In the current study, we have demonstrated that an IFN-γ–dependent anemia develops acutely in the context of diverse infections or sterile cytokine infusion. This anemia is associated with the widespread appearance of hemophagocytosis in vivo, and both processes are dependent on IFN-γ signaling in macrophage lineage cells. Furthermore, IFN-γ–triggered hemophagocytosis is characterized as a macropinocytic process. These findings are summarized diagrammatically in Fig. S6
. In addition to defining a new mechanism for the development of acute inflammation-associated cytopenias, these studies have provided the first evidence for the causal role of macrophages and the significance of hemophagocytosis in this pathological process. Critical aspects of the hemophagocytic response remain undefined, however. Future studies will focus on better understanding the receptors/ligands that are likely to govern uptake, as well as the signaling mechanisms downstream of STAT1 and IRF1 which drive hemophagocytosis. Although the current study has provided new mechanistic insights into poorly understood pathological processes, several caveats are applicable to our findings. First, although IFN-γ–driven CAI is a profound cause of infection-associated anemia in our studies, it is not likely to be the only cause of cytopenias in complex acutely ill patients. Other processes, such as blood loss, hemolysis, and/or decreased marrow output may be occurring, depending on the clinical context. Future studies will be needed to determine the relative contribution of hemophagocytosis/CAI to otherwise unexplained acute inflammation-associated cytopenias in human patients. Second, although we have demonstrated an essential role for IFN-γ in our studies, this does not rule out a potential role for other inflammatory cytokines or mediators in acute inflammation-associated cytopenias. Indeed, Milner et al. (2010)
have demonstrated that IL-4 exposure can drive hemophagocytosis and mild anemia in experimental animals. Although we have assessed IFN-γ’s role in diverse infections (viral and parasitic), future studies examining other infections or inflammatory contexts (e.g., bacterial pathogens) will be needed to define the uniqueness of IFN-γ for promoting CAI. Third, although we conclude that hemophagocytosis is an essential part of IFN-γ–driven CAI, it remains possible that other IFN-γ–driven processes are contributing to the observed anemia. We conclude that hemophagocytosis is causal for CAI based on three lines of data: (1) these processes have the common mechanism that both are abolished in the absence of macrophage IFN-γ signaling; (2) hemophagocytosis correlates kinetically with anemia, preceding it by 1 d and continuing during its development; and (3) a massive number of RBCs are eliminated (~1010
) within 5 d of IFN-γ infusion and yet the only trace of this consumptive process is found intracellularly, within macrophages. Although hemophagocytosis is clearly important for the development of CAI, these data do not formally exclude the possibility that other IFN-γ–driven macrophage-mediated processes may also contribute to CAI.
A final caveat for interpreting our data are that even though our flow cytometric assays reveal a global up-regulation of RBC uptake, the histological appearance of hemophagocytosis is notably variable in human patients with HLH (or acute inflammation-associated cytopenias). In the case of HLH, this may be a result of specific aspects of disease development. It is commonly reported that hemophagocytosis is variably found at the onset of clinical disease but is readily demonstrated at later time points during active disease (Henter et al., 1998
). Furthermore, the variable prevalence of hemophagocytosis may be related to sampling error or limitations of clinical sampling. Spleen tissue is rarely available for clinical assessment but appears to be the best tissue to examine in experimental animals. Our data also point to a third potential explanation for the variable clinical and experimental prevalence of hemophagocytosis; histologically demonstrable hemophagocytosis may only be the tip of an iceberg of cellular engulfment. In our controlled experimental studies, we find that most splenic macrophages acquire RBC antigens after IFN-γ infusion. When examined microscopically, most of these same cells appear to be laden with unidentifiable debris, whereas a subset contain probable RBCs and only a fraction contain clearly recognizable RBCs or nucleated cells ( and not depicted). This finding suggests that variations in breakdown of engulfed cells may play a major role in the histological phenomenon of hemophagocytosis.
This study has significant implications for the development of new translational therapies and for the deeper understanding of macrophage biology, immune-mediated pathologies, and hematologic processes. First, these studies provide a clear rationale for developing therapies that directly target IFN-γ–activated macrophages in patients with HLH and those with acute inflammation-associated cytopenias. Although inflammatory macrophages have been hypothesized to be important for the development of HLH and similar acute inflammatory pathologies (Abshire, 1996
), their role has not been previously demonstrated. This study demonstrates, for the first time, the causal role of IFN-γ–activated hemophagocytic macrophages in driving cytopenias. Because this IFN-γ–driven pathological process occurs in both mutant and normal mice, it appears to be a pathophysiologic process that can be triggered in any individual with sufficient immune activation. The broader relevance of these findings is underscored by the fact that in some case series, nearly one third of patients admitted to hospital intensive care units with sepsis have unexplained cytopenias at presentation (Bateman et al., 2008
), and such critically ill patients with early onset cytopenias or marrow hemophagocytosis have been found to have inferior survival rates (Strauss et al., 2004
). Thus, this study provides a rationale for assessing hemophagocytosis in these various disease states and targeting this process for potential therapeutic benefit.
