Bacterial Lipopolysaccharide Increases Prostaglandin Production by Rat Astrocytes via Inducible Cyclo-oxygenase: Evidence for the Involvement of Nuclear Factor B Giuseppa Pistritto,* Ornella Franzese,† Giacomo Pozzoli,* Cesare Mancuso,* Giuseppe Tringali,* Paolo Preziosi,* and Pierluigi Navarra* ,1 *Institute of Pharmacology, Catholic University Medical School, Rome, Italy; and †Department of Experimental Medicine and Biochemical Sciences, University of “Tor Vergata,” Rome, Italy Received August 26, 1999 This study was set to investigate the mechanisms through which bacterial lipopolysaccharide (LPS) stimulates prostaglandin (PG) production in rat astro- cytes. Primary cultures of rat hypothalamic astrocytes were established. Cells were treated with LPS alone or LPS plus antagonists of various pathways, and the subsequent changes in cyclo-oxygenase (COX) activity were monitored by measuring a COX end product, PGE2, released into the incubation medium. It was found that (i) LPS produced a concentration-depen- dent increase in PGE2 release from astrocytes. The potency of LPS was significantly increased by the ad- dition of serum into the incubation medium; (ii) after 24 h of incubation, inducible COX (COX-2) accounts for most of the LPS-stimulated PG production, as the lat- ter was markedly reduced by dexamethasone and the specific COX-2 inhibitor NS 398; and (iii) nuclear fac- tor B appears to play a role in the activation of COX-2 induced by LPS, since certain inhibitors of this tran- scription factor were able to antagonize, at least in part, the effects of LPS on PGE2 release. © 1999 Academic Press Key Words: lipopolysaccharide; prostaglandin E2; in- ducible cyclo-oxygenase; nuclear factor B; dexameth- asone; aurothiomalate; diethyldithiocarbamate; astro- cytes. Recent studies have enlightened some peculiarities in the expression and regulation of type 1 and type 2 cyclo-oxygenase (COX-1 and -2, also referred to as con- stitutive and inducible COX respectively) in the brain. Several authors have shown that both isoforms are predominantly associated with neurons, where COX-2 is constitutively expressed, whereas the contribution of glial cells to brain prostaglandin (PG) production ap- pears to be negligible (1– 4). The opposite concept emerges from in vitro studies on primary cultures of glial cells. In fact, cultured rat astrocytes produce and release much higher amounts of PGE2 compared to neurons (5). COX-2 gene expression is virtually unde- tectable in primary cultures of mouse astrocytes and rat microglia under basal conditions, but it is potently induced by such stimuli as bacterial lipopolysaccharide (LPS) or the pro-inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor- (TNF-) (6, 7). Thus, primary cultures of glial cells produce and release prostanoids with a profile similar to those of monocytes and other peripheral cells of the immune-inflammatory lineage. As a consequence, cultured glial cells are cur- rently considered a valuable in vitro model to investi- gate the inflammatory processes of the brain underly- ing such disorders as Alzheimer disease (8). In the present study, we have investigated the mech- anisms through which LPS stimulates PG production in primary cultures of neonatal rat astrocytes. PGE2 released in the incubation medium was taken as a marker of COX activity. The effect of LPS on PG pro- duction was abolished by the inhibitor of COX-2 syn- thesis, dexamethasone (DEX). However, no glucocorti- coid responsive element is present in the promoter region of COX-2 gene, suggesting that the inhibitory effect of DEX is exerted on intermediate factors in- volved in the regulation of COX-2 gene. We have there- fore investigated one of such factors, nuclear factor B (NF-B) (9), providing evidence that the latter partic- ipates into the process of PG production activated by the endotoxin. 1 To whom correspondence should be addressed at Institute of Pharmacology, Catholic University Medical School, Largo Francesco Vito 1-00168 Rome, Italy. Fax: +39 6 3050159. E-mail: pnavarra@ rm.unicatt.it. Biochemical and Biophysical Research Communications 263, 570 –574 (1999) Article ID bbrc.1999.1413, available online at http://www.idealibrary.com on 570 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.