LPS-stimulated NF-κB p65 dynamic response marks the initiation of TNF expression and transition to IL-10 expression in RAW 264.7 macrophages.
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Abstract
During injury and infection inflammation is a response by macrophages to effect healing and repair The kinetics of the responses of proinflammatory TNF anti inflammatory IL 10 and inflammatory master regulator NF B 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 I B was maximally reduced 45 min following LPS treatment but expression increased at 10 h reaching a maximum at 16 h NF B phosphorylation was significantly increased 45 min following LPS treatment maximal at 2 h and decreased to basal levels by 6 h Nuclear NF B expression was elevated 30 min following LPS treatment maximal by 45 min and returned to basal levels by 24 h Binding of nuclear NF B to consensus oligonucleotide sequences followed a similar pattern to that observed for p NF B but lasted slightly longer Following LPS treatment TNF mRNA expression began at 1 h was maximal at 6 h and decreased starting at 10 h TNF 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