Effect of cytofectins on the immune response of murine macrophages to mammalian DNA

DNA, depending on base sequence, can induce a wide range of immune responses. While bacterial DNA is stimulatory, mammalian DNA is inactive alone and can, moreover, inhibit the response to bacterial DNA. To determine whether the mode of cell entry affects the immune properties of mammalian DNA, we have investigated the effects of the cytofectin agents Fugene 6 (Roche Diagnostics Corp., Indianapolis, IN), Lipofectin and Lipofectamine (Life Technologies, Grand Island, NY) on the responses of murine macrophages to DNA from calf thymus and human placenta. Whereas calf thymus and human placenta DNA alone failed to stimulate J774 or RAW264·7 cell lines or bone marrow-derived macrophages, these DNAs in complexes with cytofectin agents stimulated macrophages to produce nitric oxide but not interleukin 12. Both single-stranded and double-stranded DNAs were active in the presence of cytofectins. Macrophage activation by the DNA–cytofectin complexes was reduced by chloroquine, suggesting a role of endosomal acidification in activation. As shown by flow cytometry and confocal microscopy, the cytofectins caused an increase in the uptake of DNA into cells. Our findings indicate that macrophages vary in their response to DNA depending on uptake pathway, suggesting that activation by DNA reflects not only sequence but also context or intracellular location

Chromatin clearance in C57Bl/10 mice: interaction with heparan sulphate proteoglycans and receptors on Kupffer cells

Chromatin is an important autoantigen in the pathogenesis of systemic lupus erythematosus (SLE) as an immunogen and as a part of nephritogenic immune complexes. Earlier studies focused on clearance of DNA. However, DNA released into the circulation from dying cells is found associated with histones in nucleosomes. The liver is the major organ involved in clearance of chromatin from the circulation of mice. Heparan sulphate proteoglycans (HSPG) have been implicated in the clearance of various charged molecules. Receptor-mediated clearance of ssDNA by the liver has also been reported. Because chromatin contains positively charged histones in addition to DNA, we wished to determine if HSPG and/or DNA receptors are involved in chromatin clearance. The rate of clearance of H1-stripped chromatin from the bloodstream of C57Bl/10 mice was markedly decreased by prior treatment of mice with Heparinase I. Clearance was also inhibited by heparin, heparan sulphate, and DNA, but not by colominic acid. DNA was the most effective inhibitor of clearance and released chromatin from sites of clearance. Depletion of Kupffer cells and splenic macrophages using liposome-encapsulated Clodronate (dichloromethylene bisphosphonate) markedly inhibited chromatin clearance. These data suggest that chromatin clearance is mediated by charge interactions with cell surface HSPG and by DNA receptors. Clearance and degradation of chromatin require functional macrophages in the liver and spleen