Internalization of hyaluronan (HA)–coated phagocytic prey is mediated by CD44. (A-D) Confocal fluorescence and differential interference contrast (DIC) images of murine peritoneal macrophages demonstrating the specific binding of anti-CD44 (CD44) antibody. The solid line represents 10 μm. (E) Quantitative analysis of phagocytosis of HA- and gelatin (control)–coated beads in the presence or absence of soluble HA, anti-CD44 antibody, or buffer control (none). Note that the macrophages internalize HA-coated beads and this is inhibited by soluble HA or by blocking anti-CD44 antibody. By contrast, uptake of gelatin-coated beads is substantially less (phagocytic index < 5). (F) Quantitative analysis of phagocytosis of IgG-opsonized erythrocytes (RBCs) in the presence or absence of soluble HA or buffer control (none). Note that uptake of IgG-opsonized erythrocytes (RBCs, mediated by Fcγ receptors) is not inhibited by soluble HA. (G-J) Confocal immunofluorescence images (G,I) and flow cytometric analysis (H,J) of RAW264.7 macrophages stained with anti-CD44 (G) or isotype control antibodies demonstrating high levels of expression of CD44 and confirming the specificity of the anti-CD44 antibody. (K) Quantitative analysis of phagocytosis of HA-coated beads by RAW macrophages demonstrating efficiency of internalization of HA-coated beads and inhibition by soluble HA or by blocking anti-CD44 antibody. Uptake of gelatin-coated (control) beads is comparatively very low. (L) Quantitative analysis of efficiency of phagocytosis of IgG-opsonized RBCs by RAW macrophages in the presence or absence of soluble HA or buffer control (none). Note that uptake of IgG-opsonized RBCs (mediated by Fcγ receptors) is not inhibited by soluble HA. *Difference from control (no blocking), P < .05, mean ± standard error (SE) of 4 separate experiments.