The plasmid pcDNA3 differentially induces production of interferon-α 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-α (IFN-α) 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-α 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-α 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-α when preincubated with lipofectin. Plasmid expression was not necessary for induction of IFN-α. Furthermore, the IFN-α 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-α 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-α 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-α 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.