Figure 2. Erythrophagocytic transformation of macrophages in RBC-heme Matrigel plugs.

(A) Subcutaneously placed FGF-enriched Matrigel plugs were used to study the effect of hemorrhage on the phenotype of invading macrophages. Plugs enriched with intact RBCs (RBC-heme) or RBC-ghosts were used to study the specific effect of hemoglobin-heme. (B) FGF-enriched Matrigel plugs were collected from the subcutaneous (s.c.) injection site 7 days after injection and classified by blinded investigators as non-hemorrhagic or hemorrhagic based on their macroscopic appearance. (C) Flow cytometry contour plots of plug-invading cells, defining F4/80⁺CD11b⁺ macrophages with lower MHC class II expression in hemorrhagic plugs. The box plots depict the mean fluorescence intensities of MHC class II expression within the CD11b⁺F4/80⁺ population (n = 6–8 plugs per condition; each dot represents 1 plug; t test). (D) RBC-heme and RBC-ghost plugs were collected 7 days after s.c. injection. (E) Flow cytometry contour plots of plug-invading cells defining F4/80⁺CD11b⁺ macrophages with lower MHC class II expression in RBC-heme than RBC-ghost plugs. The box plots depict the mean fluorescence intensities of MHC class II expression within the CD11b⁺F4/80⁺ population (n = 9 plugs per condition; each dot represents 1 plug; t test). (F) Fluorescence immunohistochemistry images of Matrigel plug sections stained with TER119 (red, RBC-heme or RBC-ghost), anti-F4/80 (yellow, macrophages), and anti-HMOX1 antibodies (cyan). Nuclei were stained with DAPI (white). Images were acquired using a PhenoImager HT (Akoya). Corresponding consecutive sections stained for iron show iron accumulation in infiltrating cells. Scale bars: 500 μm. The inset shows an iron-positive erythrophagocyte containing multiple RBC remnants.