The induction of HMGB1 release from RAW 264.7 cells by transfected DNA.

High mobility group protein 1 (HMGB1) is a non-histone nuclear protein that can activate innate immunity when in an extracellular location. As shown in in vitro studies, while polyinosinic-polycytidylic acid [poly (I:C)] and LPS, TLR3 and TLR4 ligands, respectively, can induce HMGB1 release from macrophages, CpG DNA, a TLR 9 ligand, does not. Since DNA displays distinct immunostimulatory activity when transfected into cells, we investigated whether transfected DNA can induce HMGB1 release from macrophages. In these experiments, using RAW 264.7 cells as model, we show that DNA, either natural DNA or synthetic oligonucleotides, can induce HMGB1 release when used to stimulate cells with the transfection reagent Lipofectamine 2000; release occurred irrespective of the intrinsic activity of the DNA. The induction of HMGB1 release by transfected DNA was dependent on IFN-beta as shown by the inhibitory effects of an antibody. In addition, JNK activation mediated HMGB1 release induced by a transfected phosphorothioate oligonucleotide but not by transfected natural DNA. Together, these findings indicate that transfected DNA can stimulate macrophages to release HMGB1 under conditions in which free DNA is inactive and suggest a role of DNA in inducing inflammation when bound to molecules that influence its entry into cells.