Solute carrier 11a1 (Slc11a1; formerly Nramp1) regulates metabolism and release of iron acquired by phagocytic, but not transferrin-receptor-mediated, iron uptake

Abstract

Solute carrier 11a1 (Slc11a1; formerly Nramp1; where Nramp stands for natural-resistance-associated macrophage protein) is a proton/bivalent cation antiporter that localizes to late endosomes/lysosomes and controls resistance to pathogens. In the present study the role of Slc11a1 in iron turnover is examined in macrophages transfected with Slc11a1Gly169 (wild-type) or Slc11a1Asp169 (mutant = functional null) alleles. Following direct acquisition of transferrin (Tf)-bound iron via the Tf receptor, iron uptake and release was equivalent in wild-type and mutant macrophages and was not influenced by interferon-γ/lipopolysaccharide activation. Following phagocytosis of [59Fe]Tf—anti-Tf immune complexes, iron uptake was equivalent and up-regulated similarly with activation, but intracellular distribution was markedly different. In wild-type macrophages most iron was in the soluble (60%) rather than insoluble (12%) fraction, with 28% ferritin (Ft)-bound. With activation, the soluble component increased to 82% at the expense of Ft-bound iron (< 5%). In mutant macrophages, 40–50% of iron was in insoluble form, 50–60% was soluble and < 5% was Ft-bound. Western-blot analysis confirmed failure of mutant macrophages to degrade complexes 24h after phagocytic uptake. Confocal microscopy showed that complexes were within lysosome-associated membrane protein 1-positive vesicles in wild-type and mutant macrophages at 30min and 24h, implying failure in the degradative process in mature phagosomes in mutant macrophages. NO-mediated iron release was 2.4-fold higher in activated wild-type macrophages compared with mutant macrophages. Overall, our data suggest that iron acquired by phagocytosis and degradation is retained within the phagosomal compartment in wild-type macrophages, and that NO triggers iron release by direct secretion of phagosomal contents rather than via the cytoplasm.