Chunyu Cao

Coexpression of Microsomal-Type Prostaglandin E Synthase with Cyclooxygenase-2 in Brain Endothelial Cells of Rats during Endotoxin-Induced Fever

Fever is triggered by an elevation of prostaglandin E2(PGE2) 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 PGE2 biosynthesis, and examined its induction in the rat brain after intraperitoneal injection of pyrogen lipopolysaccharide (LPS). In Northern blot analysis, mPGESmRNA 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-inducedmPGES mRNA signals were mainly associated with brain blood vessels, especially vein or venular-type ones, in the whole brain area. Immunohistochemical study demonstrated that mPGES-like 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 PGH2. Inhibition of cyclooxygenase-2 activity resulted in suppression of both PGE2 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 PGE2 production during fever by expressing COX-2 and mPGES.

Endothelial cells of the rat brain vasculature express cyclooxygenase-2 mRNA in response to systemic interleukin-1β : a possible site of prostaglandin synthesis responsible for fever

We previously showed that intraperitoneal injection of lipopolysaccharide induced cyclooxygenase-2 (COX-2) mRNA in as yet unidentified cells of blood vessels and leptomeninges in the rat brain and proposed a possible role of these cells as the source of prostaglandin E2 in the genesis of fever (Cao et al., Brain Res., 697 (1995) 187-196). In the present study, to proceed further with this line of research, we addressed the following two questions: first, does a pyrogenic dose of interleukin-1 beta (IL-1 beta), an endogenous pyrogen, induce COX-2 mRNA in the brain blood vessels and leptomeninges? Secondly, if it does, what type of cells are positive for COX-2 mRNA? Intraperitoneal injection of recombinant human IL-1 beta (30 micrograms/kg) induced fever in rats and an in situ hybridization study revealed that faint but significant COX-2 mRNA signals appeared in the blood vessels and leptomeninges at 1.5 h after the injection (the early rising phase of fever). The mRNA signals increased in number and intensity at 4 h (early plateau phase), decreased at 6.5 h (early recovery phase), and completely disappeared by 10 h after the injection (late recovery phase). The COX-2 mRNA positive cells in the blood vessels were likely to be the endothelial cells since the corresponding cells in the adjacent mirror-imaged section also expressed mRNAs for intracellular adhesion molecule-1 and the type-I interleukin-1 receptor, although those in the leptomeninges still remained unidentified. These results imply that circulating IL-1 beta acts on its receptor on the endothelial cells of the brain vasculature to induce COX-2 mRNA, which is possibly responsible for the elevated level of PGE2 seen during fever.

Induction by lipopolysaccharide of cyclooxygenase-2 mRNA in rat brain; its possible role in the febrile response

Cyclooxygenase 2 (COX-2) is a newly discovered isoform of cyclooxygenase that is inducible by lipopolysaccharide (LPS) or cytokines. This enzyme is considered to play a major role in inflammatory processes by catalyzing the production of prostaglandins. In the present study, induction of COX-2 mRNA in the rat brain by intraperitoneal injection of LPS was studied by the in situ hybridization technique with special attention paid to timing and sites of induction along with the time course of fever. In situ hybridization was carried out on sections of rat brain, 1 h (latent phase), 2.5 h (maximally febrile phase), 4 h (plateau phase), and 7 h (recovery phase) after the LPS injection, as well as on those from the brains of untreated and saline-injected rats. Injection of LPS induced COX-2 mRNA in the brain in two different constituents: neuronal cells and non-parenchymal cells of the blood vessels and leptomeninges. Induction in the neuronal cells was restricted to some telencephalic areas where the COX-2 mRNA signal was also detected in control animals. The signal was maximally enhanced by 50 to 80% over the basal level 1 h after LPS injection. The COX-2 mRNA signal was hardly detectable in neuronal and glial cells in other brain regions, including the preoptic area, either in control or LPS-injected rats. Strong COX-2 mRNA signals, however, appeared in the inner surface of blood vessels and the leptomeninges over the entire brain, including the preoptic area and its vicinity. The signals were not detectable in the brains of control rats and were most intense in the brains of rats treated with LPS for 2.5 h or 4 h. These results demonstrate that two major cell groups in the brain, neuronal cells and non-parenchymal cells, are responsible for the enhanced production of prostaglandins after systemic LPS treatment. Considering the site and timing of induction, we propose a possible role for blood vessels and leptomeninges as the source of prostaglandin E2 in the genesis of fever.

Cyclooxygenase-2 is induced in brain blood vessels during fever evoked by peripheral or central administration of tumor necrosis factor

Cyclooxygenase-2 (COX-2) is an inducible type of enzyme that is involved in prostaglandin biosynthesis. In the present study, we examined whether or not COX-2 is involved in fever that is induced by tumor necrosis factor-alpha (TNF-alpha) and, if so, where in the brain COX-2 is induced by this factor. Intraperitoneal (i.p.) injection of TNF-alpha into rats evoked a fever that started 1 h after the TNF injection, peaked 3 h after the injection, and then gradually declined. The fever was suppressed by pretreatment with a COX-2-specific inhibitor. With a time course similar to that of fever, COX-2 mRNA was induced in brain blood vessels. On the other hand, in some of the telencephalic neurons, COX-2 mRNA was constitutively expressed under the normal condition; but its level gradually decreased during the course of fever. Fever was also evoked by an intracerebroventricular (i.c.v.) injection of TNF-alpha. This febrile response was also suppressed by a COX-2 specific inhibitor and was associated with the induction of COX-2 mRNA in the brain blood vessels. On the other hand, the telencephalic neurons did not show consistent change in COX-2 mRNA level after i.c.v. injection of TNF-alpha or saline. COX-2-like immunoreactivity was found in some cells of the brain blood vessels 3 h after the TNF-alpha injection by either i.p. or i.c.v. route. Most of the COX-2-like immunoreactive cells were endothelial cells since COX-2-like immunoreactivity was colocalized with von Willebrand factor, an endothelial cell marker, in the same cells. These results suggest that the brain blood vessels are the major sites where TNF-alpha enhances PG biosynthesis after peripheral as well as after central injection, and provides further evidence supporting the hypothesis that COX-2 induced in the brain blood vessels is involved in fever.

