CD163 mediates Hp-independent uptake of free Hb in HEK293 cells. (A) CD163⁺ (CD163) and CD163^- (control) HEK293 cells were incubated with 10 μg/mL Alexa-488-labeled Hbfl or Hbfl-Hp complexes for 30 minutes (fl indicates the fluorescence protein). After washing and trypsinization, cell-associated fluorescence was determined by FACS analysis. Although only minimal Alexa-488 fluorescence was detected in the CD163^- cell line (control), significant fluorescence, above background levels, was detected in the CD163⁺ cells. Data represent mean ± SD of 3 independent experiments. (B) Confocal fluorescence microscopy images (original magnification, 400 ×)of CD163⁺ (CD163) and CD163^- (control) cells were acquired after 30-minute incubation of cells with 10 μg/mL Hbfl or Hb-Hpfl. The pattern of green Alexa-488 fluorescence demonstrates that free Hbfl and Hb-Hpfl complexes are taken up into an intracellular endosomal compartment, and this is mediated by CD163 (green, Alexa-488 Hb; blue, DAPI nuclear staining; red, actin filaments). Immunofluorescent staining of CD163 with anti-CD163 (clone 5C6-FAT) and Alexa-594 (red) goat antimouse antibody was performed to confirm receptor expression in CD163-transformed HEK293 cells (right panels). (C) CD163⁺ and CD163^- HEK293 cells were incubated for 8 hours with the indicated CD163 ligands in DMEM without added serum (to avoid nonspecific protein-protein interactions). Each ligand was applied at 3 different concentrations: 1000, 200, and 40 μg/mL. HO-1 mRNA induction relative to nontreated samples was determined by quantitative real-time RT-PCR and was corrected for differences in glyceraldehyde-3-phosphate dehydrogenase mRNA. Results are expressed as mean ± SD of 3 independent experiments. HbA₀ indicates native human hemoglobin; CN-Hb, hemoglobin blocked with cyanide; myo, myoglobin.