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The Role of Interleukin-6 in Lipopolysaccharide-Induced Fever by Mechanisms Independent of Prostaglandin E-2
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.ORCID iD: 0000-0002-2230-4174
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
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2009 (English)In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 150, no 4, 1850-1860 p.Article in journal (Refereed) Published
Abstract [en]

Fever has been shown to be elicited by prostaglandin E-2 (PGE(2)) binding to its receptors on thermoregulatory neurons in the anterior hypothalamus. The signals that trigger PGE(2) production are thought to include proinflammatory cytokines, such as IL-6. However, although the presence of IL-6 is critical for fever, IL- 6 by itself is not or only weakly pyrogenic. Here we examined the relationship between IL-6 and PGE(2) in lipopolysaccharide (LPS)-induced fever. Immune-challenged IL- 6 knockout mice did not produce fever, in contrast to wild-type mice, but the expression of the inducible PGE(2)-synthesizing enzymes, cyclooxygenase-2 and microsomal prostaglandin E synthase-1, was similarly up-regulated in the hypothalamus of both genotypes, which also displayed similarly elevated PGE(2) levels in the cerebrospinal fluid. Nevertheless, both wild-type and knockout mice displayed a febrile response to graded concentrations of PGE(2) injected into the lateral ventricle. There was no major genotype difference in the expression of IL-1 beta and TNF alpha or their receptors, and pretreatment of IL- 6 knockout mice with soluble TNF alpha receptor ip or intracerebroventricularly or a cyclooxygenase-2 inhibitor ip did not abolish the LPS unresponsiveness. Hence, although IL- 6 knockout mice have both an intact PGE(2) synthesis and an intact fever-generating pathway downstream of PGE(2), endogenously produced PGE(2) is not sufficient to produce fever in the absence of IL-6. The findings suggest that IL- 6 controls some factor(s) in the inflammatory cascade, which render(s) IL- 6 knockout mice refractory to the pyrogenic action of PGE(2), or that it is involved in the mechanisms that govern release of synthesized PGE(2) onto its target neurons.

Place, publisher, year, edition, pages
2009. Vol. 150, no 4, 1850-1860 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-17620DOI: 10.1210/en.2008-0806OAI: oai:DiVA.org:liu-17620DiVA: diva2:210917
Available from: 2009-04-07 Created: 2009-04-06 Last updated: 2017-12-13
In thesis
1. Prostaglandin E2 in Brain-mediated Illness Responses
Open this publication in new window or tab >>Prostaglandin E2 in Brain-mediated Illness Responses
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

We are unceasingly exposed to potentially harmful microorganisms. The battle against threatening infectious agents includes activation of both the innate and of the adaptive immune systems. Illness responses are elicited and include inflammation, fever, decreased appetite, lethargy and increased sensitivity to painful stimuli in order to defeat invaders. While many of these signs of disease are controlled by the central nervous system, it has remained an enigma how signals from the peripheral immune system reach the brain through its blood-brain barrier, which precludes macromolecules, including cytokines, from diffusing into the brain parenchyma.

Previous findings indicate the existence of a pathway across the blood-brain barrier, which includes binding of the cytokine interleukin-1 (IL-1) to its receptor in the brain vessels, thereby inducing the production of the prostaglandin E2 (PGE2) synthesizing enzymes cyclooxygenase-2 (Cox-2) and microsomal prostaglandin E synthase-1 (mPGES-1), which ultimately synthesize PGE2. PGE2 subsequently binds to any of the four prostaglandin E2 (EP) -receptors. Previous results from our laboratory have suggested that this pathway plays a critical role in the febrile response to infectious stimuli. The present thesis aims at further investigating the molecular events underlying immune-to-brain signalling, with special emphasis on fever, hypothalamic-pituitary-adrenal (HPA) -axis activation and anorexia and their connection to signalling molecules of the cytokine and prostaglandin families, respectively.

