Contribution of plasmid DNA to inflammation in the lung after administration of cationic lipid : pDNA complexes

Cationic lipid-mediated gene transfer to the mouse lung induces a dose-depe ndent inflammatory response that is characterized by an influx of leukocyte s and elevated levels of the cytokines interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and interferon gamma (IFN-gamma), We have examin ed the contribution of plasmid DNA (pDNA) to this observed toxicity, specif ically the role of unmethylated CpG dinucleotides, which have been previous ly shown to be immunostimulatory. We report here that complexes of cationic lipid GL-67 and unmethylated pDNA (pCF1-CAT) instilled into the lungs of B ALB/c mice induced highly elevated levels of the cytokines TNF-alpha, IFN-g amma, IL-6, and IL-12 in the bronchoalveolar lavage fluids (BALF). In contr ast, BALE of animals administered either GL-67 alone or GL-67 complexed wit h SssI-methylated pDNA contained low levels of these cytokines. Similar res ults were observed using a plasmid (pCF1-null) that does not express a tran s-gene, demonstrating that expression of chloramphenicol acetyltransferase (CAT) was not responsible for the observed inflammation. The response obser ved was dose dependent, with animals receiving increasingly higher amounts of unmethylated pDNA exhibiting progressively higher levels of the cytokine s. Concomitant with this increase in cytokine levels were also elevated num bers of neutrophils in the BALF, suggesting a possible cause-and-effect rel ationship between neutrophil influx and generation of cytokines. Consistent with this proposal is the observation that reduction of neutrophils in the lung by administration of antibodies against Mac-1 alpha and LFA-1 also di minished cytokine levels. This reduction in cytokine levels in the BALE was accompanied by an increase in transgene expression. In an attempt to abate the inflammatory response, sequences in the pDNA encoding the motif RRCGYY , shown to be most immunostimulatory, were selectively mutagenized. However , instillation of a plasmid in which 14 of the 17 CpG sites were altered in to BALF/c mice did not reduce the levels of cytokines in the BALE compared with the unmodified vector. This suggests that other unmethylated motifs, i n addition to RRCGYY, may also contribute to the inflammatory response. Tog ether, these findings indicate that unmethylated CpG residues in pDNA are a major contributor to the induction of specific proinflammatory cytokines a ssociated with instillation of cationic lipid:pDNA complexes into the lung. Strategies to abate this response are warranted to improve the efficacy of this nonviral gene delivery vector system for the treatment of chronic dis eases.