Introduction

Iron is an essential element for vertebrates, but both its deficiency and excess cause pathological conditions [1, 2]. Because there are no secretory pathways for excess iron, both genetic and iatrogenic oversupply cause refractory disease conditions. Iron is mainly recycled in hepatocytes, the parenchymal cells of the liver, and Kupffer cells of the reticuloendothelial (RE) system. These cells have the potential to store relatively large amounts of iron inside ferritin molecules [3], and then proliferate iron-rich lysosomes known as hemosiderins [4]. In hemochromatosis, in addition to the iron-laden hepatocytes and Kupffer cells, macrophages appear in the portal fibrous bands of cirrhotic livers as the third type of iron storage cell [5].

The four molecules of hemochromatosis protein (HFE), transferrin receptor 2 (TFR2), hemojuvelin (HJV), and human anti-microbial peptide (HAMP) are be involved in hemochromatosis [1]. Identification of the iron regulator hormone hepcidin [6] and its receptor ferroportin (FPN) [7, 8], which had been known as the only iron exporter, have provided new insights into iron overload syndromes. Based on the hepcidin/FPN system, genetic iron overload syndromes have been classified into 3 subtypes of pre-hepatic aceruloplasminemia (ACP) due to the CP gene, hepatic hemochromatosis of the HFE, TFR2, HJV, and HAMP genes, and post-hepatic ferroportin disease (FPD) [9]. In other words, hemosiderins, the histological symbols of iron overload syndromes, are all products of non-primary lysosomal diseases.

None of the genes or gene products responsible for the genetic iron overload syndromes have been linked to any lysosomal enzymes [10]. If this remains true, hemosiderins may consist of heterogeneous metabolites including iron complexes, copper complexes and other nutrients. This is in contrast to the fact that, in a primary lysosomal disease, the lysosomal matrices correspond with the substrates targeted by the deficient enzyme [11], for example, glycogen vs. acid alpha-glucosidase deficiency in Pompe disease [12]. In a previous study, we reported that hemosiderins of iron-rich lysosomes consist of major iron complexes of Fe, P, and O, and minor copper complexes of Cu and S under various conditions from hemochromatosis to iatrogenic iron overload [13]. We postulate that cuproproteins are adaptively induced by cytotoxic iron, following ferritin synthesis, because the iron homeostasis is tightly controlled by cuproproteins [14].

Cuproproteins appear in the secondary lysosomes of Wilson disease and chronic cholestasis such as primary biliary cholangitis and primary sclerosing cholangitis secondary to the impaired biliary secretion of copper. Toxic copper was stored in the dense bodies as detoxified SH-rich cuprothioneins regardless of the etiologies ([15] Hanaichi). Under iron overload conditions, detoxified copper-sulfur complexes in the secondary lysosomes may reflect the enhanced turnover of cuproproteins in the liver, but may be different from native enhanced cuproproteins involved in iron metabolism of the cytoplasm. Either copper regurgitated from the biliary system or copper released from super annuated cuproproteins molecules may be stored as detoxified forms of SH-rich cuprothioneins [13]. This second report was focused on the iron–copper interaction in large inclusions of aggregated hemosiderins found in the macrophages of severe iron overload syndromes. Further structure-based study on macrophagic inclusions of aggregated hemosiderins may provide new information on the defense mechanism against toxic iron.

Materials and methods

The study protocol, including diagnostic liver biopsy and gene analysis of iron overload syndromes, was approved by the Ethics Committees of Nagoya University School of Medicine (NU No. 2005-277, 2008-660, 2012-277-2) and Aichi Gakuin University School of Pharmacy (AGU No. 10). Prior to the liver biopsy and genetic test, informed consent was obtained from each patient. Five patients with livers showing heavy iron overload associated with iron-rich inclusions larger than 2.0 µm and 2 control patients with mild iron overload were selected from our database. All patients had been referred to the gene analysis center of Aichi Gakuin University School of Pharmacy for the definite diagnosis of iron overload syndromes. The 5 genes of HFE, TFR2, HJV, HAMP, and SLC40A1 were analyzed as reported previously ([10], Hattori). Their clinical features, iron parameters, and liver histology had been fully evaluated at entry. Some of them were reported in detail previously ([16] Koyama, [17] Watanabe, [18] Tsuchida, [19] Yamashita). The serum concentration of hepcidin-25 was determined by a liquid chromatography/selected reaction monitoring mass spectrometry (LC/MS/MS) assay system, as reported previously ([20], Kaneko). The liver histology included HE stain for the standard test, Berlin blue stain for iron, and rhodanine stain for copper.