In paper I, the molecular processes linking the proinflammatory cytokine interleukin-6 (IL-6) and PGE2 in the febrile response were investigated. Both IL-6 and PGE2 have been shown to be critical players in the febrile response, although the molecular connections are not known, i.e. if IL-6 exerts its effects up- or downstream of PGE2. Mice deficient in IL-6 were unable to respond to bacterial lipopolysaccharide (LPS) with a febrile response, but displayed similar induction of Cox-2 and mPGES-1, and similar concentrations of PGE2 in the cerebrospinal fluid as wild-type mice. Paradoxically, the IL-6 deficient mice responded with a dose-dependent elevation of body temperature in response to intracerebroventricularly injected PGE2. Furthermore, IL-6 per se was not pyrogenic when injected peripherally in mice, and did not cause increased levels of PGE2 in cerebrospinal fluid. IL-6 deficient mice were not refractory to the action of PGE2 because of excess production of some hypothermia-producing factor, since administration of a Cox-2 inhibitor in LPS-challenged IL-6 deficient mice did not unmask any hypothermic response, and neutralization of tumor necrosis factor α (TNFα), associated with hypothermia, did not produce fever in LPS-challenged IL-6 deficient mice. These data indicate that IL-6 rather than exerting its effects up- or down-stream of PGE2 affects some process in parallel to PGE2, perhaps by influencing the diffusion and binding of PGE2 onto its target neurons.

In papers II and III, we injected the proinflammatory cytokine IL-1β in free-fed wild-type mice, in mice with a deletion of the gene encoding mPGES-1, or in mice deficient in the EP1, EP2 and EP3. Food intake was continuously measured during their active period, revealing that mPGES-1 deficient mice were almost completely resistant to anorexia induced by IL-1β. However, all of the investigated EP receptor deficient mice exhibited a normal profound anorexic response to IL-1β challenge, suggesting that the EP4 is the critical receptor that mediates IL-1β-induced anorexia. We also investigated the role of mPGES-1 in anorexia induced by lipopolysaccharide (LPS) in mPGES-1 deficient mice. The profound anorexic response after LPS-challenge was similar in mPGES-1 deficient and wild-type mice. To further investigate the anorectic behaviour after LPS injection, we pre-starved the animals for 22 hours before injecting them with LPS. In this paradigm, the anorexia was less profound in mPGES-1 knock-out mice. Our results suggest that while the inflammatory anorexia elicited by peripheral IL-1β seems largely to be dependent on mPGES-1-mediated PGE2 synthesis, similar to the febrile response, the LPS-induced anorexia is independent of this mechanism in free-fed mice but not in pre-starved animals.

In papers IV and V, the role of prostanoids for the immune-induced HPA-axis response was investigated in mice after genetic deletion or pharmacological inhibition of prostanoid-synthesizing enzymes, including Cox-1, Cox-2, and mPGES-1. The immediate LPS-induced release of ACTH (adrenocorticotropic hormone and corticosteroids was critically dependent on Cox-1 derived prostanoids and occurred independently of Cox-2 and mPGES-1 derived PGE2. In contrast, the delayed HPA-axis response was critically dependent on immune-induced PGE2, synthesized by Cox-2 and mPGES-1, and occurred independently of Cox-1 derived enzymes. In addition, in the mPGES-1 deficient mice, the synthesis of CRH hnRNA and mRNA was decreased in the paraventricular nucleus of the hypothalamus after LPS-challenge, indicating that the delayed hormone secretion was mediated by PGE2-induced gene-transcription of CRH in the hypothalamus. The expression of the c-fos gene and Fos protein, an index of synaptic activation, was maintained in the paraventricular nucleus and its brainstem afferents both after unselective and Cox-2 selective inhibition as well as in Cox-1, Cox-2, and mPGES-1 knock-out mice. This suggests that the immune-induced neuronal activation of autonomic relay nuclei occurs independently of prostanoid synthesis and that it is insufficient for eliciting stress hormone release.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2010. 86 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1162
Keyword
prostaglandin e2, PGE2, cox-1, cox-2, mPGES-1, fever, anorexia, food intake, HPA-axis, EP-receptor, LPS, IL-1, IL-6
National Category
Medical and Health Sciences Physiology
Identifiers
urn:nbn:se:liu:diva-53701 (URN)978-91-7393-462-6 (ISBN)
Public defence
2010-02-26, Berzeliussalen, Ingång 65, Hälsouniversitetet, Campus US, Linköpings Universitet, Linköping, 13:00 (Swedish)
Opponent
Supervisors
Available from: 2010-02-11 Created: 2010-02-01 Last updated: 2010-02-11Bibliographically approved
2. The Role of Interleukin-6 in the Febrile Response
Open this publication in new window or tab >>The Role of Interleukin-6 in the Febrile Response
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Everyone who has been exposed to influenza or a bacterial infection knows how it feels to be sick. Apart from not being willing to participate in social activities, losing your appetite and experiencing pain, you have also most likely suffered from increased body temperature which defines fever, one of the most prominent signs of an acute ongoing infection. Invading the body, the infectious microorganisms are combated by the activated innate and adaptive immune systems, and the impaired balance is thus restored. While fever is an event that is controlled by the central nervous system, it has for long been debated how the inflammatory signals generated in the periphery communicate with the brain that is protected by the bloodbrain barrier which prevents large molecules such as cytokines from entering into the brain parenchyma.

