Unique cytokine production profile following stimulation with DNA in macrophages from NZB/W F1 mice.

Nucleosome is the major autoantigen in systemic lupus erythematosus (SLE). Professional antigen-presenting cells (APCs), such as macrophages (M Phis) and dendritic cells (DCs), play the central roles in the acquisition of Ag-specific immune responses and activation of such APCs is required for the efficient Ag-presentation. Therefore, adjuvant activity of DNA in nucleosomes would cause the prominent effects on the production of anti-nucleosome antibodies. In this study, we report that elicited peritoneal M Phis from New Zealand Black/White F1 (NZB/W) mice showed a unique cytokine production profile following stimulation with DNA. M Phis from 5-week old NZB/W mice produced a higher amount of IL-6 and about a half amount of TNF-alpha after stimulation with DNA complexed with cationic liposomes compared with those from control ICR mice. These results suggest that M Phis of NZB/W mice have altered responsiveness to DNA and this might elevate the antigenicity of nucleosomes to induce the production of anti-nucleosome antibodies.

DNA and its cationic lipid complexes induce CpG motif-dependent activation of murine dendritic cells.

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.

The uptake and degradation of DNA is impaired in macrophages and dendritic cells from NZB/W F(1) mice.

DNA/anti-DNA Ab immune complexes seem to play the critical roles in the development of systemic lupus erythematosus (SLE). However, little is known about the removal of DNA by MPhi and DC. We found that elicited peritoneal MPhis and BM-derived DCs from a lupus-prone strain of New Zealand Black/White F(1) (NZB/W) mice showed impaired DNA uptake and degradation compared with those from control ICR mice. The impairment was mainly observed as the reduced degradation of DNA probably in endosomal compartment and this impaired DNA degradation might, at least in part, result from the reduced DNA uptake in these phagocytic cells. In addition, these impairments was not related to the disease progression since the cells from diseased, 6-month-old NZB/W mice as well as the cells from prediseased, 5-week-old NZB/W mice also exhibited the similar impairment. We also found that the MPhis and DCs of diseased NZB/W mice showed reduced DNA binding at 4 degrees C. However, this reduced DNA binding could be restored to the control level by pretreatment with DNase. Interestingly, this pretreatment had little effect on the DNA uptake in MPhis and DCs of diseased NZB/W mice at 37 degrees C. Hence, the present results imply an impaired function of lupus MPhis and DCs of NZB/W mice to cause retained DNA clearance.

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.

Efficient uptake and rapid degradation of plasmid DNA by murine dendritic cells via a specific mechanism.

In spite of the important roles of dendritic cells in DNA-based therapies, the cellular uptake mechanism of plasmid DNA (pDNA) in dendritic cells is poorly understood. The present study was undertaken to investigate the binding and uptake of pDNA in vitro using a murine dendritic cell line, DC2.4 cells. A significant and time-dependent cellular association of [32P]pDNA with DC2.4 cells was observed at 37 degrees C and this fell markedly at 4 degrees C. The binding and uptake of [32P]pDNA were significantly inhibited by cold pDNA, polyinosinic acid (poly[I]), dextran sulfate, or heparin, but not by polycytidylic acid (poly[C]), dextran, or EDTA, suggesting that a specific mechanism mediated by a receptor like the macrophage scavenger receptor may be involved. The TCA precipitation experiments showed that DC2.4 cells rapidly endocytosed and degraded a significant amount of [32P]pDNA at 37 degrees C and released the degradation products into the medium. The pDNA degradation was also significantly inhibited by poly[I], but not poly[C]. The rate of pDNA degradation by DC2.4 cells was significantly higher than that by macrophages. A confocal microscopic study using fluorescein-labeled pDNA confirmed the rapid internalization and degradation of pDNA by the dendritic cells. Taken together, these results indicate that pDNA is efficiently taken up and rapidly digested by the dendritic cells via a specific mechanism. These findings may suggest the important role of the dendritic cells in the innate immune system for host defense.