Hsp70 mediates production of pro-inflammatory factors in a Tlr4-dependent manner. A) Differential expression of cytokines, chemokines, as well as genes encoding Nos2 and subunits of the reactive oxygen species-producing NAD(P)H oxidase was assessed in primary cultures of astrocytes and microglia isolated from wild type (WTa and WTm referred to WT astrocytes and WT microglia) and Tlr4KO animals (Tlr4KOa and Tlr4KOm referred to Tlr4KO astrocytes and Tlr4KO microglia), and treated with Hsp70 (5 μg/ml) containing 1.5 pg/ml of endotoxins and PBS (control) during a 24-hour period. For each gene, results were expressed as a percentage of the corresponding value in the wild type astrocytes (WTa) treated with PBS (**P < 0.01, n = 6). B) The amount of Tnf in culture media from astrocytes and microglia isolated from wild type and Tlr4KO mice and treated with Hsp70 were determined by using mouse Tnf-specific ELISA kits. (**P < 0.01, n = 6). C) Hsp70 induces production of the immature form of Il1b, but not the active form of Il1b in astrocytes. The astrocytes isolated from wild type and Tlr4KO animals were treated with Hsp70 (5 μg/ml) and lysed after 24 hours. Forms of Il1b were analyzed by western blot analysis. D) Heat inactivation of recombinant Hsp70 significantly reduced pro-inflammatory response of treated primary astrocytes. Primary astrocytes were treated for 24 hours with recombinant Hsp70 (5 μg/ml), heat-inactivated recombinant Hsp70 (5 μg/ml), and PBS as a control (**P < 0.01, n = 6). For each gene, results are expressed as a percentage of corresponding value in the astrocytes treated with PBS (control) (denHsp70 samples were heated for one hour at 56°C before treatment of cell cultures). Hsp70, heat shock protein 70; PBS, phosphate buffered saline; WT, wild type.