- Top of page
- Materials and methods
Two anatomical sites that are important in human iron metabolism are the liver and placenta. Liver macrophages recycle iron from erythrocytes, and the placenta transfers iron from the mother to the fetus. The cellular distribution of proteins involved in iron transport in these two sites was studied. Transferrin receptor-1 (TfR1) and Ferroportin (FPN) expression was found on the placental syncytiotrophoblast (STB) and were polarised such that TfR1 was on the apical maternal-facing membrane and FPN was on the basal fetal-facing membrane, consistent with unidirectional iron transport from mother to fetus. Ferritin was strongly expressed in the stroma, suggesting that fetal tissue can store and accumulate iron. HFE was on some parts of the basal STB and, where present, HFE clearly colocalised with FPN but not TfR1. In the stroma, both HFE and FPN were present on CD68+ Hofbauer macrophage cells. In liver, the location of HFE is controversial. Using four mouse monoclonals and two polyclonal sera we showed that the pattern of HFE expression mirrored the distribution of CD68+ macrophage Kupffer cells. FPN was also most strongly expressed by CD68+ Kupffer cells. These findings contribute to understanding how iron is transported and stored in the human placenta and liver.
Controlled transport of iron is crucial to maintaining health. The molecular mechanisms that regulate iron homeostasis are becoming clearer with the recent identification of many genes with roles in iron metabolism (Hentze et al, 2004). How the gene products act in concert is unclear, and the pathogenesis of disorders of iron metabolism, such as hereditary haemochromatosis, is similarly ill defined.
We elected to investigate the locations of proteins of iron metabolism in the liver and the placenta. During pregnancy, iron is transferred from the mother across the placenta to supply the iron requirements of the developing fetus. Iron deficiency anaemia early in pregnancy doubles the risk of preterm delivery (Scholl, 2005), while fetal anaemia affects heart development and may contribute to the development of cardiovascular disease in adulthood(Davis et al, 2005). Understanding how iron is transported through the placenta is important in this context.
Two cell layers on the chorionic villi separate the maternal and fetal circulations. In the human haemochorial placenta the outer layer is directly in contact with maternal blood and is formed by the syncytiotrophoblast (STB), a single layer of fused cells. This originates from an underlying layer of cells called the villous cytotrophoblasts. The fetal capillary endothelium lies close to the basal (fetal) side of the STB. It has been demonstrated that iron attached to maternal transferrin (Tf) binds to Tf receptors on the apical (maternal) side of the STB (McArdle & Morgan, 1982; McArdle et al, 1984). This complex is internalised into endosomes (McArdle et al, 2003) and iron is released and transferred to the cytoplasm possibly by the divalent metal transporter (DMT1/Nramp2) (Georgieff et al, 2000). The mechanism of transfer across the STB cytoplasm is unknown. It is likely that iron is exported as Fe(II) by iron-regulated protein 1 (IREG-1)/ferroportin-1 (FPN) (Abboud & Haile, 2000; Donovan et al, 2000; McKie et al, 2000) across the basolateral side of the STB. Transferrin has a higher affinity for Fe (III) compared to Fe(II); Danzeisen et al (2002) identified a copper oxidase in placental membranes, similar to hephaestin in the gut, which they suggested catalyses the required oxidation of Fe(II) to Fe(III). Once bound to fetal transferrin, the iron can be taken up by stromal cells for storage in ferritin or enter the fetal circulation for transport and uptake by other organs.
The reported localisation of proteins by immunohistochemistry or electron microscopy is broadly compatible with this model of placental iron handling. Trophoblasts in normal pregnancy express transferrin receptor (TfR1) (Galbraith et al, 1980; Bergamaschi et al, 1990) but more is expressed on STB than on cytotrophoblast (Starreveld et al, 1992). TfR1 has been localised to both the apical and basal membranes of the STB (Vanderpuye et al, 1986; Verrijt et al, 1997). However, TfR1 has been localised predominantly to the apical membrane of the STB (van Dijk et al, 1993; Petry et al, 1994; Georgieff et al, 2000). Electron microscopy studies of human placenta confirmed this, where TfR1 colocalised with β-2 microglobulin at the apical side of the STB (Leitner et al, 2002). DMT1, also known as DCT-1 and Nramp2 (Gunshin et al, 1997; Andrews, 1999), is expressed in most if not all tissue. Georgieff et al (2000) identified DMT1 at the basal side of STB. FPN is located on the basolateral membrane of STB (Donovan et al, 2000; Bradley et al, 2004). Ferritin, the iron storage protein, has been found in placental term STB (Brown et al, 1979), cytotrophoblast and fetal endothelium (Dumartin & Canivenc, 1992). The gene HFE has been identified as the site of mutation in the common form of human haemochromatosis (Feder et al, 1996). The function of HFE is not known, but Parkkila et al (1997a) showed that HFE protein was expressed in human placenta and demonstrated that HFE could bind to TfR1 in lysates of placental tissue. Immunohistochemistry appeared to localise HFE with strong expression on the apical membrane of the STB (Parkkila et al, 1997a), where TfR1 is expressed in abundance.
In the liver the expression pattern of HFE is also unclear. We previously found that in the human liver, HFE was strongly expressed by macrophage Kupffer cells and weakly by the liver sinusoidal lining cells (Bastin et al, 1998). Griffiths et al (2000), using two polyclonal antisera directed against the alpha-1 and the alpha-3 domains of HFE, also showed that HFE protein was strongly expressed by human liver Kupffer cells, but not detectably by hepatocytes. HFE is also expressed in circulating monocytes, monocyte/macrophage cell lines and macrophages (Parkkila et al, 2000; Drakesmith et al, 2002). This cellular distribution is important, as the iron deposition in iron-loaded haemochromatosis patients in the liver is usually more severe in the hepatocytes, with Kupffer cells being relatively spared (Block et al, 1965). Kupffer cells recycle iron from dying red blood cells and release the iron back into plasma for re-incorporation into red cell precursors in the bone marrow.
The expression pattern of rat Hfe mRNA and protein has been studied, and two groups have shown, relatively recently, that rat Hfe is more strongly expressed by hepatocytes compared to Kupffer cells (Holmstrom et al, 2003; Zhang et al, 2004). As a result the expression of human HFE has become thought of as being localised to hepatocytes as well.
The present study aimed to clarify the locations of proteins involved in iron metabolism in human placenta and liver. Both alkaline phosphatase anti-alkaline phosphatase (APAAP) and immunofluorescence techniques were used to stain frozen tissue sections that had been minimally interfered with. In the placenta, HFE was present on stromal CD68+ cells and occasionally on the basal (but not the apical) membrane of the STB, in contrast to previous findings (Parkkila et al, 1997a). FPN and TfR1 were expressed on opposite membranes of the STB, which lies close to but not overlapping CD31+ endothelial fetal capillaries. Using six different anti-HFE antibodies, we showed that in the liver HFE was strongly expressed by Kupffer cells and minimally detectable on hepatocytes. These findings illustrate the routes of iron transport in the placenta and suggest a species difference between rats and humans with respect to the cellular distribution of HFE in liver.