Pharmacological investigations on lipopolysaccharide-induced permeability changes in the blood– brain barrier in vitro Pieter J. Gaillard,* A. (Bert) G. de Boer, and Douwe D. Breimer Blood-Brain Barrier Research Group, Department of Pharmacology, Leiden/Amsterdam Center for Drug Research (LACDR), Leiden University, The Netherlands Received 12 September 2001 Abstract Lipopolysaccharide-induced changes in blood– brain barrier (BBB) permeability were investigated with a pharmacological approach in vitro. Lipopolysaccharide induced a concentration- and time-dependent (non)reversible opening of the BBB, and brain astrocytes make brain capillary endothelial cells (BCEC) resistant to this BBB disruption. De novo protein synthesis was essential for the recovery, because cycloheximide prevented the recovery process. Dexamethasone pretreated BCEC were more resistant to lipopolysaccharide, while no protective response was induced by heat shock nor by inhibition of P-glycoprotein. BBB opening was tempered by free radical inhibitors (i.e., pretreatment with N-acetyl-cysteine or uric acid combined with deferroxamine mesylate). No effects of modulators of prostanoid-, leukotriene-, or platelet-activating factor pathways were observed. Therefore, lipopolysaccharide-induced BBB opening seems to be primarily mediated by excessive free radical production. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Blood– brain barrier; Lipopolysaccharide; Inflammation; Brain capillary endothelial cells; Brain astrocytes Introduction Lipopolysaccharide (LPS) is an endotoxin extracted from the cell wall of gram-negative bacteria and is the primary endotoxin involved in inflammatory processes that change the permeability of the blood– brain barrier (BBB) during bacterial infections, like bacterial meningitis and sepsis (de Vries et al., 1995). In general, LPS bound to serum-derived LPS binding protein, activates CD14-posi- tive mature myeloid cells (i.e., monocytes, macrophages, and polymorphonuclear leukocytes) and CD14-negative cells (B-cell lines and endothelial cells) through an interac- tion with membrane-bound CD14 and soluble CD14, re- spectively (Pugin et al., 1995). At the cellular level, LPS has been described to exert its effects through activation of several different signal transduction pathways, in as many different cell types. In particular, LPS has been described to induce transcrip- tion of acute phase proteins and to activate protein kinases, which leads to formation or activation of several transcrip- tion factors (e.g., AP-1, NF-B, etc.) (Verma et al., 1995). These pleiotropic transcription factors have been described to regulate transcription of, e.g., proinflammatory cytokines (e.g., TNF, IL-1, IL-2, IL-6, IL-8), adhesion molecules (e.g., endothelial leukocyte adhesion molecule (ELAM), VCAM, ICAM), inducible NO synthase (iNOS), inducible cyclo-oxygenase (COX-2), and latent matrix metallopro- teinases (MMPs), but also of potentially protective proteins like NF-B inhibitory proteins (IB), IFN-, tissue inhibi- tors of metalloproteinases (TIMPs), metallothionein (MT), superoxide dismutase (SOD), P-glycoprotein (Pgp), and heat shock proteins (HSPs) (Verma et al., 1995; Feinstein et al., 1996). In addition, LPS has been described to directly activate phospholipase A2 (PLA2), and to produce arachi- donic acid leading to production of eicosanoids (i.e., pro- stanoids and leukotrienes) and platelet-activating factor * Corresponding author. Department of Pharmacology, LACDR, Lei- den University, Gorlaeus Laboratories, P.O. Box 9502, 2300 RA Leiden, The Netherlands. Fax: +31-71-527-6292. E-mail address: Gaillard@LACDR.LeidenUniv.nl (P.J. Gaillard). R Available online at www.sciencedirect.com Microvascular Research 65 (2003) 24 –31 www.elsevier.com/locate/ymvre 0026-2862/03/$ – see front matter © 2003 Elsevier Science (USA). All rights reserved. PII: S0026-2862(02)00009-2