Antagonism of immunostimulatory CpG-oligodeoxynucleotides by 4-aminoquinolines and other weak bases: Mechanistic studies

Oligodeoxynucleotides with unmethylated CpG motifs are immunostimulatory. C hloroquine and a number of structural analogs specifically and powerfully i nhibit this effect at nanomolar concentrations. We explored the mechanism o f this inhibition, with 4-aminoquinolines, quinacrine, 9-aminoacridines, an d novel dibasic analogs, many of which are fluorescent. WEHI 231 murine B-l ymphoma cells accumulated analogs up to a concentration several hundredfold higher than the medium. Uptake was rapid, nonsaturable, reversible, and pa rtially inhibited by monensin, an agent that collapses pH gradients within cells. Uptake did not correlate highly with efficacy as inhibitors of CpG-o ligodeoxynucleotide (ODN)-induced effects, suggesting that analogs act by a specific action. Confocal microscopy revealed analogs concentrating in lar ge peripheral organelles. CpG-ODN is taken up by cells into acidifed, small , perinuclear vesicles. This uptake is thought to be necessary for immunost imulatory activity. Cellular uptake of fluorescent CpG-ODN was not inhibite d by the analogs. The pH of intracellular CpG-ODN (6.4) was not affected by analogs at the concentration required for inhibition, but pH was increased by higher concentrations. UV spectroscopy revealed no binding of analogs t o CpG-ODN. Nuclear Overhauser effect spectroscopy revealed that an analog b ound to phosphatidylcholine vesicles, with the ring structure of the analog buried within the lipid and the side chain facing the aqueous environment. We conclude that the analogs do not inhibit the action of CpG-ODN by preve nting the uptake or acidification of CpG-ODN. It seems more likely that the analogs inhibit the efficacy of CpG-ODN by a specific action within acidif ied vesicles, possibly at the interface of a phospholipid membrane.