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-1beta and TNFalpha or their receptors, and pretreatment of IL-6 knockout mice with soluble TNFalpha 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.

Interleukin-6 (IL-6) is critical for the febrile response to peripheral immune challenge. However, the mechanism by which IL-6 enables fever is still unknown. To characterize the IL-6 dependent fever generating pathway, we used microarray analysis to identify differentially expressed genes in the brain of lipopolysaccharide (LPS)-treated IL-6 wild type and knock-out mice. Mice lacking IL-6 displayed two-times lower expression of the lipocalin-2 gene (lcn2), and this difference was confirmed by real-time RT-PCR. Conversely, induction of lipocalin-2 protein was observed in brain vascular cells following i.p. administration of recombinant IL-6, suggesting a direct relationship between IL-6 and lipocalin-2.Immunohistochemical analysis also revealed that LPS-induced lipocalin-2 is expressed by brain endothelial cells and is partly co-localized with cyclooxygenase-2 (Cox-2), the rate limiting enzyme for the production of inflammatory induced prostaglandin E 2 (PGE 2 ), the key mediator of fever. The direct role of lipocalin-2 in fever was examined in LPS-challenged lipocalin-2 knock-out mice. In both male and female mice, normal fever responses were observed at near-thermoneutral conditions (29-30°C), but when recorded at normal room temperature (19-20°C), the body temperature of lipocalin-2 knock-out female mice displayed an attenuated and delayed fever response compared with their wild type littermates. This difference was reflected in significantly attenuated mRNA expression of Cox-2 in the brain of lipocalin-2 knock-out female mice, but not of male mice, following challenge with peripheral LPS. Our findings suggest that IL-6 influence the expression of lipocalin-2, which in turn may be involved in the control of the formation of Cox-2, and hence the central PGE 2 -production.We have thus identified lipocalin-2 as a new factor in the pathway of inflammatory IL-6 signaling. The effect of lipocalin-2 in LPS-induced fever is gender-dependent and ambient temperature-specific, however the effects of lipocalin-2 on fever are discrete and thus Hamzic et al., p. 3 lipocalin-2 cannot be considered as a major mediator of the IL-6-dependent fever generating pathway.

We examined the expression of interleukin (IL)-1beta, IL-6 and tumour necrosis factor (TNF) alpha in mice lacking microsomal prostaglandin E synthase-1 (mPGES-1), which neither produce prostaglandin E(2), nor mount a febrile response upon immune challenge. Intraperitoneal lipopolysaccharide (LPS) injection resulted in a strongly induced expression of all three cytokines in the brain and viscera, similar to wild-type animals. Several brain regions additionally showed modest induction of receptors for these cytokines in both genotypes. Telemetric recordings of body temperature showed that the mPGES-1 deficient mice remained afebrile upon LPS challenge, in contrast to the prominent fever displayed by the wild-type mice. These data demonstrate that LPS-induced cytokine expression occurs independently of prostaglandin E(2), and imply that endogenously expressed IL-1beta, IL-6, and TNFalpha are not pyrogenic per se, supporting the role of prostaglandin E(2) as the final and obligatory mediator of LPS-induced fever.

KO background, but with intact IL-6 production in cells of hematopoietic origin, only showed a minor elevation of the body temperature after peripheral LPS injection. While they displayed significantly elevated levels of IL-6 both in plasma and CSF compared with control mice, the increase was modest compared with that seen in LPS injected mice on a WT background, the latter being approximately 20 times larger in magnitude. These results suggest that IL-6 of non-hematopoietic origin is the main source of IL-6 in LPS-induced fever, and that IL-6 produced by hematopoietic cells only plays a minor role.Hamzic et al., page 3