For the current ultrastructural study, portions of their liver tissues in paraffin blocks, after the chemical extraction of paraffin, were re-fixed in a glutaraldehyde solution. Then, a few pieces of the liver specimen, with and without post-osmic acid fixation, were embedded in epoxy resin, and ultra-thin sections mounted on gold grids were examined under an electron microscope (EM) with an energy dispersive X-ray detector (EDX), JEM 1400 Plus (JEOL, Tokyo, Japan), as previously reported [13].

First, the TEM mode was applied for visualizing the inclusions and other organella in the ultra-thin sections stained twice by uranyl and lead solutions. Second, 2-dimensional analysis using the STEM mode was performed for element distribution in the electron-dense particles of unstained sections. The accumulation of each element in the particles was determined by the positive imaging of its specific X-ray with the standard 25-sweep-cycle analysis. This ultrastructural study was focused on the individual hemosiderins of lysosomal sizes, and larger inclusions of aggregated hemosiderins in the liver.

Results

The clinical features of the 5 patients and 2 controls are summarized in Tables 1, 2, and 3. Three male and 2 female patients were enrolled in the study. Their ages ranged among 40 and 79 years. The etiologies of their iron overloads varied from genetic to iatrogenic backgrounds. All the patients were biochemically affected by markedly heavy iron overloading of serum ferritin concentrations of more than 5000 ng/mL. The serum levels of hepacidin25 were variable, ranging from below the detection limit of HJV-hemochromatosis to the highest of 186 ng/mL of FPD-B-like disease. A 33-year-old female patient with iatrogenic iron overload and a 44-year-old male patient with ACP served as the controls, and their serum ferritin levels were 2076 and 961 ng/mL, respectively.

Table 1 Clinical features of patients with inclusion bodies of aggregated hemosiderins in the liver and controls
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Table 2 Biochemical parameters of patients with inclusion bodies of aggregated hemosiderins in the liver and controls
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Table 3 Histology and iron overload of the livers with inclusion bodies of aggregated hemosiderins and controls
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The liver specimens of the 5 patients showed portal fibrosis or cirrhosis associated with inflammatory reactions and heavy iron deposits in the macrophages of expanded portal tracts, and in Kupffer cells and hepatocytes of the nodular parenchyma. Iron-filled inclusions that were round in shape and larger than 2 µm in size appeared in periportal areas (Fig. 1). There were no inclusions in the control livers with mild iron overload. Copper staining was negative in all 7 liver specimens.

Fig. 1
figure 1

Macrophages with inclusions in peri-portal parenchyma of the pre-cirrhotic liver fibrosis of TFR2 hemochromatosis stained by Berlin blue. Iron overload is parenchymal hepatocyte-dominant, but a few macrophages with dark-blue stained inclusions (arrows) are scattered in the parenchyma. Kupffer cells look normal in size except for iron deposits in their cytoplasm (in circles). There are many iron-rich cells presenting with transitional forms between hepatocytes and macrophages. The short bar indicates 2.0 µm, while the long bar indicates 50 µm

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Under EM, inclusions appeared in the macrophages characterized by scanty mitochondria and glycogen in their cytoplasm (Fig. 2). Usually, the inclusions that resembled organelles of membrane-bound aggregated hemosiderins co-existed with a few individual hemosiderins in the macrophages. Based on an X-ray microanalyzer, the major components of the inclusions were the iron complexes of Fe, O, and P, and minor components were copper complexes of Cu and S (Fig. 3). The copper element was iron-dependent in its distribution within the mosaic matrices of the inclusions. Hepatocytes of the control livers contained a large number of individual hemosiderins, but no large inclusions of aggregated hemosiderins. Some individual hemosiderins of the control livers were also visualized in Cu-X-ray images as well as Fe images.

