Fever is triggered by an elevation of prostaglandin E 2 (PGE 2 ) in the brain. However, the mechanism of its elevation remains unanswered. We herein cloned the rat glutathione-dependent microsomal prostaglandin E synthase (mPGES), the terminal enzyme for PGE 2 biosynthesis, and examined its induction in the rat brain after intraperitoneal injection of pyrogen lipopolysaccharide (LPS). In Northern blot analysis, mPGES mRNA was weakly expressed in the brain under the normal conditions but was markedly induced between 2 and 4 hr after the LPS injection. In situ hybridization study revealed that LPS-induced mPGES mRNA signals were mainly associated with brain blood vessels, especially vein or venular-type ones, in the whole brain area. Immunohistochemical study demonstrated that mPGESlike immunoreactivity was expressed in the perinuclear region of brain endothelial cells, which were identified as von Willebrand factor-positive cells. Furthermore, in the perinuclear region of the endothelial cells, mPGES was colocalized with cyclooxygenase-2 (COX-2), which is the enzyme essential for the production of the mPGES substrate PGH 2 . Inhibition of cyclooxygenase-2 activity resulted in suppression of both PGE 2 level in the CSF and fever (Cao et al., 1997), suggesting that the two enzymes were functionally linked and that this link is essential for fever. These results demonstrate that brain endothelial cells play an essential role in the PGE 2 production during fever by expressing COX-2 and mPGES.

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Cyclooxygenase-2 (COX-2), a key enzyme in the biosynthesis of prostaglandins, is induced in brain blood vessels by pyrogens, and its essential role in fever has been hypothesized. In this study, we determined (1) the type of cells that express cyclooxygenase-2 in brain blood vessels of lipopolysaccharide-treated rats, and (2) the precise relationship between the time course of fever and that of cyclooxygenase-2 protein expression in these cells. Five hours after the lipopolysaccharide injection (100 microg/kg, i.p.), cyclooxygenase-2-like immunoreactive cells were found in the parenchymal and subarachnoidal blood vessels. In these blood vessels, the cyclooxygenase-2-like immunoreactivity was restricted to the perinuclear region of the endothelial cells as revealed by a laser confocal microscopy, double-immunofluorescence staining with an endothelial marker, and immunoelectron microscopy. On the other hand, the cyclooxygenase-2-like immunoreactive cells were distinct from microglia or perivascular/meningeal macrophages as revealed by double immunostaining with macrophage/microglia-specific antibodies. Cyclooxygenase-2-like immunoreactive cells were first found at 1.5 hr after the lipopolysaccharide injection, at which time the fever had not been developed. After that, the number of cyclooxygenase-2-like immunoreactive cells and fever followed a similar time course, both being highest at 5 hr after the lipopolysaccharide injection and both returning to the baseline by 24 hr. These results demonstrate that brain endothelial cells are the primary sites where the activation of arachidonic acid cascade takes place during fever after intraperitoneal injection of lipopolysaccharide.

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We previously showed that a febrile dose of lipopolysaccharide (LPS) in rats resulted in induction of cyclooxygenase-2 (COX-2) mRNA in brain blood vessels/leptomeninges and telencephalic neurons. To elucidate the causal link between fever and LPS-induced COX-2 mRNA, we experimentally modified one or the other of these parameters and examined their relation. 1) LPS-induced fever was suppressed by pretreatment with a COX-2-specific inhibitor. 2) Levels of COX-2 mRNA in the neurons and blood vessels 2.5 h after LPS administration were even higher in the inhibitor-pretreated rats (afebrile) than in vehicle-pretreated ones (febrile). 3) After repeated administration of LPS, rats became tolerant to LPS, in which state LPS induced neither fever nor COX-2 mRNA in blood vessels/leptomeninges. When rats had not completely established LPS tolerance, they showed various degrees of fever that were closely correlated with the level of COX-2 mRNA in blood vessels but not with that in neurons. 4) Urethan anesthesia reduced basal as well as LPS-induced COX-2 mRNA in telencephalic neurons, but the rats still responded to LPS with fever and induction of COX-2 mRNA in the blood vessels/leptomeninges. These results suggest that COX-2 induced in brain blood vessels/leptomeninges is involved in the molecular mechanism of LPS-induced fever.