Preexposure of murine macrophages to CpG-containing oligonucleotides results in nuclear factor κB p50 homodimer-associated hyporesponsiveness

Long Jin, Daniel P. Raymond, Traves D. Crabtree, Shawn J. Pelletier, Christine K. Rudy, Timothy L. Pruett, Robert G. Sawyer

Research output: Contribution to journalArticlepeer-review

10 Scopus citations


Background. DNA containing the CpG motif is associated with immunomodulation of the innate immune response. Preexposure of macrophages to CpG DNA elicits a hyporesponsiveness to subsequent lipopolysaccharide (LPS) stimulation. We tested the hypothesis that this effect is due to decreased nuclear translocation of nuclear factor κB (NK-κB). Methods. Murine macrophage-like RAW 264.7 cells were incubated with 1.5 μg/mL CpG-containing oligonucleotides (CpG ODN) for 0.5 to 9 hours followed by restimulation with 1 μg/mL LPS for 20 minutes. Some cells were cotransfected with an NF-κB sensitive luciferase reporter construct and a control β-gal plasmid. Cytoplasmic and nuclear extracts were assayed for NF-κB by electrophoretic mobility shift assay and supershift assays, for NF-κB, IκB and phospho-IκB by Western blot, for luciferase activity, and for p38, c-Jun NH2-terminal kinase, and extracellular signal-related kinase activity assay. Results. NF-κB functional activity was decreased as demonstrated by luciferase activity assay in the prolonged CpG ODN pretreatment groups. Unlike endotoxin tolerance, CpG ODN preexposure increased cytoplasmic phospho-IκBα and did not abrogate mitogen-activated protein kinase activity. Conclusions. In macrophages, exposure to CpG DNA increases expression of the inhibitory p50 NF-κB homodimer and decreases NF-κB activity without inhibition of IκB kinases. Mitogen-activated protein kinase activity remains intact. Understanding these interactions between different toll receptor ligands may provide insight into novel therapeutic modalities.

Original languageEnglish (US)
Pages (from-to)245-251
Number of pages7
Issue number2
StatePublished - Aug 2002


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