Hp complex formation enhances CD163-Hb interactions. (A) CD163⁺ HEK293 cells were incubated for 30 minutes with Hb-Hpfl (fl indicates the fluorescence protein) in the presence or absence (control) of potential competitor proteins. Whereas both Hb and Hb-Hp significantly inhibited the uptake of fluorescent Hb-Hpfl, none of the control proteins (myoglobin, albumin, and free haptoglobin) significantly inhibited fluorescent-ligand uptake when added in a 100-fold excess concentration. Data shown represent the mean ± SD of 3 replicate values from one representative experiment. (B-C) Native Hb (•) and Hb-Hp (○) competitively inhibited the uptake of fluorescent Hbfl (B) and Hb-Hpfl complexes (C). CD163⁺ HEK293 cells were incubated for 30 minutes with fluorescent ligand (50 nM) and the unlabeled competitor at the concentrations indicated. Irrespective of the fluorescent ligand used, the Hb-Hp complexes inhibited CD163-mediated ligand uptake approximately 10 times more efficiently than free Hb. Data shown are the means of 2 independent experiments performed in duplicate. Molar concentrations are indicated with respect to the amount of Hb dimer.