Second, these studies have significant implications for a better understanding of how IFN-γ influences macrophage biology. Although IFN-γ has for many years been described as a classical activator of macrophages, our data suggest that prolonged in vivo exposure triggers an additional unique and unexpected phenotype. Although hemophagocytosis was not triggered by a single dose of IFN-γ, continuous exposure over 24 h led to widespread uptake of RBCs. Although these kinetics will need to be studied in further detail, they suggest that new gene expression or other complex feedback mechanisms (Hu and Ivashkiv, 2009
) may be driving the hemophagocytic response. Future studies will be needed to better define such processes. The current studies also have significant implications for better understanding of how inflammation influences apoptotic cell uptake by macrophages. The process of hemophagocytosis bears similarities to efferocytosis (uptake of apoptotic cells) in that they are both macropinocytic processes. Also, because IFN-γ has recently been demonstrated to increase apoptotic cell uptake by macrophages (Fernandez-Boyanapalli et al., 2010
), it is possible that IFN-γ plays a significant role in altering apoptotic cell uptake in inflammatory environments. Although the proximal signals triggering cell uptake during hemophagocytosis are not known, engulfment is generally limited by “don’t eat me” signals that need to be actively circumvented or outweighed by “eat me” signals such as opsonization or phosphatidylserine exposure on the target cell (Oldenborg et al., 2000
; Gardai et al., 2005
). Induction of hemophagocytosis may involve alterations of macrophage receptiveness to don’t eat me signals, which may also alter apoptotic cell uptake. Future studies examining these signals, as well as macrophage membrane/cytoskeletal dynamics (such as microtubule formation; Binker et al., 2007
) may deepen our understanding of the hemophagocytic response and other fundamental biological processes.
Third, the current study provides significant new insight into understanding how inflammation drives destructive pathologies. The current study has defined a new form of immune-mediated pathology, CAI, and defined its critical underlying mediators, signaling mechanisms, and cell types. Although our studies illustrate the pathological potential of IFN-γ–driven up-regulation of macrophage pinocytosis and hemophagocytosis, future studies may define the nonpathological role of this process in the immune response. IFN-γ generally acts locally (not as a hormone) and is secreted directionally from T cells toward interacting cells (Schroder et al., 2004
; Huse et al., 2006
). This cytokine may commonly be sustained at high levels in inflammatory microenvironments (undoubtedly above the threshold level for CAI we defined with systemic infusion), triggering localized changes in macrophage endocytosis. Perhaps only when such levels are sustained systemically, as occurs with certain infections or in individuals with HLH, the process becomes pathological. Although IFN-γ appears to be uniquely capable of driving CAI (Jordan et al., 2004
), further study will be required to determine whether other inflammatory cytokines may have similar effects or whether they may modify the effects of IFN-γ. However, because IFN-γ is both necessary for induction of anemia in the context of acute infection and sufficient to induce anemia (and other cytopenias) in uninfected animals, this cytokine may be contributing to pathological cytopenias in a variety of clinical conditions.
Finally, these studies have increased our understanding of hematologic dynamics. CAI is a unique process that is distinct from the anemia of chronic inflammation because the former is an acute and consumptive process, whereas the latter involves alterations of iron metabolism and chronic suppression of hematopoiesis (Agarwal and Prchal, 2009
). Even though IFN-γ may suppress hematopoiesis in certain contexts, our studies do not suggest that marrow suppression plays a significant role in the cytopenias we observed after short-term IFN-γ exposure. The brisk reticulocytosis we observed after 5 d of IFN-γ infusion also suggests that significant hemophagocytosis may be occurring more commonly than is appreciated in various clinical contexts. Gene expression studies examining peripheral blood from patients with HLH or those with systemic onset juvenile idiopathic arthritis have revealed a unique primitive erythropoietic signature (Hinze et al., 2010
; Sumegi et al., 2011
).This finding suggests that significant compensation is occurring for (sometimes occult) hemophagocytosis. Over 60 yr ago, Alexander et al. (1956)
found that red blood cell life spans were significantly shortened in patients with rheumatoid arthritis. Remarkably, this shortened half-life correlated with a marker of systemic inflammation (erythrocyte sedimentation rate), both among patients and across time in individual patients. Although the anemia of chronic disease may also play a role in this patient population, such findings and our current data suggest that old assumptions about the physiology and pathology of the hematopoietic system should be reexamined.