LPS-stimulated NF-κB p65 dynamic response marks the initiation of TNF expression and transition to IL-10 expression in RAW 264.7 macrophages

Abstract

During injury and infection, inflammation is a response by macrophages to effect healing and repair. The kinetics of the responses of proinflammatory TNFα, anti-inflammatory IL-10, and inflammatory master regulator NF-κB elicited by lipopolysaccharide (LPS) may be critical determinants of the inflammatory response by macrophages; however, there is a lack of homogeneous kinetic data in this pathway. To address this gap, we used the RAW 264.7 macrophage cell line to define intracellular signaling kinetics and cytokine expression in cells treated with LPS for 15 min to 72 h. The abundance of IκBα was maximally reduced 45-min following LPS treatment, but expression increased at 10-h, reaching a maximum at 16 h. NF-κB phosphorylation was significantly increased 45-min following LPS treatment, maximal at 2-h, and decreased to basal levels by 6-h. Nuclear NF-κB expression was elevated 30-min following LPS treatment, maximal by 45-min, and returned to basal levels by 24-h. Binding of nuclear NF-κB to consensus oligonucleotide sequences followed a similar pattern to that observed for p-NF-κB, but lasted slightly longer. Following LPS treatment, TNFα mRNA expression began at 1-h, was maximal at 6-h, and decreased starting at 10-h. TNFα protein secretion in conditioned growth medium began at 4-h and was maximal by 16-h. IL-10 mRNA expression was induced by LPS at 10-h, and was maximal at 16-h. IL-10 protein secretion was induced at 16-h and was maximal at 24-h. Our data reveal the temporal kinetics of pro- and anti-inflammatory signaling events that may be important therapeutic targets for inflammatory diseases.