title: The molecular basis for the lack of immunostimulatory activity of vertebrate DNA.
abstract: Macrophages and B cells are activated by unmethylated CpG-containing sequences in bacterial DNA. The lack of activity of self DNA has generally been attributed to CpG suppression and methylation, although the role of methylation is in doubt. The frequency of CpG in the mouse genome is 12.5% of Escherichia coli, with unmethylated CpG occurring at approximately 3% the frequency of E. coli. This suppression of CpG alone is insufficient to explain the inactivity of self DNA; vertebrate DNA was inactive at 100 micro g/ml, 3000 times the concentration at which E. coli DNA activity was observed. We sought to resolve why self DNA does not activate macrophages. Known active CpG motifs occurred in the mouse genome at 18% of random occurrence, similar to general CpG suppression. To examine the contribution of methylation, genomic DNAs were PCR amplified. Removal of methylation from the mouse genome revealed activity that was 23-fold lower than E. coli DNA, although there is only a 7-fold lower frequency of known active CpG motifs in the mouse genome. This discrepancy may be explained by G-rich sequences such as GGAGGGG, which potently inhibited activation and are found in greater frequency in the mouse than the E. coli genome. In summary, general CpG suppression, CpG methylation, inhibitory motifs, and saturable DNA uptake combined to explain the inactivity of self DNA. The immunostimulatory activity of DNA is determined by the frequency of unmethylated stimulatory sequences within an individual DNA strand and the ratio of stimulatory to inhibitory sequences.
authors.affiliation: Cooperative Research Center for Chronic Inflammatory Diseases, Institute for Molecular Bioscience, School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Australia. k.stacey@imb.uq.edu.au
authors.forename: Katryn J
authors.initials: KJ
authors.lastname: Stacey
authors.forename: Greg R
authors.initials: GR
authors.lastname: Young
authors.forename: Francis
authors.initials: F
authors.lastname: Clark
authors.forename: David P
authors.initials: DP
authors.lastname: Sester
authors.forename: Tara L
authors.initials: TL
authors.lastname: Roberts
authors.forename: Shalin
authors.initials: S
authors.lastname: Naik
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Sweet
authors.forename: David A
authors.initials: DA
authors.lastname: Hume
mesh_terms: D000276:Adjuvants, Immunologic / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D000818:Animals; D001483:Base Sequence / Q000187:drug effects / Q000502:physiology; D001667:Binding, Competitive / Q000276:immunology; D002417:Cattle; D002460:Cell Line; D002999:Clone Cells; D018899:CpG Islands / Q000187:drug effects / Q000276:immunology; D004247:DNA / Q000187:drug effects / Q000378:metabolism / Q000494:pharmacology* / Q000502:physiology*; D019175:DNA Methylation / Q000187:drug effects; D004269:DNA, Bacterial / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D016680:Genome, Bacterial; D006801:Humans; D007166:Immunosuppressive Agents / Q000378:metabolism / Q000494:pharmacology; D008262:Macrophage Activation / Q000187:drug effects / Q000276:immunology*; D051379:Mice; D009838:Oligodeoxyribonucleotides / Q000037:antagonists & inhibitors / Q000494:pharmacology
chemical_list: D000276:Adjuvants, Immunologic; C408982:CPG-oligonucleotide; D004269:DNA, Bacterial; D007166:Immunosuppressive Agents; D009838:Oligodeoxyribonucleotides; D004247:DNA; C427203:calf thymus DNA