HMGB1 potentiates the cytokine response to CpG-ODNs. (A) CpG-ODN triggers the release of HMGB1. BMDCs (3 × 10⁶ cells/mL) and BMDMs (2 × 10⁶ cells/mL) were treated with CpG-ODN (10 μg/mL) for the indicated time periods. The medium bathing the cells (40 μL) was subjected to SDS-PAGE and immunoblotted (IB) with anti-HMGB1 or anti-LDH antibody. As a positive control, 2 μg macrophage whole cell lysates were used. Cell viability was determined by trypan blue exclusion. (B-D) Extracellular HMGB1 enhances induction of cytokines by CpG-DNA in a TLR9-dependent manner. Cells were seeded at 1 to 2.5 × 10⁵/well in a 96-well plate (in triplicate) and then treated with CpG-ODN, PGN, or LPS, or left untreated. The LPS inhibitor polymyxin B (10 μg/mL) was used in all treatments except LPS. (B) BMDMs were treated with CpG-ODN (10 μg/mL), LPS (0.2 or 1 μg/mL), or PGN (2.5 μg/mL) in the presence or absence of rHMGB1 (50 ng/mL), or left untreated for 24 hours. IL-6 secretion was assessed by ELISA (averages of triplicates ± SD). Experiments were replicated 3 times. (C) BMDCs were treated with CpG-ODN (10 nM to 1000 nM) in the presence or absence of rHMGB1 (1 μg/mL) or left untreated for 24 hours. Levels of IL-6, IL-12, and TNFα secretion were assessed by ELISA (averages of triplicates ± SD). Experiments were replicated 3 times. (D, upper panel) BMDMs from wild-type (wt), Tlr9^−/−, or Myd88^−/− mice were treated for 24 hours with LPS (0.2 μg/mL), CpG-ODN (10 μg/mL) plus or minus rHMGB1 (50 ng/mL), or rHMGB1 alone (50 ng/mL); IL-6 secretion was assessed by ELISA. Bars represent the average of 6 independent experiments done in triplicate plus or minus SD (**P < .001, Student t test). (D, lower panels) BMDCs from wt, Tlr4m, or Tlr2^−/− mice were treated with LPS (0.1 μg/mL), PGN (10 μg/mL), or CpG-ODN (10 μg/mL) plus or minus rHMGB1 (50 ng/mL). IL-6 secretion was assessed by ELISA. (E) rHMGB1 does not effect CpG-ODN uptake by BMDCs. Cells were treated with CpG-ODN-Cy5 in the presence or absence of rHMGB1 (250 ng/mL) for 1 hour as indicated. Cells were trypsinized and Cy5-positive cells were determined by fluorescence-activated cell sorting (FACS) analysis. (F) TLR9 was immunoprecipitated from the lysates of WEHI-231 cells that were treated with CpG-ODN (10 μg/mL). The presence of CpG-ODN in the TLR9 immunoprecipitate (IP) was detected by PCR. Levels of immunoprecipitated TLR9 for each reaction were assessed by immunoblotting (IB). (G) rHMGB1 speeds up the formation of the CpG-ODNs/TLR9 complex. WEHI-231 cells were treated with CpG-ODN (10 μg/mL) alone or preincubated for 1 hour with rHMGB1 (50 ng). TLR9 was immunoprecipitated and the levels of CpG-ODN in the TLR9 complex were assessed by PCR.