(A) Knockdown of CD163 impairs endocytosis of haptoglobin and HMGB1 complexes. The endocytosis of HMGB1 and haptoglobin complexes was measured by flow cytometric analysis. Primary human macrophages were transduced with CD163 shRNA lentiviral or vector alone (control) and incubated with FITC-labeled HMGB1 (1 μg/ml) plus haptoglobin (3 μg/ml) for 30 minutes. Endocytosis of FITC-HMGB1 was evaluated by measuring the fluorescence intensity of FITC-labeled protein as a function of CD163-PE signals. Data are representative of 5 experiments. (B) Overexpression of CD163 enhances endocytosis of haptoglobin and HMGB1 complexes. HEK 293 cells were transduced with human CD163 plasmid and incubated with FITC-HMGB1 alone (1 μg/ml) or a mixture of FITC-HMGB1 (1 μg/ml) and haptoglobin (3 μg/ml) for 30 minutes at 37°C. Endocytosis of FITC-HMGB1 was evaluated as described above, and data are representative of 4 repeats. (C) Dynasore (DYN) inhibits endocytosis of HMGB1 and haptoglobin complexes. Human macrophages on cover slips were incubated with Alexa Fluor 555–labeled HMGB1 alone (1 μg/ml) or in complex with haptoglobin (3 μg/ml) for 2 hours at 37°C. Dynasore (8 μM) was preincubated with cells for 30 minutes before the addition of HMGB1 and/or haptoglobin. Upper: Endocytic uptake of HMGB1 was visualized via Carl Zeiss fluorescence microscope (red). Nuclei were counterstained with DAPI (blue). Lower: Corresponding phase contrast image of cells. Scale bars: 10 µm. Data are representative from 8 independent experiments.