Fig. 2
figure 2

Ultrastructure of a macrophage with a variety of aggregated and isolated hemosiderins. Some of them may be visualized as inclusions under a light microscope. Note the scant cytoplasm of other organella such as mitochondria and scant presence of glycogen. Bar indicates 2.0 µm

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Fig. 3
figure 3

Two-dimensional analysis of a macrophage with inclusions of aggregated hemosiderins. The macrophage contains large inclusions and isolated hemosiderins. Their matrices consist of major complexes with iron, oxygen, and phosphorus and minor ones with copper and sulfur, indicating that a large amount of iron and trace amount of copper are stored as detoxified forms in the secondary lysosomes of iron overloaded livers. Note the P-X-ray image of a nucleus

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Discussion

This is the second report on the updated terminology for hemosiderins and related organelles in the liver with iron overload [13]. Because hemosiderins are iron-rich lysosomes that appear in secondary lysosomal diseases [10], they should be membrane-bound electron-dense bodies that contain the major component of iron complexes and minor ones of copper complexes and a variety of nutrients. Iron-rich inclusion bodies larger than 2 µm were visualized under EM as organelles of membrane-bound aggregated hemosiderins isolated in the macrophages. Their structures were characterized by a variety of aggregated and individual hemosiderins, and scanty mitochondria and glycogen, different from cytoplasm of either hepatocyte or Kupffer cells. In contrast, the control livers contained individual hemosiderins consisting of major iron complexes and minor copper ones, but they did not contain the large inclusions of membrane-bound aggregated hemosiderins.

Cellular defense systems for the potential iron have been reported as ferritin synthesis and hemosiderin proliferation [2]. The ferritin shell and its ferroxidase center have the potential to store a large amount of stabilized ferric ions in the core [3]. With a more severe iron load, hemosiderins of lysosomal origin of about 0.5 µm in diameter appear and proliferate in number to isolate toxic molecules in the membrane-bound organelle [4]. In a previous study [13], we reported that iron-rich particles in iron overload syndromes consisted of major components of Fe, O, and P, and minor components of Cu and S, reflecting the enhanced induction of cuproproteins in iron overloaded cells.

The distinct co-existence of copper with iron in the iron-rich hemosiderins and inclusions of aggregated hemosiderins may be a novel mechanism different from the copper retention known in Wilson disease and chronic cholestasis [13, 15]. Physiologically, lysosomes store a small amount of copper and recycle it between aged and renewed organelles [21, 22]. Iron-dependent copper accumulation in the inclusions of aggregated hemosiderins may reflect cytoprotective reactions in the cytosol to stabilize an activated redox state under iron-overload conditions. Candidates for induced cuproproteins include Cu/Zn superoxide dismutase and multi-copper oxidases [23, 24]. However, considering the observation that copper always co-exists with sulfur in lysosomal dense bodies of Wilson disease, chronic cholestasis, and iron overload conditions [13, 15], either copper released from superannuated cuproprotein molecules or copper regurgitated from the biliary system may be stored as detoxified forms of SH-rich cuprothioneins.

Large iron inclusions have been reported under pathological conditions. They include nuclear ferritin bodies in ferritinopathy [25] and inclusions of aggregated hemosiderins in ACP [26]. The nuclear ferritin bodies were different from the inclusion bodies in the macrophages with regard to the original organella and etiological background. The nuclear inclusions of a large amount of ferritin grains appeared in the brain and other organs associated with a mutation in the ferritin light polypeptide gene [27]. The inclusions of the ACP brain may be similar to the hepatic inclusions of our patients. Both inclusions of lysosomal origin consisted of the major iron complexes and minor copper ones. If they both appear in macrophages, it is likely that, regardless of the organs, the iron-induced macrophages have the potential to form large inclusion bodies of membrane-bound aggregated hemosiderins in the presence of marked iron overload.

In conclusion, the large inclusion body formation observed in such macrophages may be the forth defense mechanism against toxic iron, following ferritin synthesis, cuproprotein induction, and hemosiderin proliferation.