CD163 mediates Hp-independent macrophage uptake of αα crosslinked Hb into an endosomal compartment. (A) Internalization of Alexa-647 αα-DBBF Hb in macrophages was confirmed by deconvoluted fluorescence microscopy. A high degree of intracellular colocalization (panel iii, yellow) was found after coincubation with αα-DBBF Hb (panel i, Alexa-647) and the early endosomal marker transferrin (panel ii, Alexa-488) for 15 minutes at a concentration of 20 μg/mL. Alternatively, the same endosomal distribution was revealed by anti-Hb immunofluorescence staining of macrophages after incubation with nonlabeled αα-DBBF Hb (panel iv; red, Hb; magenta, Alexa-647 phalloidin stain of actin cytoskeleton; blue, DAPI nuclear stain) (original magnification, 1000 ×). (B) Macrophage-associated fluorescence was determined after 30-minute incubation with 10 μg/mL Alexa-633-labeled αα-DBBF Hb (control). Nonspecific binding was determined by the addition of a 300-fold excess of unlabeled Hb (open curves). Uptake of αα-DBBF Hb was inhibited by a blocking rabbit polyclonal IgG (poly) but not by an equal concentration of a nonblocking monoclonal anti-CD163 antibody (5C6-FAT).