Hp independence of CD163-mediated Hb endocytosis revealed by αα-DBBF-Hb. (A) Molecular schematic of the β-Cys93 modification in αα-DBBF-Hb MPC. (B) CD163-HEK293 cells were incubated with 10 μg/mL Alexa-488 Hp in the presence or absence (control) of equimolar concentrations of HbA0 or αα-DBBF Hb. Unlike HbA0, αα-DBBF-Hb did not support the uptake of fluorescent Hp in CD163-HEK293 cells. (C) Uptake of Hb-Hpfl complexes (50 nM) by CD163-HEK293 cells was determined in the presence of increasing competitor concentrations of unlabeled human HbA0 (▪), αα-DBBF Hb (♦), and αα-DBBF Hb-MPC (○). (D) Identical experiments were performed with human HbA0 (▪), αα-DBBF Hb-MPC (○), and Hb-Hp complexes (▾) (Hp phenotype 2-2) as competitors. Although αα-DBBF-Hb has a slightly higher capacity to compete for fluorescent Hb-Hp uptake by CD163+ cells than native HbA0, the αα-DBBF-Hb MPC variant displayed ligand properties equal to those displayed by the Hb-Hp complexes. Data represent mean ± SD of 3 independent experiments. (E) HbA0, αα-DBBF-Hb (DBBF), and αα-DBBF-Hb MPCs (MPC) were all labeled with Alexa-633, and uptake by CD163-HEK293 was determined after incubation for 30 minutes at a concentration of 5 μg/mL (▪). Parallel samples were incubated in the presence of 20 μg/mL human Hp 2-2 (□) or 50 μg/mL polyclonal rabbit anti-human CD163 IgG (right bar of each group). Data represent mean ± SD of triplicate well samples from one representative experiment. (F) Uptake of Alexa-633-labeled αα-DBBF Hb (5 μg/mL) by CD163-HEK293 cells was determined after 30-minute incubation with or without added Hp 2-2 (10 μg/mL; Hp), polyclonal rabbit anti-human CD163 IgG (50 μg/mL; poly), or mouse anti-human CD163 monoclonal antibodies RM3/1 or 5C6-FAT (each at 50 μg/mL; RM3/1 and 5C6, respectively; filled curves). Open curves represent the fluorescence of a control sample obtained after concurrent incubation with a 300-fold excess of unlabeled HbA0.
