Microglia produce TNFα following poly(I:C) stimulation. A:Microglia (MG), astrocytes (AST) or oligodendrocytes (OL) were treated with or without poly(I:C) (PIC; 50 μg/ml) for 24 h and TNFα levels were determined by ELISA. Results are means ± SEM of 3 independent experiments. B:Expression of TLR3 (top), TNFα, IL-1β, IL-6, Iba-1 and GFAP (bottom) was determined by RT-PCR from monocultures treated with PBS or poly(I:C) for 24 h. Results are from 2 independent experiments and are representative of 3 independent experiments for each product. C:Kinetic analysis of TNFα production following stimulation with varying doses of poly(I:C) in microglia (left) and astrocyte (right) monocultures. Results are combined means ± SEM of triplicate wells and are representative of 3 independent experiments. D, E:Intracellular cytokine staining of mixed glial cultures treated with vehicle (sterile PBS; 5 μl/ml), poly(I:C) or LPS for 8 h. TNFα immunoreactivity did not localize to GFAP+ astrocytes (D), but instead to Iba-1+ microglia (E). Results are representative of 3 independent experiments. F:Expression of TNFα, CCL2 and CCL5 was determined by RT-PCR from astrocytes treated with vehicle (sterile PBS; 5 μl/ml) or poly(I:C) (50 μg/ml) for 3-6 h (left) or increasing concentrations (right) of poly(I:C) for 6 h. Results are representative of 2 independent experiments. G-J:Both CCL2 (G, I) and CCL5 (H, J) expression were dose- and time-dependently increased upon poly(I:C) stimulation. Results are means ± SEM from duplicate (CCL2) or triplicate (CCL5) samples and are representative of 3 independent experiments. *, p < 0.05, **, p < 0.01, ***, p < 0.001.