The online version of this article has a Supplementary Appendix. BackgroundMacrophages play a key role in iron homeostasis. In peripheral tissues, they are known to polarize into classically activated (or M1) macrophages and alternatively activated (or M2) macrophages. Little is known on whether the polarization program influences the ability of macrophages to store or recycle iron and the molecular machinery involved in the processes. Design and MethodsInflammatory/M1 and alternatively activated/M2 macrophages were propagated in vitro from mouse bone-marrow precursors and polarized in the presence of recombinant interferon-γ or interleukin-4. We characterized and compared their ability to handle radioactive iron, the characteristics of the intracellular iron pools and the expression of molecules involved in internalization, storage and export of the metal. Moreover we verified the influence of iron on the relative ability of polarized macrophages to activate antigen-specific T cells. ResultsM1 macrophages have low iron regulatory protein 1 and 2 binding activity, express high levels of ferritin H, low levels of transferrin receptor 1 and internalize -albeit with low efficiencyiron only when its extracellular concentration is high. In contrast, M2 macrophages have high iron regulatory protein binding activity, express low levels of ferritin H and high levels of transferrin receptor 1. M2 macrophages have a larger intracellular labile iron pool, effectively take up and spontaneously release iron at low concentrations and have limited storage ability. Iron export correlates with the expression of ferroportin, which is higher in M2 macrophages. M1 and M2 cells activate antigen-specific, MHC class II-restricted T cells. In the absence of the metal, only M1 macrophages are effective. ConclusionsCytokines that drive macrophage polarization ultimately control iron handling, leading to the differentiation of macrophages into a subset which has a relatively sealed intracellular iron content (M1) or into a subset endowed with the ability to recycle the metal (M2).Key words: macrophages, iron, inflammation. 95(11):1814-1822 doi:10.3324/haematol.2010 This is an open-access paper. Polarization dictates iron handling by inflammatory and alternatively activated macrophages. Haematologica Polarization dictates iron handling by inflammatory and alternatively activated macrophages

Inherited microcytic-hypochromic anemias in rodents and zebrafish suggest the existence of corresponding human disorders. The zebrafish mutant shiraz has severe anemia and is embryonically lethal because of glutaredoxin 5 (GRLX5) deletion, insufficient biogenesis of mitochondrial iron-sulfur (Fe/S) clusters, and deregulated iron-regulatory protein 1 (IRP1) activity. This leads to stabilization of transferrin receptor 1 (TfR) RNA, repression of ferritin, and ALA-synthase 2 (ALAS2) translation with impaired heme synthesis. We report the first case of GLRX5 deficiency in a middle-aged anemic male with iron overload and a low number of ringed sideroblasts. Anemia was worsened by blood transfusions but partially reversed by iron chelation. The patient had a homozygous (c.294A>G) mutation that interferes with intron 1 splicing and drastically reduces GLRX5 RNA. As in shiraz, aconitase and H-ferritin levels were low and TfR level was high in the patient's cells, compatible with increased IRP1 binding. Based on the biochemical and clinical phenotype, we hypothesize that IRP2, less degraded by low heme, contributes to the repression of the erythroblasts ferritin and ALAS2, increasing mitochondrial iron. Iron chelation, redistributing iron to the cytosol, might relieve IRP2 excess, improving heme synthesis and anemia. GLRX5 function is highly conserved, but at variance with zebrafish, its defect in humans leads to anemia and iron overload. (Blood.

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Mitochondrial ferritin (FtMt) is a nuclear-encoded iron-sequestering protein that specifically localizes in mitochondria. In mice it is highly expressed in cells characterized by high-energy consumption, while is undetectable in iron storage tissues like liver and spleen. FtMt expression in mammalian cells was shown to cause a shift of iron from cytosol to mitochondria, and in yeast it rescued the defects associated with frataxin deficiency. To study the role of FtMt in oxidative damage, we analyzed the effect of its expression in HeLa cells after incubation with H(2)O(2) and Antimycin A, and after a long-term growth in glucose-free media that enhances mitochondrial respiratory activity. FtMt reduced the level of reactive oxygen species (ROS), increased the level of adenosine 5'triphosphate and the activity of mitochondrial Fe-S enzymes, and had a positive effect on cell viability. Furthermore, FtMt expression reduces the size of cytosolic and mitochondrial labile iron pools. In cells grown in glucose-free media, FtMt level was reduced owing to faster degradation rate, however it still protected the activity of mitochondrial Fe-S enzymes without affecting the cytosolic iron status. In addition, FtMt expression in fibroblasts from Friedreich ataxia (FRDA) patients prevented the formation of ROS and partially rescued the impaired activity of mitochondrial Fe-S enzymes, caused by frataxin deficiency. These results indicate that the primary function of FtMt involves the control of ROS formation through the regulation of mitochondrial iron availability. They are consistent with the expression pattern of FtMt observed in mouse tissues, suggesting a FtMt protective role in cells characterized by defective iron homeostasis and respiration, such as in FRDA.

Parkinson's disease is a neurodegenerative disorder characterized by oxidative stress and CNS iron deposition. Ceruloplasmin is an extracellular ferroxidase that regulates cellular iron loading and export, and hence protects tissues from oxidative damage. Using two-dimensional electrophoresis, we investigated ceruloplasmin patterns in the CSF of human Parkinson's disease patients. Parkinson's disease ceruloplasmin profiles proved more acidic than those found in healthy controls and in other human neurological diseases (peripheral neuropathies, amyotrophic lateral sclerosis, and Alzheimer's disease); degrees of acidity correlated with patients' pathological grading. Applying an unsupervised pattern recognition procedure to the two-dimensional electrophoresis images, we identified representative pathological clusters. In vitro oxidation of CSF in two-dimensional electrophoresis generated a ceruloplasmin shift resembling that observed in Parkinson's disease and co-occurred with an increase in protein carbonylation. Likewise, increased protein carbonylation was observed in Parkinson's disease CSF, and the same modification was directly identified in these samples on ceruloplasmin. These results indicate that ceruloplasmin oxidation contributes to pattern modification in Parkinson's disease. From the functional point of view, ceruloplasmin oxidation caused a decrease in ferroxidase activity, which in turn promotes intracellular iron retention in neuronal cell lines as well as in primary neurons, which are more sensitive to iron accumulation. Accordingly, the presence of oxidized ceruloplasmin in Parkinson's disease CSF might be used as a marker for oxidative damage and might provide new insights into the underlying pathological mechanisms.