are mobilized at defined levels of flow resistance. Perfusion of the beta-adrenergic blocker propranolol either alone or in combination with NE lowered splenic leukocyte outflow clearly beyond the number of cells expected at the corresponding flow resistance. No comparable effects were observed when the alpha-adrenergic blocker phentolamine was perfused. When the vasoconstrictor effect of NE was counteracted by papaverine, splenic cell outflow was significantly higher than expected for the level of flow resistance attained. Furthermore, when NE was perfused together with endotoxin, which does not inhibit the vasoconstriction induced by catecholamines, splenic cell mobilization was severalfold higher than expected at increased flow resistance. Propranolol abrogated this effect to a large extent. Furthermore, perfusion of the beta-agonist isoproterenol stimulated lymphoid cell outflow from the spleen despite increased flow resistance. These studies show a dual effect of NE on cell mobilization from the spleen: cell retention by decreasing blood flow and stimulation of cell output by a beta-adrenergically mediated, smooth muscle-independent mechanism.

Cytokines mediate protective stimulation of glucocorticoid output during autoimmunity: involvement of IL-1.

Endogenous glucocorticoid levels are increased during experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Although this endocrine response is essential for survival, the mechanism that triggers the stimulation of glucocorticoid output during the disease remains unknown. We report here that 1) after immunization with the encephalitogenic antigen myelin basic protein (MBP), increased blood glucocorticoid levels are not only observed in Lewis rats, but also in PVG rats, which do not develop EAE; 2) immune cells obtained from animals with EAE and stimulated in vitro with MBP produced mediators that increased glucocorticoid levels when administered to naive recipients; and 3) acute in vivo blockade of interleukin-1 (IL-1) receptors inhibited, to a large extent, the increase in corticosterone levels during EAE. These results show that the increase in corticosterone levels after immunization with MBP can be dissociated from the stress of the paralytic attack that characterizes EAE. Furthermore, they indicate that an endocrine response, which is decisive for the prevention or moderation of EAE, is mainly the result of the stimulation of the hypothalamic-pituitary-adrenal axis by cytokines produced during the immune response that induces the autoimmune disease.

A neuromodulatory role of interleukin-1beta in the hippocampus.

It is widely accepted that interleukin-1beta (IL-1beta), a cytokine produced not only by immune cells but also by glial cells and certain neurons influences brain functions during infectious and inflammatory processes. It is still unclear, however, whether IL-1 production is triggered under nonpathological conditions during activation of a discrete neuronal population and whether this production has functional implications. Here, we show in vivo and in vitro that IL-1beta gene expression is substantially increased during long-term potentiation of synaptic transmission, a process considered to underlie certain forms of learning and memory. The increase in gene expression was long lasting, specific to potentiation, and could be prevented by blockade of potentiation with the N-methyl-D-aspartate (NMDA) receptor antagonist, (+/-)-2-amino-5-phosphonopentanoic acid (AP-5). Furthermore, blockade of IL-1 receptors by the specific interleukin-1 receptor antagonist (IL-1ra) resulted in a reversible impairment of long-term potentiation maintenance without affecting its induction. These results show for the first time that the production of biologically significant amounts of IL-1beta in the brain can be induced by a sustained increase in the activity of a discrete population of neurons and suggest a physiological involvement of this cytokine in synaptic plasticity.

Interleukin-1, but not stress, stimulates glucocorticoid output during early postnatal life in mice.

The development of neuroendocrine functions depends not only on genetically determined mechanisms but also on phenotypic signals. Some of these signals may derive from the immune system. For example, interleukin-1 beta (IL-1 beta) stimulates glucocorticoid output during the early postnatal period, and administration of this cytokine at birth induces permanent alterations in the HPA axis in adulthood. We have extended these studies and found that the glucocorticoid response elicited in 5-day-old mice by a low dose of IL-1 beta is not desensitized by previous exposure to the cytokine. We have also compared the magnitude of the increase in corticosterone levels induced by IL-1 in 3-day-old and adult mice to that caused by acute stress. IL-1 beta and acute stress caused a comparable increase in corticosterone levels in adult mice. In newborn mice, however, IL-1 beta, but not restraint or cold stress, stimulated corticosterone output. Thus, IL-1 beta can elicit a corticosterone response during the postnatal stress-hyporesponsive period. Furthermore, when the corticosterone levels attained following IL-1 beta administration were compared to the basal levels of the hormone at a given age, the increase in plasma corticosterone levels was several fold higher in newborn than in adult animals. These data, together with the long-lasting endocrine effects of cytokine exposure at birth, suggest an important role of immune cytokines in the programming of neuroendocrine functions during ontogeny.

Administration of interleukin-1 at birth affects dopaminergic neurons in adult mice.

