7 cells. The cellular uptake of ODN1668 was highly dependent on the concentration of ODN1668 after a 4-h-incubation of ODN. The addition of ODN1720 or DNase I-treated ODN1720 hardly altered the cellular uptake of ODN1668 (Fig. 5A). Thus, the cellular uptake of ODN1668 was not affected by DNase I-treated

ODN1720, so it would not be involved in the mechanism of increased TNF-α production by DNase I-treated DNA. Next, we focused on the stability of ODN1668 against DNases, because the presence RAD001 chemical structure of DNA or DNA fragments could increase the stability of ODN1668, which would result in increased cytokine production. To evaluate the effect of DNase-treated DNA on the stability of ODN1668 against DNases, ODN1668 was incubated with DNase I or DNase II in the presence of DNase-treated ODN1720. Unexpectedly, the degradation of ODN1668 by DNase I was markedly accelerated by the addition of DNase I-treated ODN1720 (Fig. 5B). Similar experiments were performed at lower DNase I concentrations of 0.1 and 0.5 U/mL, which MK-1775 manufacturer could better reflect the situation of cultured macrophages. Under the DNase I concentration of 0.5 U/mL,

the degradation of ODN1668 by DNase I was also accelerated by the addition of DNase I-treated ODN1720, whereas no significant degradation of ODN1668 was observed at a concentration of 0.1 U/mL DNase I for the experimental period of 4.5 h (Supporting Information Fig. 3). Therefore, it was postulated that the increased CpG motif-induced TNF-α production by DNase

I-treated DNA was not mediated by the increase in the stability of CpG DNA against DNase I. On the other hand, the degradation of ODN1668 by DNase II was retarded by the addition of DNase I-treated ODN1720 (Fig. 5C) or DNase II-treated ODN1720 (Fig. 5D). Taking into consideration that the DNase II-treated ODN1720 did not increase the ODN1668-induced TNF-α production (Fig. 3B), it seems that the ODN stabilization to DNase II did not contribute to the increase in TNF-α production by ODN1668. Therefore, the effects of DNase I-treated ODN1720 on the degradation of ODN1668 by DNase II would not be important for the ODN1668-induced TNF-α production. To evaluate whether DNase I-treated DNA increases the CpG DNA-induced inflammatory Oxalosuccinic acid response in vivo, ODN1668 was subcutaneously injected with intact or DNase I-treated ODN1720 into the footpad of the right hind leg of mice. The injection of ODN1668 alone did not induce significant footpad swelling (Fig. 6A), and the co-injection of ODN1720 had little effect on it. However, co-injection of DNase I-treated ODN1720 significantly increased the footpad swelling. Moreover, the infiltration of mononuclear cells and neutrophils into the footpad was evaluated using the paraffin sections of the footpad of mice receiving a subcutaneous injection of ODN1668 (Fig. 6B).