Specific endocytosis and degradation of naked DNA in the endocardial cells of cod (Gadus morhua L.)

SUMMARY DNA vaccines are administered in the form of plasmid DNA (pDNA) carrying a strong promoter and the gene of interest. In this study we investigated the tissue distribution, cellular uptake and the fate of intravenously (i.v.) and intramuscularly (i.m.) injected pDNA in Atlantic cod (Gadus morhua L.). The anatomical distribution of pDNA was determined using both morphological and radiotracing methods. Cellular uptake and receptor specificity were studied in cultures of cod atrial endocardial endothelial cells (aEEC) and head kidney leukocytes. The short-term fate of the endocytosed pDNA in vivo and in vitro was investigated by Southern blot. Expression of the pDNA (R70pRomiLuc)-derived gene was investigated in cod tissues and cultures of cod aEEC by means of real-time RT-PCR and luciferase activity assay. 125I-labelled pDNA was rapidly eliminated from the blood by the aEEC of the cod heart atrium and ventricle. Co-injection of trace amounts of 125I-labelled pDNA with excess amounts of non-labelled pDNA or formaldehyde-treated albumin (FSA), a ligand for the cod EEC scavenger receptor, significantly inhibited the accumulation of the radiotracer in the heart. The organ to blood ratio of radioactivity after inhibition of the cod EEC scavenger receptor demonstrated that the radioactivity not taken up by the EEC remained in the blood. Fluorescence microscopy of tissue sections from cod injected with fluorescein-labelled pDNA confirmed intracellular uptake of pDNA by the endocardial cells of the atrium and ventricle. In purified cultures of cod aEEC the fluorescein-labelled pDNA was taken up in structures reminiscent of endosomal/lysosomal vesicles. Uptake of 125I-labelled pDNA in cultures of cod aEEC was specific. Incubation of cultures with 125I-labelled pDNA together with excess amounts of FSA and fucoidan, which are molecules also known to bind to the scavenger receptors, reduced the uptake of the pDNA by at least 70%. Mannan, a ligand for the mannose receptor, did not inhibit the uptake of 125I-labelled pDNA. Despite, low uptake of 125I-fluorescein-pDNA in the kidney of the cod, the uptake of pDNA in cultured cod head kidney leukocytes was significant. Southern blot analysis of cod tissues after injection of pDNA and culture of aEEC given 10 μg pDNA per 106 cells demonstrated the presence of degradation products in tissues and in the cell cultures. Real-time RT–PCR studies showed expression of luciferase mRNA only at the injection site 168 h after injection. Neither expression of luciferase mRNA nor luciferase activity was present in cod aEEC incubated for 48 h with 10μ g pDNA. These results suggest that the EEC are very important for removal of blood borne pDNA in cod and that the uptake by these cells was mediated in a scavenger–receptor-like manner. Uptake of pDNA by head kidney leukocytes was only observed in vitro. The endocytosed DNA was subjected to intracellular degradation and was not expressed by the cod EEC. Despite the low amount of radioactivity found in the head kidney after i.v. injection of 125I-labelled pDNA, the head kidney leukocytes seem to have a high capacity for uptake of 125I-labelled pDNA in vitro.