Iron metabolism in normal and hemochromatotic macrophages.

Certain metabolic pathways of iron were studied in macrophages (cultured human monocytes) obtained from normal and hemochromatotic subjects. The relative abilities of the hydrophobic ferrous chelator 2,2' bipyridine and the hydrophilic ferric chelators desferrioxamine (DFO) and diethylenetriaminepenta-acetic acid (DTPA) to release iron from normal and hemochromatotic macrophages which had previously been loaded with diferric transferrin were tested but there were no differences between the two groups. The relative affinity of the macrophages for diferric transferrin was next studied. Although the hemochromatic macrophages had a somewhat lower affinity for diferric transferrin iron than normal macrophages (Kd 4.7 x 10(-8) M vs. 3.0 x 10(-8)M) the difference did not reach statistical significance (t = 2.01013; P less than 0.07). In a further experiment there was no evidence that apotransferrin was directly involved in the release of iron from hemochromatotic macrophages. A clue to the nature of postendocytotic trans-membrane transport of iron was provided by the finding that it was inhibited by the hydrophobic ferrous chelator 2,2' bipyridine. However, the degree of inhibition was similar in both normal and hemochromatotic macrophages. In summary, none of the metabolic processes examined in the present study was abnormal in cultured human blood monocytes from hemochromatotic subjects.

Effect of ferrous and ferric chelators on transferrin-iron-macrophage interactions.

A study was done to evaluate the effect of ferrous and ferric chelators on the interaction between transferrin-iron and cultured human blood monocytes. This interaction has been previously shown to involve a specific receptor and vesicle protonation. Transferrin-iron uptake was significantly inhibited by the hydrophobic ferrous chelator 2,2' bipyridine, and the inhibition was shown not to be a consequence of the mobilisation of intracellular iron by the chelator. Chase experiments and prolonged incubation studies suggested that the chelator prevented the iron released from transferrin from negotiating the unit membrane. The iron and transferrin then appeared to be returned independently to the incubation medium. In contrast, a hydrophilic ferrin chelator, desferrioxamine, had only a very modest effect on the interaction. These findings are compatible with the hypothesis that transferrin-iron is reduced to the ferrous state during its uptake by the culture human blood monocyte.

Transferrin iron interactions with cultured hepatocellular carcinoma cells (PLC/PRF/5).

Hepatocellular carcinoma cells of the PLC/PRF/5 cell line had 1.9 x 10(5) transferrin receptors per tumor cell with a Kd of 1.5 x 10(-8) M. At high concentrations of transferrin the binding was not saturable. Transferrin internalization by hepatoma cells was shown by time and temperature-dependent binding studies and by pronase experiments. Transferrin recycling was confirmed by the demonstration of a progressive increase in the cellular molar ratios of iron to transferrin and by chase experiments. Ammonium chloride interfered with iron unloading. The vinca alkaloid vincristine inhibited iron and transferrin uptake. The hepatocarcinoma cells appeared to lack asialoglycoprotein receptors and therefore internalized partially desialated transferrin by the regular route. Iron uptake from transferrin was markedly inhibited by the hydrophobic ferrous chelator 2,2' bipyridine but was relatively unaffected by the hydrophilic ferric chelator desferroxamine. The implication that ferrous iron was involved in postendocytic transvesicular membrane iron transport was supported by a study in which hepatoma cells were shown to take up large amounts of ferrous iron suspended in 270 mM sucrose at pH 5.5. The interaction at this pH between surface labeled hepatoma cell extracts and ferrous iron on a Sephacryl S-300 column suggested that the postendocytic transvesicular transport of iron through the membrane was in part protein mediated. The endocytosed iron in hepatoma cells was found in association with ferritin (33%), transferrin (31%) and a low molecular weight fraction (21%).

Apotransferrin receptors and the delivery of iron from cultured human blood monocytes.

A study was done to find out whether apotransferrin receptors are involved in the release of iron from reticuloendothelial cells. To this end, human macrophages which had been obtained by culturing blood monocytes for 7 days were incubated with either diferric or apotransferrin at the physiological pH of 7.4 or at an acidic pH (6.0). While specific diferric transferrin receptors (Kd 1.3 X 10(-8) M) were demonstrated at pH 7.4, no apotransferrin receptors were found. In contrast, both diferric receptors (Kd 2.1 X 10(-8) M) and apotransferrin receptors (Kd 6.8 X 10(-9) M) were found at pH 6.0. The findings of specific apotransferrin binding at acidic pH fits in with the current understanding of iron uptake by cells, in which the iron-transferrin complex is endocytosed and the iron is released at acidic pH. The present results suggest that the apotransferrin remains attached to its receptor in the endocytosed vesicle at this acidic pH but that it becomes detached at the cell surface where the pH is neutral. No evidence was found to indicate that iron is transported out of macrophages via apotransferrin receptors at the physiological pH.

Transferrin receptors and transferrin iron uptake by cultured human blood monocytes.

Transferrin receptors have been previously found on human macrophages and it has also been shown that transferrin iron is taken up by these cells. It has therefore been inferred that the uptake is receptor mediated and involves an endocytic pathway. The subject was addressed directly in the present study in which the transferrin-iron-receptor interaction was characterized in cultured human blood monocytes. Specific, saturable diferric transferrin binding was demonstrated, with a kDa of 3.6 X 10(-8) M and a calculated receptor density of 1.25-2.5 X 10(5) receptors per cell. Incubation at 4 degrees C markedly reduced transferrin binding and completely inhibited iron uptake. Chase experiments confirmed progressive cellular loading of iron, with concomitant loss of transferrin. Inhibitors of endocytic vesicle acidification (ammonium chloride and 2,4-dinitrophenol) inhibited iron unloading from endocytosed diferric transferrin, while microtubular inhibitors (colchicine and vindesine) and a microfilament inhibitor (cytochalasin B) reduced diferric transferrin uptake but had little effect on the iron unloading pathway. A similar effect was noted with a calcium ion antagonist (verapamil) and with 2 calmodulin antagonists (chlorpromazine and imipramine). These latter findings suggest the importance of cytoskeleton-membrane interactions via a calcium, calmodulin and protein kinase C mediated system. Endocytosed iron accumulated progressively as ferritin within the cultured monocytes.