The contribution of HMGB1 to TLR9 activation.

<p>C57BL/6 BMDCs were stimulated with <i>L. major</i> or vertebrate DNA (20 µg) complexed with rHMGB1 (1 µg) or alone (as control and reference) for 6 h. (<b>A</b>) Cytokines mRNA were detected by real-time PCR. The data are expressed as the n-fold increase with the expression in stimulated BMDCs by <i>L. major</i> or vertebrate DNA alone. The mRNA expression levels were normalized to the expression of the HPRT gene. nd: not detectable. (<b>B</b>) IL-6 and TNFα production was measured in supernatants by ELISA. (<b>C</b>) Time-course analysis of HMGB1 in the cytoplasm and nucleus of BMDCs not stimulated (NS) or stimulated by CpG 1826 (1 µg), <i>L. major</i> or vertebrate DNA (20 µg). <b><i>(left)</i></b> Analysis was performed by Western Blot at 30 and 60 min post-induction. <b><i>(right)</i></b> Quantification of HMGB1 in cytoplasmic extracts was normalized to that of actin and expressed as the n-fold difference with the unstimulated BMDCs. The mean and SEM of six independent experiments, each including both 30 min and 60 min extracts, is represented by the histogram. (<b>D</b>) C57BL/6 BMDCs were stimulated with <i>L. major</i> DNA alone (20 µg) or with glycyrrhizin (10 or 20 µg/ml) for 6 h. Percentage (%) of inhibition = [100-{cytokines production by <i>L. major</i> DNA with glycyrrhizin/cytokines production by <i>L. major</i> DNA alone}]×100. The data represent three independent experiments: one representative for C and the mean and SEM in A, B, D (*p<0,05, **p<0,01).