Previous studies from our group have provided evidence in support of the existence of a pathway across the blood-brain barrier by demonstrating that proinflammatory cytokine interleukin-1 transfers the inflammatory message to the brain through binding to its receptors situated in the brain vessels. This will subsequently trigger the production of the prostaglandin E2 (PGE2) that enters the brain and exerts its effect by binding to the receptors located on the thermoregulatory neurons. Interleukin-6 (IL-6) is another cytokine essential for fever signaling; however, the mechanism has not yet been identified. The research on which this thesis is based aimed at elucidating the role of IL-6 in inflammatory induced fever.

In paper I, we demonstrated that mice incapable of producing inflammatory PGE2 still responded with an intact cytokine production in the brain upon peripheral lipopolysaccharide (LPS) stimulation. Thus, although the mice had induced expression of inflammatory cytokines in the brain, this was not sufficient for a fever response without simultaneous production of PGE2. The relationship between IL-6 and PGE2, both essential for fever, was further investigated in paper II, focusing on clarifying the mechanism by which IL-6 controls fever. We demonstrated that mice deficient in IL-6 did not respond with fever upon peripheral LPS administration despite an intact expression of PGE2 in the brain. In contrast, upon intracerebroventricular administration of PGE2 into the brain, a dose-dependent fever response was monitored in IL-6 deficient mice. Thus, we suggest that IL-6 exerts its effect neither up- nor downstream from PGE2, and propose instead that IL-6 may act alongside the PGE2 and regulate the process that deals with the transport of and binding of PGE2 onto its receptors. To further investigate the elusive role of IL-6 in fever, we performed a microarray analysis to identify the genes that were differentially expressed in the brain of LPS-challenged IL-6 deficient mice compared to wild type mice (paper III). We demonstrated that mice lacking IL-6 displayed two-times lower expression of lipocalin-2 in the hypothalamus. IL-6 and lipocalin-2 were directly related to each other since peripherally administrated IL-6 induced the expression of lipocalin-2 in cells associated with the brain vessels. Lipocalin-2 induced by LPS was expressed by brain endothelial cells and partly co-localized with cyclooxygenase-2, one of the enzymes essential for inflammatory PGE2 production in the endothelial cells. We also demonstrated that lipocalin-2 in a sex-dependent and ambient temperature-specific manner may be implicated in thermogenesis. We have thus identified a new factor in the IL-6 regulated fever pathway, but the pathway is still not understood. On important question that remained to be answered was in which  compartment IL-6 was needed for the signaling. This question was studied further in paper IV, where we investigated the role of hematopoietically produced IL-6 in fever by constructing chimeric mice. We concluded that IL-6 produced by cells of non-hematopoietic origin is critical for the LPSinduced fever while hematopoietically produced IL-6 plays only a minor role in contributing to fever.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2013. 92 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1349
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-87453 (URN)978-91-7519-722-7 (ISBN)
Public defence
2013-01-30, Berzeliussalen, ingång 64, plan 9, Campus US, Linköpings universitet, Linköping, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2013-01-18 Created: 2013-01-18 Last updated: 2016-05-04Bibliographically approved

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Nilsberth, CamillaElander, LouiseHamzic, NamikLönn, JohannaEngström, LindaBlomqvist, Anders

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