title: The plasmid pcDNA3 differentially induces production of interferon-alpha and interleukin-6 in cultures of porcine leukocytes.
abstract: An adjuvant effect of invertebrate DNA has been attributed to its relative high frequency of unmethylated CpG dinucleotides. Here we describe the interferon-alpha (IFN-alpha) and interleukin-6 (IL-6) inducing properties of a commonly used eukaryotic expression vector, pcDNA3, in porcine leukocytes. The magnitude of the cytokine response was compared to that induced by the synthetic ds RNA analogue poly(I):poly(C), inactivated preparations of Aujeszky's disease virus (ADV) and the Gram-negative bacteria Actinobacillus pleuropneumoniae. The plasmid, as well as poly(I):poly(C), required lipofectin to induce IFN-alpha production whereas both preparations induced IL-6 irrespective of preincubation with lipofectin. However, the nucleic acid-induced levels of IL-6 were low compared to those induced by A. pleuropneumoniae. The IFN-alpha response elicited by pcDNA3 in the presence of lipofectin was as high as, or higher than that induced by ADV. Interestingly, also A. pleuropneumoniae induced a substantial production of IFN-alpha when preincubated with lipofectin. Plasmid expression was not necessary for induction of IFN-alpha. Furthermore, the IFN-alpha inducing capacity of pcDNA3 was not reduced when the two predicted immunostimulatory sequences 5'AACGTT3' were deleted. Nor did the ability of the plasmid to induce IFN-alpha production decrease when the ampicillin resistance (ampR) gene was replaced with the kanamycin resistance (kanR) gene. However, methylation of all cytidines in CpG dinucleotides of pcDNA3 abolished the IFN-alpha inducing capacity. These in vitro results indicate an immunomodulatory role of bacterial DNA also in the pig. Unmethylated CpG dinucleotides are crucial for induction of IFN-alpha by the plasmid, but other CpG motifs than those within the 5'AACGTT3' sequences of the ampR gene contribute to this induction in porcine cells.
authors.affiliation: Department of Veterinary Microbiology, Division of Immunology, Swedish University of Agricultural Sciences, BMC, Box 588, S-751 23, Uppsala, Sweden. mattias.magnusson@vmm.slu.se
authors.forename: M
authors.initials: M
authors.lastname: Magnusson
authors.forename: E
authors.initials: E
authors.lastname: Johansson
authors.forename: M
authors.initials: M
authors.lastname: Berg
authors.forename: M L
authors.initials: ML
authors.lastname: Eloranta
authors.forename: L
authors.initials: L
authors.lastname: Fuxler
authors.forename: C
authors.initials: C
authors.lastname: Fossum
mesh_terms: D016977:Actinobacillus pleuropneumoniae / Q000276:immunology; D000818:Animals; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D018076:DNA, Complementary / Q000235:genetics / Q000276:immunology; D011558:Herpesvirus 1, Suid / Q000276:immunology; D016898:Interferon-alpha / Q000096:biosynthesis* / Q000276:immunology; D015850:Interleukin-6 / Q000096:biosynthesis* / Q000276:immunology; D007962:Leukocytes / Q000276:immunology* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010714:Phosphatidylethanolamines / Q000276:immunology; D010957:Plasmids / Q000235:genetics / Q000276:immunology*; D013047:Specific Pathogen-Free Organisms; D013552:Swine / Q000097:blood / Q000276:immunology*; D019444:Vaccines, DNA / Q000235:genetics / Q000276:immunology
chemical_list: D018076:DNA, Complementary; D016898:Interferon-alpha; D015850:Interleukin-6; D010714:Phosphatidylethanolamines; D019444:Vaccines, DNA; C020888:1,2-dielaidoylphosphatidylethanolamine