The initiation of fever has been a matter of controversy. Based on observations of little or no induction of prostaglandin synthesizing enzymes in the brain during the first phase of fever it was suggested that fever is initiated by prostaglandin released into the circulation from cells in the liver and lungs. Here we show in the mouse that prostaglandin synthesis is rapidly induced in the brain after immune challenge. These data are consistent with our recent findings in functional experiments that prostaglandin production in brain endothelial cells is both necessary and sufficient for the generation of all phases of fever.

Fever is known to be elicited by prostaglandin E2 acting on the brain, but its origin has remained disputed. We show in mice that selective deletion of prostaglandin synthesis in brain endothelial cells, but not in neural cells or myeloid cells, abolished fever induced by intravenous administration of lipopolysaccharide and that selective rescue of prostaglandin synthesis in brain endothelial cells reinstated fever. These data demonstrate that prostaglandin production in brain endothelial cells is both necessary and sufficient for eliciting fever. 

We examined the signaling route for fever during localized inflammation in male and female mice, elicited by casein injection into a preformed air pouch. The localized inflammation gave rise to high concentrations of prostaglandins of the E species (PGE(2)) and cytokines in the air pouch and elevated levels of these inflammatory mediators in plasma. There were also elevated levels of PGE(2) in the cerebrospinal fluid, although there was little evidence for PGE(2) synthesis in the brain. Global deletion of the PGE(2) prostaglandin E receptor 3 (EP3) abolished the febrile response as did deletion of the EP3 receptor in neural cells, whereas its deletion on peripheral nerves had no effect, implying that PGE(2) action on this receptor in the CNS elicited the fever. Global deletion of the interleukin-1 receptor type 1 (IL-1R1) also abolished the febrile response, whereas its deletion on neural cells or peripheral nerves had no effect. However, deletion of the IL-1R1 on brain endothelial cells, as well as deletion of the interleukin-6 receptor a on these cells, attenuated the febrile response. In contrast, deletion of the PGE(2) synthesizing enzymes cyclooxygenase-2 and microsomal prostaglandin synthase-1 in brain endothelial cells, known to attenuate fever evoked by systemic inflammation, had no effect. We conclude that fever during localized inflammation is not mediated by neural signaling from the inflamed site, as previously suggested, but is dependent on humoral signaling that involves interleukin actions on brain endothelial cells, probably facilitating PGE(2) entry into the brain from the circulation and hence representing a mechanism distinct from that at work during systemic inflammation.

Fever is a cardinal sign of inflammation and is evolutionary conserved. Fever is known to be beneficial during acute inflammation, but over time and if very high it can be detrimental. The signaling pathways by which fever is initiated by the brain and the peripheral mechanisms through which the temperature increase is generated were studied from several point of views. Fever is known to be dependent on prostaglandin E₂ (PGE₂) binding to its receptors in the median preoptic nucleus of the hypothalamus, which signals to the brainstem and through sympathetic nerves to heat conserving and heat producing effector organs. This thesis focuses on identifying the cells that produce the PGE₂ critical for the fever response; showing where in the brain the critical PGE₂ production takes place; demonstrating how peripheral inflammation activates these cells to produce PGE₂; and finally, identifying the effector mechanisms behind the temperature elevation in fever. By using a newly developed specific antibody we showed that the enzyme responsible for the terminal step in the production of PGE₂, microsomal prostaglandin E-synthase 1 (mPGES-1), is expressed in endothelial cells of brain blood vessels in mice where it is co-expressed with the enzyme cyclooxygenase-2 (Cox-2), which is known to be induced in these cells and to be rate limiting for the PGE₂ production. The mPGES-1 enzyme was also expressed in several other cell types and structures which however did not express Cox-2, such as capillary-associated pericytes, astroglial cells, leptomeninges, and the choroid plexus. The role of the mPGES-1 in these other cells/structures remains unknown. Next, by using mice with selective deletion of Cox-2 in brain endothelial cells, we showed that local PGE₂ production in deep brain areas, such as the hypothalamus, is critical for the febrile response to peripheral inflammation. In contrast, PGE₂ production in other brain areas and the overall PGE₂ level in the brain were not critical for the febrile response. Partly restoring the PGE₂ synthesizing capacity in the anterior hypothalamus of mice lacking such capacity with a lentiviral vector resulted in a temperature elevation in response to an intraperitoneal injection of bacterial wall lipopolysaccharide (LPS). The data show that the febrile response is dependent on the local release of PGE2 onto its target neurons, possibly by a paracrine mechanism. Deletion of the receptor for the pyrogenic cytokine IL-6 on brain endothelial cells, but not on neurons or peripheral nerves, strongly attenuated the febrile response to LPS and reduced the induction of the Cox-2 expression in the hypothalamus. Furthermore, mice deficient of the IL- 6Rα gene in the brain endothelial cells showed a reduced SOCS3 mRNA induction, whereas IκB mRNA-levels were unaffected, suggesting that the IL-6 signaling occurs via STAT3 activation and not signaling through the transcription factor NF-κB. This idea was confirmed by the observation of attenuated fever in mice deficient of STAT3 in brain endothelial cells. These data show that IL-6, when endogenously released during systemic inflammation, is pyrogenic by binding to IL-6R on brain endothelial cells to induce prostaglandin synthesis in these cells. Finally, we demonstrate that mice with genetic deletion of uncoupling protein-1 (UCP-1), hence lacking functional brown adipose tissue, had a normal fever response to LPS, and that LPS caused no activation of brown adipose tissue in wild type mice. However, blocking peripheral cutaneous vasoconstriction resulted in a blunted fever response to LPS, suggesting that heat conservation, possibly together with shivering or non-shivering thermogenesis in the musculature, is responsible for the generation of immune-induced fever, whereas brown adipose tissue thermogenesis is not involved.  

