PII: S0361-9230(02)00951-6 Brain Research Bulletin, Vol. 59, No. 6, pp. 447–452, 2003 Copyright © 2002 Elsevier Science Inc. All rights reserved. 0361-9230/03/$–see front matter Endothelial activation is an intermediate step for peripheral lipopolysaccharide induced activation of paraventricular nucleus Ning Quan, 1,2,3∗ Lingli He 1 and Wenmin Lai 1 1 Department of Oral Biology, Health Science Center, Ohio State University, Columbus, OH, USA; 2 Institute for Behavioral Medicine Research, Columbus, OH, USA; and 3 Neuroscience Graduate Studies Program, Ohio State University, Columbus, OH, USA [Received 1 August 2002; Revised 30 September 2002; Accepted 7 October 2002] ABSTRACT: Peripheral injection of bacterial endotoxin lipopoly- saccharide (LPS) activates the paraventricular nuclei of the hypothalamus (PVN), and consequently the hypothalamus- pituitary adrenal axis. Inflammatory cytokine interleukin-1 (IL-1) has been considered as a key mediator that translates the peripheral LPS stimulation into neuronal activation in the PVN. Several studies attempting to localize the expression of receptors for IL-1 (IL-1R), however, have failed to detect IL-1R on PVN neurons. It remains unclear, therefore, how IL-1 might stimulate the neurons of the PVN. In this study, we traced the cellular responsiveness to IL-1 by measuring the mRNA production of the cytokine responsive gene Iκ Bα in the PVN. After either peripheral injection LPS or intracerebroventricular (i.c.v.) injection of IL-1β,Iκ Bα mRNA was found mostly in en- dothelial cells of the brain with the highest level of expression in PVN blood vessels. In addition, both injections also induced the expression of cyclooxygenase-2 in brain endothelial cells. Pretreatment with indomethacin, a cyclooxygenase inhibitor, blocked LPS and IL-1 induced neuronal activation in the PVN, but did not reduce the induction of Iκ Bα in PVN endothelium. These results show that IL-1 acting on the endothelial cells of the brain, particularly in the PVN, may be an intermediate step relating peripheral immune signals to the brain. © 2002 Elsevier Science Inc. All rights reserved. KEY WORDS: IL-1, Blood–brain barrier, Iκ B, COX-2, HPA axis. INTRODUCTION Activation of PVN neurons by interleukin-1 (IL-1) was docu- mented by the first set of studies that established a clear link between the immune system and the central nervous system. In these studies, peripherally administered IL-1 was found to stim- ulate the secretion of corticosterone by increasing the level of corticotropin releasing hormone (CRH) production from the PVN [1,19]. Later, it was shown that activation of the immune system by bacterial endotoxin lipopolysaccharide (LPS) similarly activated the hypothalamus–pituitary–adrenal (HPA) axis, and this activa- tion can be blocked by intracerebral, but not peripheral, injection of interleukin-1 receptor antagonist (IL-1ra) [9]. It has been pos- tulated that IL-1, induced by peripheral immune stimulation, acts in the CNS to activate the HPA axis. It is not clear, however, whether IL-1 can act directly on PVN neurons. An early study showed that radiolabeled IL-1 bond di- rectly to neurons of the hypothalamus and suggested that there may be IL-1 receptors (IL-1R) on PVN neurons [7]. The functional iso- type of the IL-1 receptor, IL-1RI, however, has not been detected in PVN neurons by either in situ hybridization [5] or immunohisto- chemistry [8]. Interestingly, IL-1RI was found to localize primar- ily on endothelial cells of the brain [5]. In addition, IL-1 and LPS stimulate the production of the inducible cyclooxygenase, COX-2, also in brain endothelial cells [3,16]. Because PVN activation in- duced by either LPS or IL-1 can be attenuated by inhibitors of COX [2,10], it is possible that endothelial cells are a critical inter- mediate for the activation of PVN. In this study, we determined the cell type that responded directly to cytokine stimulation in the PVN by examining the expression of the cytokine responsive gene Iκ Bα and investigated whether endothelial cells within the PVN mediate IL-1 induced activation of PVN neurons. MATERIAL AND METHODS Animals Male Sprague–Dawley rats (175–200 g, Harlan, IN) were group housed (three per cage) with food and water available ad lib. in a light (0600 to 1800 h) and temperature-controlled environment (20–22 ◦ C). All procedures were approved by the Ohio State Uni- versity Animal Care and Use Committee. Experimental Procedures The animals were divided into eight experimental groups (n = 4 in each group). For fresh frozen tissue collection, all animals were sacrificed by decapitation and their brains removed for analysis. Experiment 1 comprised two groups. Animals were injected in- traperitoneally (i.p.) with phosphate-buffered saline (PBS, Group 1) or 1 mg/kg of LPS (Escherichia coli, serotype 055 Group 2). They ∗ Address for correspondence: Dr. Ning Quan, Department of Oral Biology, Health Science Center, Ohio State University, 2214 Postle Hall, 305 W. 12th Avenue, Columbus, OH 43210, USA. Fax: +1-614-292-6087; E-mail: quan.14@osu.edu 447