Inhibition of lipopolysaccharide-induced cyclooxygenase-2, tumor necrosis factor-a and [Ca 2+ ] i responses in human microglia by the peripheral benzodiazepine receptor ligand PK11195 Hyan B. Choi,* C. Khoo,* Jae K. Ryu,* Edo van Breemen,* Seung U. Kim  , à and James G. McLarnon* *Department of Pharmacology and Therapeutics, and  Division of Neurology, Faculty of Medicine, The University of British Columbia, Vancouver, British Columbia, Canada àBrain Disease Research Center, Ajou University, Suwon, South Korea Abstract The anti-inflammatory actions of the mitochondrial peripheral benzodiazepine receptor (PBR) agonist PK11195 [1-(2-chloro- phenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carbox- amide] were investigated in human microglia. Application of the microglial inflammatory stimulus lipopolysaccharide (LPS, at 100 ng/mL for 3 h), induced enhancement of the expres- sions of the inducible enzyme, cyclooxygenase-2 (COX-2) and the pro-inflammatory cytokine, tumor necrosis factor-a (TNF-a). PK11195 (at 50 lM) significantly inhibited the LPS- induced up-regulation of both inflammatory factors; at a lower concentration of PK11195 (2 lM) expression of TNF-a, but not COX-2, was reduced. Production of both factors, using immunocytochemistry for COX-2 and ELISA for TNF-a, was markedly reduced with 50 lM of PK11195 added to LPS solution. Acute application of LPS induced a transient increase in intracellular Ca 2+ [Ca 2+ ] i exhibiting both a slow development and recovery in kinetic behavior. This increase in [Ca 2+ ] i consisted primarily of a Ca 2+ influx component accompanied by a smaller mobilization from intracellular Ca 2+ stores. In the presence of PK11195, the amplitude of the [Ca 2+ ] i response induced by LPS was reduced by 54%. Another mitochondrial agent cyclosporin A (CsA), which also acts at the permeability transition pore (PTP) of mitochondrial membrane but at a site different from the PBR, was ineffective in reducing either the LPS-induced expression of COX-2 and TNF-a or the endotoxin increase in [Ca 2+ ] i . These results indicate that the mitochondrial effector PK11195 is a specific and effective agent for inhibiting LPS-induced microglial expressions of COX-2 and TNF-a and that modulation of Ca 2+ -mediated signaling pathways could be involved in the anti-inflammatory actions. Keywords: cyclooxygenase-2, human microglia, intracellular calcium, lipopolysaccharide, mitochondrial effector, PK11195, tumor necrosis factor-a. J. Neurochem. (2002) 83, 546–555. Microglia are resident cells of the CNS that serve a functional role as scavenger cells similar to that of peripheral macrophages. These cells have been implicated as key mediators of inflammation in the CNS, during which microglia proliferate and contribute to the inflammatory process by phagocytosis and secretion of cytokines, eicosa- noids and other agents (McGeer and McGeer 1995). Although inflammation of the CNS is intended as a homeostatic means to contain damage to neural tissue, such damage may occur from the inflammatory process itself (McGeer and McGeer 1995; Rogers and Griffin 1998). In this case microglial responses to inflammatory stimuli in the brain may actually exacerbate, rather than inhibit, neuronal damage. Received May 29, 2002; accepted June 18, 2002. Address correspondence and reprint requests to Dr James G. McLarnon, Department of Pharmacology and Therapeutics, Faculty of Medicine, 2176 Health Sciences Mall, The University of British Columbia, Vancouver, BC Canada, V6T 1Z3. E-mail: Mclarnon@interchange.ubc.ca Abbreviations used: [Ca 2+ ] i , intracellular calcium concentration; COX-2, cyclooxygenase-2; CsA, cyclosporin A; DAPI, 4,6-diamino- dino-2-phenylindole; DMEM, Dulbecco’s modified Eagle’s medium; ER, endoplasmic reticulum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LPS, lipopolysaccharide; PBS, phosphate-buffered saline; PBR, peripheral benzodiazepine receptor; PK11195, [1- (2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxa- mide; PSS, standard physiological solution; PTP, permeability transition pore; SOC, store-operated channel; TNF-a, tumor necrosis factor-a. Journal of Neurochemistry , 2002, 83, 546–555 546 Ó 2002 International Society for Neurochemistry, Journal of Neurochemistry , 83, 546–555