Sickness responses to lipopolysaccharide (LPS) were examined in mice with deletion of the interleukin (IL)-1 type 1 receptor (IL-1R1). IL-1R1 knockout (1(0) mice displayed intact anorexia and HPA-axis activation to intraperitoneally injected LPS (anorexia: 10 or 120 mu g/kg; HPA-axis: 120 mu g/kg), but showed attenuated but not extinguished fever (120 g/kg). Brain PGE2 synthesis was attenuated, but Cox-2 induction remained intact. Neither the tumor necrosis factor-alpha (TNF alpha) inhibitor etanercept nor the IL -6 receptor antibody tocilizumab abolished the LPS induced fever in IL -1R1 KO mice. Deletion of IL -1R1 specifically in brain endothelial cells attenuated the LPS induced fever, but only during the late, 3rd phase of fever, whereas deletion of IL-1R1 on neural cells or on peripheral nerves had little or no effect on the febrile response. We conclude that while IL-1 signaling is not critical for LPS induced anorexia or stress hormone release, IL-1R1, expressed on brain endothelial cells, contributes to the febrile response to LPS. However, also in the absence of IL-1R1, LPS evokes a febrile response, although this is attenuated. This remaining fever seems not to be mediated by IL-6 receptors or TNFa, but by some yet unidentified pyrogenic factor. 

Fever is a hallmark of inflammatory and infectious diseases. The febrile response is triggered by prostaglandin E-2 synthesis mediated by induced expression of the enzymes cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase 1 (mPGES-1). The cellular source for pyrogenic PGE(2) remains a subject of debate; several hypotheses have been forwarded, including immune cells in the periphery and in the brain, as well as the brain endothelium. Here we generated mice with selective deletion of COX-2 and mPGES1 in brain endothelial cells. These mice displayed strongly attenuated febrile responses to peripheral immune challenge. In contrast, inflammation-induced hypoactivity was unaffected, demonstrating the physiological selectivity of the response to the targeted gene deletions. These findings demonstrate that PGE(2) synthesis in brain endothelial cells is critical for inflammation-induced fever.

Previous studies in rats have demonstrated that microsomal prostaglandin E synthase-1 (mPGES-1) is induced in brain vascular cells that also express inducible cyclooxygenase-2, suggesting that such cells are the source of the increased PGE2 levels that are seen in the brain following peripheral immune stimulation, and that are associated with sickness responses such as fever, anorexia, and stress hormone release. However, while most of what is known about the functional role of mPGES-1 for these centrally evoked symptoms is based on studies on genetically modified mice, the cellular localization of mPGES-1 in the mouse brain has not been thoroughly determined. Here, using a newly developed antibody that specifically recognizes mouse mPGES-1 and dual-labeling for cell-specific markers, we report that mPGES-1 is constitutively expressed in the mouse brain, being present not only in brain endothelial cells, but also in several other cell types and structures, such as capillary-associated pericytes, astroglial cells, leptomeninges, and the choroid plexus. Regional differences were seen with particularly prominent labeling in autonomic relay structures such as the area postrema, the subfornical organ, the paraventricular hypothalamic nucleus, the arcuate nucleus, and the preoptic area. Following immune stimulation, mPGES-1 in brain endothelial cells, but not in other mPGES-1-positive cells, was coexpressed with cyclooxygenase-2, whereas there was no coexpression between mPGES-1 and cyclooxygenase-1. These data imply a widespread synthesis of PGE2 or other mPGES-1-dependent products in the mouse brain that may be related to inflammation-induced sickness symptom as well as other functions, such as blood flow regulation.

