Hemophagocytosis causes a consumptive anemia of inflammation

Abstract

Cytopenias of uncertain etiology are commonly observed in patients during severe inflammation. Hemophagocytosis, the histological appearance of blood-eating macrophages, is seen in the disorder hemophagocytic lymphohistiocytosis and other inflammatory contexts. Although it is hypothesized that these phenomena are linked, the mechanisms facilitating acute inflammation-associated cytopenias are unknown. We report that interferon γ (IFN-γ) is a critical driver of the acute anemia observed during diverse microbial infections in mice. Furthermore, systemic exposure to physiologically relevant levels of IFN-γ is sufficient to cause acute cytopenias and hemophagocytosis. Demonstrating the significance of hemophagocytosis, we found that IFN-γ acts directly on macrophages in vivo to alter endocytosis and provoke blood cell uptake, leading to severe anemia. These findings define a unique pathological process of broad clinical and immunological significance, which we term the consumptive anemia of inflammation.

Figure 1.

Sustained elevation of IFN-γ induces a severe consumptive anemia in physiological contexts and doses. (a) Blood hemoglobin concentrations were determined in WT, Prf1^−/−, and Prf1^−/−Ifng^−/− mice before or 15 d after infection with LCMV. Data are mean values ± SEM. n = 5–9 mice per group combined from three experiments. *, P < 0.0001, compared with uninfected controls. (b) WT mice were left uninfected or were infected with T. gondii and, 4–5 d later, given either IFN-γ–neutralizing or isotype control antibody. 9 or 10 d after infection, hemoglobin concentrations were determined. Data are mean values ± SEM. n = 8–9 mice per group combined from two experiments. *, P < 0.0001, compared with all other points; #, P < 0.0001, compared with uninfected controls. (c) Mice were infused with various amounts of IFN-γ to achieve a range of serum levels of IFN-γ (x axis) for 5 d before blood hemoglobin concentrations were determined. The leftmost trend line includes control infusion mice (as 0 values). Each point represents an individual mouse. n = 53 mice. (d) WT mice were infused with saline or IFN-γ via osmotic pumps (to maintain levels >2,500 pg/ml in the IFN-γ group) and hemoglobin levels were assessed at the indicated times. Data are mean values ± SEM. n = 107 mice combined from five experiments. Zero time point represents mice that received no infusion. *, P ≤ 0.001, compared with control infusion.

Figure 2.

Systemic IFN-γ exposure induces hemophagocytosis in vivo. (a and b) Brightfield micrographs of H&E-stained sections from mice infused with or without IFN-γ for 5 d of spleen sections (a; bars, 200 µm) or cytospun F4/80^hi spleen cells (b; bars, 10 µm). (c, Top) mice were given 5-d IFN-γ infusions, 5-d control infusions, or injected with anti-RBC antibody 1 d before analyzing spleen cells. Spleen cells were permeabilized and stained intracellularly for RBC antigen (after blocking surface-exposed antigen with unlabeled antibody). (c, Bottom) Alternatively, RBCs were labeled with PKH26 and injected or not intravenously 24 h before spleen cell analysis. Dot plots are representative samples with mean percentages of n = 5–6 mice per group from two combined experiments (TER-119 staining) or n = 6–8 mice per group from three combined experiments (PKH26 labeling). Numbers represent the mean percentage TER⁺ or PKH⁺ of F4/80^hi spleen cells ± SEM. (d) Mice were infused with IFN-γ for the indicated times and spleen cells were stained intracellularly (as in c) for RBC antigen. Data are mean values ± SEM. n = 6–11 mice per group combined from two experiments. *, P < 0.001 compared with control infusion.

Figure 3.

IFN-γ acts directly on macrophages to induce hemophagocytosis and CAI. (a) WT and MIIG mice, which selectively lack IFN-γ signaling in macrophage-lineage cells, were given IFN-γ or control infusions for 5 d and intracellular RBC antigen was assessed in splenic F4/80^hi macrophages by flow cytometry. Histograms are representative of three experiments and are gated onF4/80^hi cells. Bar graph data are mean values ± SEM. n = 5–14 mice per group combined from three experiments. (b) WT, Fcer1g^−/−, and MIIG mice were challenged with IFN-γ infusion (for 5 d) or anti-RBC antibody, and blood hemoglobin concentration was determined. Data are mean values ± SEM. n = 7–18 mice per group combined data from two or more experiments. *, P < 0.0001 compared with control infusion.

Figure 4.

IFN-γ–driven CAI is STAT1 and IRF-1 dependent but largely NOS2 and Cybb independent. WT, Stat1^−/−, and Irf1^−/−, Nos2^−/−, and Cybb^−/− mice were infused with IFN-γ over 5 d and blood hemoglobin concentrations were determined. Data are mean values ± SEM. n = 3–13 per group combined from two or more experiments. *, P ≤ 0.001 compared with control infusion

Figure 5.

IFN-γ infusion leads to increased size, membrane protrusions, F-actin assembly, and pinocytosis by macrophages. (a) 5 d after control or IFN-γ infusion, F4/80^hi gated spleen cells were assessed by flow cytometry for forward/side scatter. Dot plots are representative of three experiments. (b) 3 d after control or IFN-γ infusion, mouse peritoneal cells were cytospun and stained for H&E. Bars, 10 µm. (c) 3 d after control or IFN-γ infusion, F-actin was measured in F4/80⁺ peritoneal cells by flow cytometric phalloidin-A647 staining. Histograms are representative of three experiments. (d) WT mice infused with IFN-γ for 1 d were injected with a fluid-phase marker (BSA-A647) and spleen cells were assessed 1 d later (while infusion continued) for uptake of this marker. Bar graph represents mean values ± SEM. n = 8–11 mice per group combined from three experiments. *, P < 0.001 compared with all conditions. (e) Mice infused with IFN-γ for 1 d were injected intravenously with PKH26-labeled RBCs along with BSA-A647 (as in d). 1 d later (while infusion continued), F4/80^hi gated spleen cells were assessed for combined RBC and fluid-phase uptake by flow cytometry. Dot plots are representative two experiments. Numbers represent mean percentage of BSA⁺ of hemophagocytic (PKH⁺) F4/80^hi spleen cells. n = 3–6 mice per group combined from two experiments.

Figure 6.

IFN-γ–induced hemophagocytosis is a macropinocytic process. (a) Depictions of fluid-phase exclusion (classical ligand-driven phagocytosis) versus fluid-phase inclusion (macropinocytosis). PKH26-labeled apoptotic cells or RBCs (with or without opsonizing antibody), along with a fluid-phase marker (soluble Alexa Fluor 647), were injected i.p. into mice which had been infused for 48 h with saline or IFN-γ. 45 min after injection, peritoneal cavities were lavaged and F4/80^hi cells were assessed for colocalization of fluid-phase marker and target cells. (b) Example images of peritoneal macrophages. Bar, 10 µm. (c) Histograms (gated on F4/80^hi PKH⁺ A647⁺ cells) quantitating fluid-phase inclusion (median intensity) in target cell–containing endosomes (PKH minimum intensity mask, expanded 1 pixel), representative of three experiments. Bar graphs represent percentage of colocalization ± SD of the mean of fluid-phase and target cells, combining two experiments. *, P = 0.03, one-tailed Student’s t test.