SUMMARYThe production of in¯ammatory cytokines from macrophages (Mf), upon stimulation with plasmid DNA (pDNA) containing CpG motifs, is a critical process for DNA-based therapies such as DNA vaccination and gene therapy. We compared Mf activation, following stimulation with naked pDNA, based on the production of cytokines from cell lines (RAW264.7 and J774A1) and peritoneal Mfs in primary culture. The Mf cell lines RAW264.7 and J774A1 produced a signi®cant amount of tumour necrosis factor-a (TNF-a) upon stimulation with naked pDNA and this response required endosomal acidi®cation. On the other hand, peritoneal Mfs (both resident and elicited) in primary culture did not secrete TNF-a or interleukin-6, although they contain the mRNA of toll-like receptor-9 (TLR-9) and are able to respond to CpG oligodeoxynucleotides. This unresponsiveness was not a result of impaired cellular uptake of pDNA because the primary cultured Mfs showed a higher uptake of pDNA than the RAW264.7 and J774A1 cell lines. These ®ndings have important implications for Mf activation by naked pDNA as it has been generally assumed that pDNA that contains CpG motifs is a potent agent for inducing in¯ammatory cytokines in vivo, based on evidence from in vitro studies using Mf cell lines.
Summary
Unmethylated CpG motifs in bacterial DNA, but not in vertebrate DNA, are known to trigger an inflammatory response of antigen‐presenting cells (APC). In this study, we investigated the cytokine release from murine dendritic cells (DC) by the addition of various types of DNA in the free or complexed form with cationic lipids. Naked plasmid DNA and Escherichia coli DNA with immunostimulatory unmethylated CpG motifs induced pro‐inflammatory cytokine secretion from granulocyte–macrophage colony‐stimulating factor (GM‐CSF)‐cultured bone marrow‐derived DC and the DC cell‐line, DC2.4 cells, though vertebrate calf thymus DNA (CT DNA) with less CpG motifs did not. These characteristics differed from mouse peritoneal resident macrophages that do not respond to any naked DNA. The amount of cytokines released from the DC was significantly increased by complex formation with cationic lipids when CpG‐motif positive DNAs were used. Unlike murine macrophages or Flt‐3 L cultured DC, GM‐CSF DC did not release inflammatory cytokines in response to the addition of CT DNA/cationic lipid complex, suggesting that the activation is completely dependent on CpG motifs. Taken together, the results of the present study demonstrate that murine DC produce pro‐inflammatory cytokines upon stimulation with CpG‐containing DNAs and the responses are enhanced by cationic lipids. These results also suggest that DC are the major cells that respond to naked CpG DNA in vivo, although both DC and macrophages will release inflammatory cytokines after the administration of a DNA/cationic lipid complex.
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