The cytokine IL-6, which is released upon peripheral immune challenge, is critical for the febrile response, but the mechanism by which IL-6 is pyrogenic has remained obscure. Herewegenerated mice with deletion of themembranebound IL-6 receptor alpha (IL-6R alpha) onneural cells, on peripheral nerves, on fine sensory afferent fibers, and on brain endothelial cells, respectively, and examined its role for the febrile response to peripherally injected lipopolysaccharide. We show that IL-6R alpha on neural cells, peripheral nerves, and fine sensory afferents are dispensable for the lipopolysaccharide-induced fever, whereas IL-6R alpha in the brain endothelium plays an important role. Hence deletion of IL-6R alpha on brain endothelial cells strongly attenuated the febrile response, and also led to reduced induction of the prostaglandin synthesizing enzyme Cox-2 in the hypothalamus, the temperature-regulating center in the brain, as well as reduced expression of SOCS3, suggesting involvement of the STAT signaling pathway. Furthermore, deletion of STAT3 in the brain endothelium also resulted in attenuated fever. These data show that IL-6, when endogenously released during systemic inflammation, is pyrogenic by binding to IL-6R alpha on brain endothelial cells to induce prostaglandin synthesis in these cells, probably in concerted action with other peripherally released cytokines.

The proinflammatory cytokine interleukin-1 beta (IL-beta) plays a major role in the signal transduction of immune stimuli from the periphery to the central nervous system, and has been shown to be an important mediator of the immune-induced stress hormone release. The signaling pathway by which IL-1 beta exerts this function involves the blood-brain-barrier and induced central prostaglandin synthesis, but the identity of the blood-brain-barrier cells responsible for this signal transduction has been unclear, with both endothelial cells and perivascular macrophages suggested as critical components. Here, using an irradiation and transplantation strategy, we generated mice expressing IL-1 type 1 receptors (IL-1 RI) either in hematopoietic or non-hematopoietic cells and subjected these mice to peripheral immune challenge with IL-beta. Following both intraperitoneal and intravenous administration of IL-beta, mice lacking IL-1R1 in hematopoietic cells showed induced expression of the activity marker c-Fos in the paraventricular hypothalamic nucleus, and increased plasma levels of ACTH and corticosterone. In contrast, these responses were not observed in mice with IL-1R1 expression only in hematopoietic cells. Immunoreactivity for IL-1R1 was detected in brain vascular cells that displayed induced expression of the prostaglandin synthesizing enzyme cyclooxygenase-2 and that were immunoreactive for the endothelial cell marker CD31, but was not seen in cells positive for the brain macrophage marker CD206. These results imply that activation of the HPA-axis by IL-1 beta is dependent on IL-1R1 s on non-hematopoietic cells, such as brain endothelial cells, and that IL-1R1 on perivascular macrophages are not involved.

Acetaminophen is one of the world's most commonly used drugs to treat fever and pain, yet its mechanism of action has remained unclear. Here we tested the hypothesis that acetaminophen blocks fever through inhibition of cyclooxygenase-2 (Cox-2), by monitoring lipopolysaccharide induced fever in mice with genetic manipulations of enzymes in the prostaglandin cascade. We exploited the fact that lowered levels of a specific enzyme make the system more sensitive to any further inhibition of the same enzyme. Mice were immune challenged by an intraperitoneal injection of bacterial wall lipopolysaccharide and their body temperature recorded by telemetry. We found that mice heterozygous for Cox-2, but not for microsomal prostaglandin E synthase-1 (mPGES-1), displayed attenuated fever, indicating a rate limiting role of Cox-2. We then titrated a dose of acetaminophen that did not inhibit the lipopolysaccharide-induced fever in wild-type mice. However, when the same dose of acetaminophen was given to Cox-2 heterozygous mice, the febrile response to lipopolysaccharide was strongly attenuated, resulting in an almost normalized temperature curve, whereas no difference was seen between wild-type and heterozygous mPGES-1 mice. Furthermore, the fever to intracerebrally injected prostaglandin E₂ was unaffected by acetaminophen treatment. These findings reveal that acetaminophen, similar to aspirin and other non-steroidal anti-inflammatory drugs, is antipyretic by inhibiting cyclooxygenase-2, and not by inhibiting mPGES-1 or signaling cascades downstream of prostaglandin E₂.