AMIS-inducing antibodies do not trigger significant macrophage phagocytosis of RBCs. PKH67+HOD-RBC were sensitized with TER119, which induces a robust level of phagocytosis.32 In comparison, additional RBCs were sensitized with anti-HOD antibodies (pIgG anti-HEL, or anti-Duffy CBC-512 IgG2a, or MIMA29 IgG2a), a mouse IgG2a isotype control, mouse pIgG nonspecific, or kept nonsensitized (PBS). RAW macrophages were stimulated with nonsensitized or sensitized RBC for 30 minutes at 37°C and 5% CO2. External, nonphagocytosed RBCs were removed by hypotonic (water) lysis. (A) Brightfield microscopy images of macrophages from each condition are shown. The white arrows indicate an example of macrophages with phagocytosed RBCs. Pictures are representative of 5 independent experiments. (B) The phagocytic index was calculated as the number of RBCs engulfed per 100 macrophages. Data represent the mean ± SEM of 5 independent experiments with total number of samples per group (n = 5-10). Statistical analyses of the data represented in the graph of panel B were performed by one-way ANOVA with Sidak multiple comparisons post hoc test (∗∗∗∗P = .0001).
