The Role of Nramp1 in Erythrophagocytosis.

Abstract Erythrocytes contain the majority of the body’s iron in the form of heme, a component of the red blood cell pigment hemoglobin. Erythrocytes transport oxygen bound to hemoglobin from the lungs to the tissues, and become progressively more damaged as they age. Macrophages are responsible for the detection and degradation of these senescent red blood cells, and play a central role in the recycling of hemoglobin-derived iron via transferrin to the bone marrow in order for erythropoiesis to occur. This recycling process, and the influence of inflammatory mediators on it, are poorly understood. Nramp1 is a protein expressed within macrophages, and is an iron transporter discovered to be mutated in mice susceptible to tuberculosis and leprosy infections. As Nramp1 localizes to phagosomal membranes following phagocytosis, we speculate that Nramp1 may play a role in the recycling of hemoglobin-derived iron following the internalization of effete erythrocytes. To test this possibility, we have utilized a model of erythrophagocytosis using both Nramp1 replete and deficient RAW264.7 murine macrophages, and macrophages elicited from wild type and Nramp1-knockout 129SV mice. We have found that Nramp1 protein is increased in primary macrophages following internalization of opsonized murine erythrocytes. This increase peaks at 6 hours of erythrocyte exposure, and decreases to baseline levels overnight. Our model of erythrophagocytosis did not induce macrophage activation as no nitric oxide production was observed. This therefore discounts the possibility that inflammatory factors are the cause of the increase in Nramp1 protein levels. Using the fluorescent iron-binding probe, calcein, we monitored changes in the macrophage labile iron pool (LIP) following erythrocyte loading. This transit pool of loosely bound, chelator-accessible iron is thought to reflect the overall iron status of the cell. We have found that, following erythrophagocytosis of opsonized murine erythrocytes, Nramp1-replete RAW264.7 macrophages experience a greater-fold increase in labile iron as compared to Nramp1-deficient macrophages. Furthermore, we have also found that primary peritoneal macrophages elicited from wild type 129SV mice phagocytose more erythrocytes when compared to macrophages elicited from their Nramp1-knockout counterparts. Interestingly, we have also observed that treatment with erythropoietin, the hormone responsible for the promotion of erythrocyte precursor survival and ultimately the increase in red blood cell production, resulted in a modest increase in Nramp1 protein levels. Our observations show distinct differences in iron metabolism between Nramp1 replete and deficient macrophages undergoing erythrophagocytosis. We speculate that Nramp1 may serve to promote a more efficient intracellular transport of iron following red blood cell engulfment.