Sara Matson

Unmethylated CpG dinucleotides (CpG motif) in bacterial DNA or synthetic oligodeoxynucleotides (CpG DNA) rapidly activate murine B cells to secrete IL-6 and IgM, as well as to proliferate. Within 30 min after CpG DNA stimulation in vivo, IL-6 mRNA levels were increased in liver, spleen, and thymus cells. Serum IL-6 protein was markedly increased within 1 h of stimulation. Treatment of a B cell line with CpG DNA led to an increase in the transcriptional activity of the IL-6 promoter. This CpG DNA-induced IL-6 production was not mediated via either a protein kinase C (PKC)-, protein kinase A (PKA)-, or nitric oxide (NO.)-dependent pathway but was inhibited by an antioxidant. In addition, the level of intracellular reactive oxygen species was increased within 20 min after CpG DNA, but not control non-CpG DNA, treatment. These results suggest that CpG DNA-induced IL-6 production is mediated through a reactive oxygen intermediate-dependent pathway. CpG DNA-mediated IL-6 production was enhanced by simultaneous signals delivered through the Ag receptor. The addition of neutralizing Abs against IL-6 to B cell cultures along with CpG oligodeoxynucleotides essentially abolished the CpG DNA-induced increased IgM secretion but had no significant effect on the B cell proliferation induced by the CpG motif. Our results suggest that the induction of IL-6 expression in response to CpG motifs in bacterial DNA may be an important immune defense mechanism that facilitates a rapid response to microbial infection.

The effects of phosphorothioate (S-oligonucleotide) or terminal phosphorothioate-phosphodiester (S-O-oligonucleotides) or methylphosphonate-phosphodiester (MP-O-oligonucleotides) modifications on mouse spleen cell surface binding, uptake, and degradation were studied using fluorescein (FITC)-conjugated oligonucleotides. S-oligonucleotides had the highest cell binding and uptake, followed by S-O-, O-, and MP-O-oligonucleotides. Competition studies indicated that S-oligonucleotides have an increased affinity for cell membrane oligonucleotide binding sites, because they could completely block O-oligonucleotide binding at a molar ratio of just 0.1. Uptake of all oligonucleotides was higher in B cells than T cells and was increased by stimulation with the B-cell mitogen, lipopolysaccharide. Although our cells had been purified using conventional techniques to eliminate dead cells, there remained about 5% of cells that were dead or dying, as determined by flow cytometry using propidium iodide staining. Of note, oligonucleotide association with dead cells was approximately 50-fold greater than that with living cells. Confocal microscopy confirmed that the oligonucleotides in living cells were intracellular, and indicated little nuclear uptake by 4 h. While extensive degradation of intracellular O-oligonucleotides was apparent by 4 h, there was no detectable degradation of S-, S-O, or MP-O-oligonucleotides.

Phosphodiester oligodeoxynucleotides bearing a 5' cholesteryl (chol) modification bind to low density lipoprotein (LDL), apparently by partitioning the chol-modified oligonucleotides into the lipid layer. Both HL60 cells and primary mouse spleen T and B cells incubated with fluorescently labeled chol-modified oligonucleotide showed substantially increased cellular association by flow cytometry and increased internalization by confocal microscopy compared to an identical molecule not bearing the chol group. Cellular internalization of chol-modified oligonucleotide occurred at least partially through the LDL receptor; it was increased in mouse spleen cells by cell culture in lipoprotein-deficient medium and/or lovastatin, and it was decreased by culture in high serum medium. To determine whether chol-modified oligonucleotides are more potent antisense agents, we titered antisense unmodified phosphodiester and chol-modified oligonucleotides targeted against a mouse immunosuppressive protein. Murine spleen cells cultured with 20 microM phosphodiester antisense oligonucleotides had a 2-fold increase in RNA synthesis, indicating the expected lymphocyte activation. Antisense chol-modified oligonucleotides showed an 8-fold increase in relative potency: they caused a 2-fold increase in RNA synthesis at just 2.5 microM. The increased efficacy was blocked by heparin and was further increased by cell culture in 1% (vs. 10%) fetal bovine serum, suggesting that the effect may, at least in part, be mediated via the LDL receptor. Antisense chol-modified oligonucleotides are sequence specific and have increased potency as compared to unmodified oligonucleotides.