Pyrogenic cytokines injected into the rat cerebral ventricle induce cyclooxygenase‐2 in brain endothelial cells and also upregulate their receptors

Peripheral immunological insults induce interleukin (IL)‐1β and IL‐6 in the brain. To elucidate the mechanism(s) of fever evoked by these brain‐derived cytokines, and possible interactions between them, we examined in rats: (i) whether cyclooxygenase‐2 is responsible for fever evoked by central injection of these cytokines; (ii) if so, where in the brain cyclooxygenase‐2 is induced; (iii) where the receptors for these cytokines are located; and (iv) how the expression of these receptors is influenced by the cytokines. Intracerebroventricular injection of these cytokines evoked fever that was suppressed by a cyclooxygenase‐2 inhibitor. Brain endothelium was the site of cyclooxygenase‐2 induction by these cytokines. IL‐1 receptor (IL‐1R) was constitutively expressed in brain endothelium, and its mRNA was further upregulated by either cytokine. IL‐6R mRNA was constitutively expressed in the cerebral cortex, and was newly induced in as yet unidentified cells in brain blood vessels by either cytokine. Messenger RNAs for cyclooxygenase‐2, IL‐1R, and IL‐6R were often observed in the same blood vessels. These results suggest that COX‐2 induced in brain endothelium is, at least in part, involved in the fever evoked by these cytokines, and that one possible interaction between these two cytokines is mutual upregulation of their receptors in the endothelium or perivascular cells, resulting in augmentation of their actions.

Chapter 15 Prostaglandin system in the brain: sites of biosynthesis and sites of action under normal and hyperthermic states

Publisher Summary This chapter reviews the recent progress in research on the brain prostaglandin (PG) system, with special attention on the in vivo location of enzymes involved in their biosynthesis and of their specific receptors under physiological, as well as pathological conditions. The recent biochemical studies, especially those that employed molecular biological techniques, have provided a great deal of information on the molecular nature of individual components of the PG system. Fever and hyperthermia are essentially distinct patho-physiological states, the former representing regulated elevation of the body temperature caused by immunological challenge and the latter representing its passive elevation caused by excessive heat load. In fact, inhibitors of PG synthesis, such as indomethacin, suppress fever but are not effective against hyperthermia. The release of PGs is dramatically increased by ischemia/reperfusion of the brain or by traumatic injury of the central nervous and treatment with a cyclooxygenase inhibitor, such as indomethacin, improves the neuronal injury. The onset and progression of Alzheimers disease may be also slowed by the treatment with cyclooxygenase inhibitors. Thus, PGs are the key molecules involved in pathological changes in the central nervous system.

Induction of cyclooxygenase-2 in the brain by cytokines.

The present study revealed that (1) a pyrogenic dose of IL-1 beta induced COX-2 mRNA in the brain vasculature and leptomeninges, and (2) the cells positive for COX-2 mRNA in the blood vessels were endothelial cells that possess receptors for IL-1. These results imply that circulating IL-1 beta acts on its receptor on the endothelial cells of the brain vasculature to induce COX-2 mRNA, which is possibly responsible for the elevated level of PGE2 seen during fever. In this sense, the endothelial cells in the brain vasculatures seem to play a role as an interface between the blood borne substance and the brain. The nature of the COX-2 mRNA-positive cells in the leptomeninges will be identified in our future study.

Possible Role of Cyclooxygenase‐2 in the Brain Vasculature in Febrile Response

These results support the hypothesis that the brain vasculature is the site of PGE2 production responsible for LPS-induced fever. LPS seems to increase the PGE2 level in the entire brain via the induction of COX-2. Fever may be mediated by PGE2 which is produced in the blood vessels in the preoptic area or which is produced in other parts of the brain and transported to the preoptic area through the ventricular system.

1002 Causal relationship between cyclooxygenase-2 induction and fever by lipo-polysaccharide

IL-IIJ is one of key mediators of pathological CNS symptoms associated with infection, such as fever, anorexia, slowwave sleep and activation of HPA-axis. We have previously shown that peripheral administration of IL-1B or lipopolysaccharide (LPS) induce rapid and transient expression of c-fos, which might contribute to the manifestation of symptoms, The same treatment will also cause the synthesis of IL-1B in the brain, but how the brain-derived IL-18 contributes to the progress of illness is still obscure. In the present study we intended to elucidate the time course and the localization of IL-18 mRNA expression in response to peripheral infection. We injected 3-5 mg/kg of LPS intraperitoneally into male SD rats (150-200 g body weight). Control animals received injection of saline. These animals were killed by decapitation 5h, 8h and 24h after the injection. Frozen sections of the brain were processed for in situ hybridization using an oligonucleotide probe. Signals for IL-la mRNA were first detected 8h after the injection in glia-like cells in discrete brain regions such as the OVLT, choroid plexus, septal nuclei, med. & lat. habenula, hypothalamic area including PVH and arcuate nucleus, central amygdaloid nucleus, nucleus tractus solitarius etc. After 24 h, signals in these areas were reduced. These findings suggest that the synthesis of IL-If3 in the CNS is slowly activated mainly through humoral pathway during peripheral infection.