The plasmid pcDNA3 differentially induces production of interferon-alpha and interleukin-6 in cultures of porcine leukocytes

An adjuvant effect of invertebrate DNA has been attributed to its relative high frequency of unmethylated CpG dinucleotides. Here we describe the inte rferon-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 Auje szky's disease virus (ADV) and the Gram-negative bacteria Actinobacillus pl europneumoniae. The plasmid, as well as poly(I):poly(C), required lipofecti n to induce IFN-alpha production whereas both preparations induced IL-6 irr espective of preincubation with lipofectin. However, the nucleic acid-induc ed 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 w as as high as, or higher than that induced by ADV. Interestingly, also A. p leuropneumoniae induced a substantial production of IFN-alpha when preincub ated 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. Thes e 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. (C) 2001 Elsevier Science B.V. All rights reserved.