Involvement of Specific Mechanism in Plasmid DNA Uptake by Mouse Peritoneal Macrophages

Abstract

The binding and uptake of plasmid DNA encoding luciferase reporter gene (pCMV-Luc) were studied in vitro using cultured mouse peritoneal macrophages. A significant and time-dependent cellular association of [32P]pCMV-Luc with resident macrophages was observed at 37 °C and this decreased at 4 °C. The binding at 4 °C was saturable and a Scatchard plot gave a maximum binding capacity of 0.81 μg/mg-protein and a dissociation constant of 0.30 μg/ml. The binding of [32P]pCMV-Luc was inhibited by polyinosinic acid, dextran sulfate and salmon sperm DNA, but not by polycytidylic acid, dextran and EDTA. A confocal microscopic study demonstrated that fluorescein-labeled pCMV-Luc was internalized at 37 °C while only cell surface binding occurred at 4 °C. No significant luciferase gene expression was obtained after incubation with a high concentration (100 μg/ml) of pCMV-Luc. These data suggest that plasmid DNA is taken up by macrophages via a mechanism mediated by a receptor like the macrophage scavenger receptor.