Interleukin-1 beta (IL-1 beta), a cytokine that plays a relevant role during inflammatory and immune processes, can also affect brain neurotransmitters and the activity of peripheral sympathetic nerves. Because both brain and peripheral catecholaminergic systems in mice are not fully developed at birth, we speculated that the development of these systems may be susceptible to modifications when mice are exposed to IL-1 beta early in life. Here we report that the administration to mice of a low dose of IL-1 beta during the first days of life results in a decreased dopamine content in the hypothalamus in adulthood. We also show that the dopamine content of the superior cervical sympathetic ganglia was reduced in adult mice that were treated with IL-1 beta at birth. No changes in noradrenaline content nor in its metabolite MHPG were detected in the brain and peripheral sympathetic ganglia of these animals. This indicates that central and probably also peripheral dopaminergic neurons are preferentially affected by IL-1 beta treatment at birth. Collectively, these results indicate that an increased production of IL-1 beta during infectious or inflammatory processes in the perinatal period may induce long-lasting, probably permanent, alterations in central and peripheral neurotransmitter systems.

Central and peripheral mechanisms contribute to the hypoglycemia induced by interleukin-1.

The impact that neuroendocrine effects of cytokines have on general host homeostasis is reflected by the profound metabolic changes observed in parallel. The effect of interleukin-1 beta (IL-1 beta) on glucose blood levels serves as an example. Although IL-1 beta stimulates glucocorticoid output and decreases hepatic glycogen content, hypoglycemia is concomitantly detected in adult and newborn mice. This effect is observed even during fasting and is probably due to increased glucose transport into tissues. Even after a glucose load, IL-1-treated animals remain hypoglycemic, suggesting that central mechanisms that control the set point of glucose homeostasis are affected. Low doses of IL-1 beta injected i.c.v. can also induce hypoglycemia. Furthermore, central blockade of IL-1 receptors partially inhibits the hypoglycemia induced by peripheral administration of IL-1 beta. On the other hand, central depletion of catecholamines exacerbates IL-1-induced hypoglycemia. IL-1-mediated effects on glucose levels might be directed at providing more energy supply to tissues during processes with high metabolic demands.

Inhibition of tumor necrosis factor-alpha action within the CNS markedly reduces the plasma adrenocorticotropin response to peripheral local inflammation in rats.

The present study tested the hypothesis that the cytokine tumor necrosis factor-alpha (TNF-alpha) is an important CNS mediator of the hypothalamo-pituitary-adrenal (HPA) axis response to local inflammation in the rat. Recombinant murine TNF-alpha administered directly into the cerebroventricles of normal rats produced a dose-dependent increase in plasma adrenocorticotropin (ACTH) concentration. Local inflammation induced by the intramuscular injection of turpentine (50 microl/100 gm body weight) also produced an increase in plasma ACTH, peaking at 160-200 pg/ml at 7.5 hr after injection (compared with 10-30 pg/ml in controls). Intracerebroventricular pretreatment with either 5 microl of anti-TNF-alpha antiserum or 1-50 microg of soluble TNF receptor construct (rhTNFR:Fc) reduced the peak of the ACTH response to local inflammation by 62-72%. In contrast, intravenous treatment with the same doses of anti-TNF-alpha or rhTNFR:Fc had no significant effect on the ACTH response to local inflammation. Although these data indicated an action of TNF-alpha specifically within the brain, no increase in brain TNF-alpha protein (measured by bioassay) or mRNA (assessed using either in situ hybridization histochemical or semi-quantitative RT-PCR procedures) was demonstrable during the onset or peak of HPA activation produced by local inflammation. Furthermore, increased passage of TNF-alpha from blood to brain seems unlikely, because inflammation did not affect plasma TNF-alpha biological activity. Collectively these data demonstrate that TNF-alpha action within the brain is critical to the elaboration of the HPA axis response to local inflammation in the rat, but they indicate that increases in cerebral TNF-alpha synthesis are not a necessary accompaniment.

Alterations in the pituitary-adrenal axis of adult mice following neonatal exposure to interleukin-1.

Interleukin-1 (IL-1), a cytokine mainly derived from activated cells of the macrophage lineage, can stimulate the hypothalamus-pituitary-adrenal (HPA) axis. Acute and long-lasting effects on the HPA axis were induced by the administration of low doses of IL-1 to mice during the first 5 days of life. In 5-day-old mice, corticosterone blood levels were markedly elevated 2 h following the last injection of IL-1. IL-1-treated mice grew normally. When studied during adulthood, however, these animals showed a reduction in morning values of corticosterone and the ACTH/corticosterone ratio was increased. Furthermore, an inverse correlation between ACTH and corticosterone levels in blood and between ACTH content in the pituitary gland and corticosterone levels was observed in IL-1-treated mice. Lower blood levels of corticosterone were not due to a reduced sensitivity of the adrenal glands, because these animals responded normally to exogenous ACTH. Another alteration observed in IL-1-exposed adult mice was a reduction in ACTH-like immunoreactivity in the pituitary gland following acute cold and restraint stress. It is concluded that exposure of mice to IL-1 early in life causes long-lasting alterations in the HPA axis. Spleen cells from adult mice treated with IL-1 at birth also developed a stronger response to allogeneic antigens than did cells from control mice. This observation indicates the relevance of immune-neuroendocrine interactions during development.