Preexposure of murine macrophages to CpG-containing oligonucleotides results in nuclear factor kappaB p50 homodimer-associated hyporesponsiveness.
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 kappaB (NF-kappaB).


METHODS
Murine macrophage-like RAW 264.7 cells were incubated with 1.5 microg/mL CpG-containing oligonucleotides (CpG ODN) for 0.5 to 9 hours followed by restimulation with 1 microg/mL LPS for 20 minutes. Some cells were cotransfected with an NF-kappaB sensitive luciferase reporter construct and a control beta-gal plasmid. Cytoplasmic and nuclear extracts were assayed for NF-kappaB by electrophoretic mobility shift assay and supershift assays, for NF-kappaB, IkappaB and phospho-IkappaB by Western blot, for luciferase activity, and for p38, c-Jun NH(2)-terminal kinase, and extracellular signal-related kinase activity assay.


RESULTS
NF-kappaB 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-IkappaB-alpha and did not abrogate mitogen-activated protein kinase activity.


CONCLUSIONS
In macrophages, exposure to CpG DNA increases expression of the inhibitory p50 NF-kappaB homodimer and decreases NF-kappaB activity without inhibition of IkappaB kinases. Mitogen-activated protein kinase activity remains intact. Understanding these interactions between different toll receptor ligands may provide insight into novel therapeutic modalities.
Jin
Long
L
Surgical Infectious Disease Laboratory, Department of Surgery, University of Virginia, Charlottesville 22906, USA.
Raymond
Daniel P
DP
Crabtree
Traves D
TD
Pelletier
Shawn J
SJ
Rudy
Christine K
CK
Pruett
Timothy L
TL
Sawyer
Robert G
RG
D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D018899:CpG Islands / Q000276:immunology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D019281:Dimerization; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000737:chemistry / Q000378:metabolism*; D009841:Oligonucleotides / Q000494:pharmacology*
D000276:Adjuvants, Immunologic; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; D016328:NF-kappa B; C000606205:Nfkbia protein, mouse; D009841:Oligonucleotides; D000072000:NF-KappaB Inhibitor alpha; D020928:Mitogen-Activated Protein Kinases