Iron and macrophages in atherogenesis

Sammanfattning: Oxidation of low density lipoprotein (LDL) may result in its uptake by macrophages and ensuing foam cell formation. Thus, oxidised LDL may play an important role in atherogenesis. Extensive in vitro evidences are in favour of the notion that LDL oxidation by cells present in atherosclerotic plaques requires the presence of transition metals. It has been confirmed repeatedly that atherosclerotic lesions contain significant amounts of copper and iron. The mechanism by which LDL becomes oxidised in vivo, though, remains unknown.In the first part of the present study we wanted to learn about how iron is involved in the process of LDL oxidation by human macrophages; whether iron may be exocytosed after cellular exposure to different iron compounds; and if such exocytosis would affect LDL oxidation and its uptake by macrophages. Human monocyte-derived macrophages (HMDMs) were firstly exposed to different iron compounds (100μM), or haemoglobin (25 or 50 μg/ml) for 24 hours. Following rinsing LDL (50 or 150 µg/ml) was added in fresh culture medium without serum. After another 24 hours the concentrations of iron and thiobarbituric acid-reactive substances (TBARS), as well as the electrophoretic mobility of LDL in medium, were found increased, while the cells showed only minimal signs of decreased viability. Neutral lipids and phospholipids accumulated in a granular, lysosome-like, pattern and the cells acquired a foam cell-like morphology.The second part of the study was designed (i) to establish a model of erythrophagocytosis by macrophages, and (ii) to study iron-sequestration within secondary lysosomes, and exocytosis by these cells following the degradation of erythrocytes. The binding and uptake of UV-irradiated red blood cells (UV-RBC) by human macrophages and J-774 cells were greatly stimulated compared to that of native erythrocytes. The uptake resulted in lysosomal accumulation of iron in a low-molecular-weight form, as shown by autometallography. Following the exposure to UV-RBC and ferric iron a much enhanced amount of cytosolic ferritin was demonstrated in macrophages by immunocytochemistry. Ensuing exocytosis of iron to the culture medium was demonstrated by atomic absorption spectroscopy.The third part of the study aimed to investigate oxidative stress-induced lipofuscinogenesis in human macrophages as well as in a test-tube system of mitochondria and lysosomes from rat liver. Firstly, control HMDMs, and HMDMs exposed to different iron compounds (100 µM Fe3+) or Hb (25 or 50 µg/ml), were incubated for 48 hours with LDL. Lipofuscin-specific autofluorescence was markedly increased in all LDL-exposed cells. A linear correlation was found between lipofuscin formation and the concentration of FeCl3 to which the HMDMs earlier had been pre-exposed. Secondly, endogenous iron in lysosomal-mitochondrial fraction (LMF) homogenates (545 µg/1, about 10 µM) was detected by atomic absorption spectrophotometry. After incubation of LMF with different concentrations of cystein for different periods of time a time- and dose-dependent TBARS-yield was observed. The peroxidation was completely inhibited by the addition of desferrioxamine or butylated hydroxytoluen (BHT). Under the same conditions the carbonyls of the trichloroacetic acid (TCA) precipitable protein of LMF were analysed. They were found increased, but only after a slight initial decrease. Following a sharp initial decrease, the normal tryptophan-tyrosine (protein) autofluorescence remained stable. In contrast, after a lag period of a few days, a lipofuscin-type autofluorescence was also observed.In conclusion: A. Lysosomal iron may be exocytosed from HMDMs, following a previous uptake of simple iron compounds or Hb promoting oxidation and uptake of LDL and thus induce foam cell formation. B. Macrophage erythrophagocytosis is a useful model for the study of the lysosomal sequestration of iron. Iron is accumulated within the macrophage acidic vacuolar apparatus arid subsequently exocytosed. C. Lipofuscin forms in secondary lysosomes as a result of iron-catalyzed oxidative reactions involving autophagocytosed materials D. LDL and iron may both play important roles in lipofuscinogenesis within atherosclerotic lesions.