To confirm these similarities, the effect of “K” ODN on the upregulation of mRNA encoding IFN-β, IL-6, IL-23A, and TNF-α by both cell types was compared. As seen in Figure 1, the response of CAL-1 cells to CpG ODN followed the same kinetics as primary human pDCs. Although the absolute magnitude of these responses differed, their pattern of cytokine production (including IL-23, a cytokine made abundantly by pDCs) were quite similar, reinforcing the conclusion that CAL-1 cells mimic the response of human pDCs to “K” ODN stimulation. Subsequent studies focused on identifying the signals are involved in the regulation of IFN-β and IL-6 by CAL-1 cells, as those genes are representative

of the dominant antiviral and pro-inflammatory responses induced when human pDCs are stimulated with “K” ODN. Most IRFs are stored in latent form in the cytoplasm and MK-8669 translocate to the nucleus when activated and phosphorylated [29]. To evaluate the effect of CpG ODN on the behavior of IRFs, CAL-1 cells were incubated with “K” ODN and cytoplasmic and nuclear lysates were examined by immunoblot (Fig. 2A and B and Supporting Information Fig. 1A). The first change observed was a significant rise in intranuclear IRF-5 levels within 1 h of stimulation. This was followed by a significant rise in nuclear IRF-1

at 3 h. In contrast, no translocation of IRFs 3, 7, or 8 from the cytoplasm to the nucleus was observed (Fig. 2A and B and Supporting Information Fig. 1A). SB203580 in vitro CAL-1 cells were stimulated

for 1–9 h with “K” ODN to examine whether the accumulation of IRF-1 and IRF-5 protein in the nucleus was associated with corresponding changes in the level of mRNA expression. As seen in Figure 2C, IRF-1 and IRF-7 (a known IFN-stimulated gene) were upregulated at 6 and 9 h (Fig. 2C). When antibody against the type 1 IFN receptor (anti-IFNR) was added, this upregulation was inhibited, suggesting that the effect was dependent upon feedback by type 1 IFN. By comparison, mRNA encoding IRF-5 and IRF-8 did not vary over time. Together, Clomifene these results suggest that “K” ODN stimulation triggers the translocation of IRF-5 from the cytoplasm to the nucleus while subsequently increasing the expression of mRNA encoding several IRFs. Members of the NF-κB transcription factor family are actively sequestered in the cytoplasm by IκB proteins. IκB proteins are phosphorylated and degraded upon TLR stimulation, resulting in the translocation of NF-κB complexes to the nucleus [30]. Although NF-κB activation has been studied in mice, data on NF-κB behavior in CpG-stimulated human cells is limited. Analysis of nuclear lysates from “K” ODN treated CAL-1 cells showed that both p50 and p65 translocated from the cytoplasm to the nucleus within 1 h (Fig. 2D). The cytoplasmic levels of these proteins did not change (Supporting Information Fig. 1B).