Restricted cytokine production from mouse peritoneal macrophages in culture in spite of extensive uptake of plasmid DNA

The production of inflammatory cytokines from macrophages (Mphi), 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 Mphi activation, following stimulation with naked pDNA, based on the production of cytokines from cell lines (RAW264.7 and J774A1) and peritoneal Mphis in primary culture. The Mphi cell lines RAW264.7 and J774A1 produced a significant amount of tumour necrosis factor-alpha (TNF-alpha) upon stimulation with naked pDNA and this response required endosomal acidification. On the other hand, peritoneal Mphis (both resident and elicited) in primary culture did not secrete TNF-alpha 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 Mphis showed a higher uptake of pDNA than the RAW264.7 and J774A1 cell lines. These findings have important implications for Mphi activation by naked pDNA as it has been generally assumed that pDNA that contains CpG motifs is a potent agent for inducing inflammatory cytokines in vivo, based on evidence from in vitro studies using Mphi cell lines.