HRG and the copurified IgG function cooperatively to facilitate phagocytosis of necrotic cells via a FcγR-dependent mechanism. Phagocytic assays were performed using the monocytic cell line THP-1 and necrotic Jurkat T cells labeled with PKH26 or CFSE, respectively. THP-1 cells were mixed with necrotic cells at a ratio of 1:10 and incubated at 37°C for 60 minutes before analysis by flow cytometry. (A) Ability of HRG^P (100 μg/mL) to enhance the phagocytosis of necrotic cells by THP-1 cells. Representative flow cytometric plots are shown for phagocytic assays performed in the presence or absence of HRG^P. Values in each gated area represent percentage of cells in the assay. (B) Effect of temperature (4°C or 37°C) and the cytoskeleton disruptor Cyto-D (25 μM) on the ability of HRG^P (100 μg/mL) to mediate the uptake of necrotic cells by THP-1 cells. OVA (100 μg/mL) is included as a negative control protein. (C) Effect of HRG^P or HRG^PID (100 μg/mL), and IgG^HRG (2 μg/mL), a human IgG2κ myeloma (2 μg/mL), or pooled normal human IgG (2 μg/mL) either alone or in the presence of HRG^PID (100 μg/mL) on the phagocytosis of necrotic cells by THP-1 cells. (D) Effect of a human IgG1κ myeloma on HRG^P (75 μg/mL)–mediated uptake of necrotic cells, with OVA being included as a negative control. Effect of (E) a human FcγRI blocking mAb (mouse F(ab′)₂ anti–human FcγRI mAb, clone 10.1) or (F) a human FcγRIIA blocking mAb (mouse F(ab′) anti–human FcγRIIA mAb, clone IV.3) on the ability of HRG^P (75 μg/mL) to enhance the phagocytosis of necrotic cells. The level of phagocytosis was determined as the percentage of PKH26-positive THP-1 cells that had ingested CFSE-positive necrotic cells. Data in panels B-F are expressed as fold difference in the level of phagocytosis relative to the necrotic cells only control, with error bars representing SEM (n = 3). NS indicates not significant. *P < .05; **P < .01; ***P < .001.