MACROPHAGES INGEST AND ARE ACTIVATED BY BACTERIAL-DNA

Recent evidence suggests that bacterial DNA activates immune responses . Here we showed that TNF-alpha mRNA was induced in bone marrow-derive d macrophages and the macrophage cell line RAW 264 by plasmid DNA, but not by DNasel-digested plasmid, plasmid methylated on CpG dinucleotid es, or by vertebrate genomic DNA, which is naturally largely methylate d on these sequences. Synthetic polynucleotides poly d(I-C) and poly I -poly C also induced TNF-alpha, IL-1 beta and plasminogen activator in hibitor-2 mRNAs were induced by plasmid DNA, and IFN-gamma-pretreated macrophages responded to DNA with induction of inducible nitric oxide synthase. The HIV-1 long terminal repeat was activated by exogenous DN A in a manner similar to TNF-alpha, and was also activated by a CpG-co ntaining oligonucleotide. Transcription factor nuclear factor-kappa B (NF-kappa B) is involved in regulation of the HIV-1 long terminal repe at and many inflammatory response genes. NF-kappa B binding activity w as increased by plasmid DNA. An important question is whether these ef fects involve DNA binding to a cell surface receptor that signals to t he interior, or whether internalization is necessary. Here we found th at plasmid was taken up by RAW 264 cells and remained sufficiently int act to code for luciferase protein. Results suggest that DNA is taken up by macrophages and characteristic bacterial DNA sequences, which in clude an un methylated CpG sequence, activate a signaling cascade lead ing to activation of NF-kappa B and inflammatory gene induction. Relev ance to DNA vaccination, gene therapy, antisense, and transfection stu dies is discussed.