Characterization of plasmid DNA binding and uptake by peritoneal macrophages from class A scavenger receptor knockout mice.
OBJECTIVE
Plasmid DNA (pDNA) has become an important class of macromolecular agent suitable for non-viral gene therapy as well as DNA vaccination. Our recent study has suggested that pDNA is taken up by mouse peritoneal macrophages via a specific mechanism mediated by a receptor similar to the scavenger receptor (SR). This study was designed to further characterize the pDNA uptake by macrophages in order to elucidate the mechanism.


METHODS
The binding and uptake of pDNA labeled with 32P or a fluorescent marker were studied in vitro using cultured Chinese hamster ovary (CHO) cells expressing the class A scavenger receptor (SRA) and peritoneal macrophages from SRA-knockout mice.


RESULTS
pDNA binding and uptake by CHO(SRA) cells were minimal and almost identical to that by wild-type CHO cells. Macrophages from the knockout mice showed pronounced pDNA binding and uptake as did the control macrophages. In both types of macrophage, pDNA binding was significantly inhibited by cold pDNA, polyinosinic acid and dextran sulfate but not by polycytidylic acid or Ac-LDL. These results provide direct evidence that SRA is not responsible for the significant binding and subsequent uptake of pDNA by mouse peritoneal macrophages. Further binding experiments revealed that, in addition to polyinosinic acid and dextran sulfate, heparin was a potent inhibitor among a variety of polyanionic compounds such as polynucleotides, anionic polysaccharides and modified proteins including Ox-LDL.


CONCLUSIONS
The present study suggest that pDNA binding and uptake by mouse peritoneal macrophages are mediated by a specific mechanism to some defined polyanions not by scavenger receptors. The finding would be an important basis for further studies to elucidate the mechanism(s) of pDNA uptake by macrophages.
Takakura
Y
Y
Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, Japan.
Takagi
T
T
Hashiguchi
M
M
Nishikawa
M
M
Yamashita
F
F
Doi
T
T
Imanishi
T
T
Suzuki
H
H
Kodama
T
T
Hashida
M
M
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003587:Cytomegalovirus / Q000235:genetics; D004279:DNA, Viral / Q000493:pharmacokinetics*; D005456:Fluorescent Dyes; D017737:Macrophages, Peritoneal / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D010761:Phosphorus Radioisotopes; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*; D051116:Receptors, Scavenger; D051117:Scavenger Receptors, Class A
D004279:DNA, Viral; D005456:Fluorescent Dyes; C495140:Msr1 protein, mouse; D010761:Phosphorus Radioisotopes; D011971:Receptors, Immunologic; D051116:Receptors, Scavenger; D051117:Scavenger Receptors, Class A