Modulation of interleukin-12 synthesis by DNA lacking the CpG motif and present in a mycobacterial cell wall complex

A mycobacterial cell wall complex prepared from the non-pathogenic microorg anism Mycobacterium phlei, where mycobacterial DNA is preserved and complex ed to cell wall fragments, possesses anticancer and immunomodulatory activi ty. DNA from a number of prokaryotes has been found to modulate the immune system and to induce cytokine synthesis. We have therefore determined wheth er the DNA associated with this complex has the ability to induce the synth esis of interleukin-12 (IL-12), a potent anticancer cytokine. Mycobacterial DNA complexed with cell wall fragments or DNA purified from M. phlei induc ed IL-12 synthesis by murine and human monocytes and macrophages in vitro, and was capable of inducing IL-12 synthesis in vivo in mice following i.p. administration. Neutralization of DNA with cationic liposomes or digestion with DNase I significantly decreased the ability of the cell wall complex t o-induce IL-12. CpG methylation of DNA extracted from these cell walls or f rom M. phlei did not affect the induction of IL-12 synthesis by monocytes a nd macrophages. In contrast, CpG methylation of DNA from Escherichia coli a bolished its;ability to induce IL-12 synthesis. These results demonstrate t hat unmethylated CpG motifs present in M. phlei DNA are not a prerequisite for the induction of IL-12 synthesis. The size of the mycobacterial DNA, in the range of 5 bp to genomic DNA, did not influence its capacity to induce IL-12. Our results emphasize that M. phlei DNA associated with the cell wa ll complex makes a significant contribution to the overall immunomodulatory and anticancer activity of this mycobacterial cell wall preparation and th at these activities are not correlated with the presence of CpG motifs.