Kinetics of lipopolysaccharide-induced transcription factor activation/inactivation and relation to proinflammatory gene expression in the murine spleen

Abstract

Bacterial lipopolysaccharide (LPS) elicits inflammation and endotoxic shock by inducing proinflammatory cytokine gene expression. The purpose of this study was to test the hypothesis that differential activation of transcription factor binding in the spleen correlates with proinflammatory cytokine gene expression in mice exposed to LPS. When proinflammatory cytokine expression in spleen was evaluated in mice injected ip with 4 mg/kg LPS over an 8-h period, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 mRNAs were elevated up to 5-, 6-, and 300-fold, respectively, over vehicle controls. Both TNF- α and IL-6 mRNA peaked at 2 h and begin to decline thereafter, whereas IL-1β mRNA remained elevated from 2 to 8 h. The capacities of splenic nuclear proteins to bind to six different consensus transcriptional control motifs associated with proinflammatory cytokine promoters were also measured over 8 h. Electrophoretic mobility shift assay (EMSA) revealed that binding activity was markedly increased at 0.5 to 8 h for activator protein-1 (AP-1) as were CCAAT enhancer-binding protein (C/EBP) and nuclear factor κB (NF-κB) at 0.5 to 1.5 h. At 0.5 h, cyclic AMP response element (CRE)-binding protein (CREB) and binding was slightly elevated, whereas activator protein- 2 (AP-2) and specificity protein 1 (Sp1) binding were not affected. Antibody supershift EMSA and Western blot analysis confirmed that increased binding of these factors correlated with LPS-induced increases in nuclear concentrations of AP-1 (c-Jun, phosphorylated c-Jun, Jun D, and Jun B), C/EBPβ, NF-κB (p50, p65, and c-Rel), CREB (CREB-1, CREB-2, and ATF-2), and AP-2α proteins. Remarkably, after 8 h, C/EBP, CREB, AP-2, and Sp1 binding activities were greatly depleted relative to both naive and corresponding vehicle controls. When mice were exposed to a second dose of LPS, 8 h after a 4 mg/kg priming dose, TNF-α and IL-6 mRNA responses were markedly impaired, suggesting that the mice were endotoxin tolerant at this time point. Taken together, the quiescent, active, and suppressive phases of transcription factor binding observed in this model were highly consistent with the rapid transient nature of LPS-induced proinflammatory cytokine expression in vivo as well as tolerance to secondary LPS exposure.