Figure 5.

Guanosine-rich oligodeoxynucleotide (G-ODN) effects are not mediated by inhibition of cellular uptake of CpG-ODN. (a) RAW 264.7 cells were stimulated overnight with 0·1 µm CpG, CpG-G-ODN or CpG-G*-ODN. In the case of CpG stimulation, cells were additionally co-incubated with 30 nm of the indicated inhibitory and control ODN. Tumor necrosis factor-α (TNF-α) was measured in the supernatant. (b) RAW264.7 cells were incubated overnight with 0·1 µm CpG-ODN alone or co-incubated with G-ODN and G-pentamere (G5), respectively. (c) Bone marrow-derived dendritic cells (BMDC) were stimulated with 1 µm CpG in the presence or absence of 0·3 µm G-ODN for the indicated time. Cell lysates were tested for phosphorylation of p38-mitogen-activated protein (MAP) kinase as well as expression of actin by western blotting. (d) RAW264.7 cells were stimulated overnight with 1 µm phosphodiester (PO)-CpG delivered with DOTAP. Cells were co-incubated, as indicated, with G-ODN. (e) RAW264.7 cells were incubated with the indicated amounts of fluorescein isothiocyanate (FITC)-labelled ODN for 3 hr and cellular uptake was quantified as described in the Materials and methods. Uptake of 1 µm CpG-ODN was set as 100%. (f) RAW264.7 cells were incubated with 1 µm FITC-labelled CpG-ODN in the presence or absence of unlabelled G-ODN and GC-ODN, respectively. Ratios of FITC-CpG-ODN and unlabelled ODN are indicated. Uptake was quantified by fluorescence-activated cell sorter (FACS) analysis and normalized to the uptake of 1 µm FITC-labelled CpG-ODN. (g) HEK293 cells were transiently transfected with murine Toll-like receptor 9 (TLR-9) or TLR-9N4C together with a nuclear factor-κB (NF-κB)-dependent luciferase reporter plasmid. Cells were stimulated for 6 hr with 0·1 µm CpG-ODN 1668 in the presence or absence of G-ODN or GC-ODN, respectively. Stimulation was determined by measuring luciferase activity. cps, counts per second.