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Keywords:

  • iron metabolism;
  • haemochromatosis

Summary

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgements
  8. References

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.

Materials and methods

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgements
  8. References

Tissues

Placentas were obtained from the Obstetrics and Gynaecology Department of the John Radcliffe Hospital (using procedures with ethical approval number: c00.147). There were five term and three first trimester placental samples. Villous tissue free from haematoma was sampled from the maternal–fetal interface, thoroughly washed to eliminate fetal and maternal blood cells and snap frozen in liquid nitrogen. Livers were obtained from the Histopathology Department of the John Radcliffe Hospital, Oxford (using procedures with ethical approval number: c02.162). The livers were morphologically normal by expert assessment and tissue samples were snap frozen in liquid nitrogen and stored at −70°C.

Tissue processing and staining for fluorescence

Cryostat tissue sections of 8 μm were cut, left to dry for 6–18 h, fixed in acetone at room temperature for 10 min, air dried, foil wrapped in pairs back-to-back and stored at −20°C until needed. Sections were removed from the freezer and allowed to reach room temperature before unwrapping. Primary antibodies (either singly or in double combinations) were applied to the sections at appropriate dilutions ranging from 10 to 20 μg/ml and incubated for 30 min at room temperature. The slides were washed for 5 min in Tris-buffered saline (TBS) and incubated with a mixture of fluorescein isothiocyanate-labelled affinity purified goat anti-rabbit Ig antibody (Sigma) at 1:50 dilution and Alexa Fluor 568 goat anti-mouse IgG antibody (Molecular Probes, Eugene, OR, USA) at 1:200 dilution for 30 min at room temperature in the dark. Therefore, the rabbit polyclonal antisera were labelled green and the murine monoclonal antibodies were labelled red on the section. The slides were washed in TBS for 5 min and mounted in Vectashield mounting medium with 4′, 6-diamidino-2-phenylindole (DAPI) to counterstain for nuclear DNA. Images were captured using an immunofluorescence Axiovert S100 (Hal100) camera (Hamamatsu, Hamamatsu City, Japan) and processed using openlab software.

Peroxidase and APAAP staining of sections

Tissue section preparations were fixed in acetone. Monoclonal or polyclonal antibody was added to the dry preparation and incubated for 30 min. The slides were washed for 5 min in TBS and incubated with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit (Dako, Ely, UK) for 30 min. The reaction was developed using diaminiobenzidene (Sigma FastTabs, Poole, UK). For APAAP staining, the procedure followed was as described by Cordell et al (1984). The slides were mounted in Aquamount.

Antibodies

Rabbit polyclonal antisera were affinity purified as described (Bastin et al, 1998). In our laboratory, sera were prepared to the 19 C-terminal amino acids of FPN (as described by Donovan et al, 2000) and the 16 C-terminal amino acids of HFE (Parkkila et al, 1997b; Bastin et al, 1998). An additional serum was raised to a peptide from the alpha-3 domain (residues 246–260) of HFE as described (Feder et al, 1997). Antisera to ferritin, Tf and Helicobacter pylori (negative control) were obtained from Dako. Antiserum to Tf receptor was obtained from Santa Cruz (Santa Cruz, CA, USA).

Monoclonal antibodies used were KP1, which recognises macrophages (CD68) (Pulford et al, 1989), Kato 16C (Turley, 1995) and Ber T9 (Boeker & Werfel, 1995), which both recognise TfR1 (CD71), anti-cytokeratin 7 for STB (Dako), anti-CD31 (vascular endothelium) (Dako), anti-hepatocyte (Dako), anti-ferritin (Biogenex, San Ramon, CA, USA), MR12 (Dako) recognises rabbit Ig and was used as a negative control. We used four different mouse monoclonals raised against HFE; JB1 from our lab raised against bacterially expressed and refolded HFE (Bastin et al, 1998; Townsend, 2003), 8C10 and 2F5, raised by injecting mice with mouse cells expressing human HFE (Ben-Arieh et al, 2001) and 10G4, raised against recombinant soluble HFE expressed by Drosophila melanogaster SC2 cells (Salter-Cid et al, 1999).

Results

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgements
  8. References

Placenta staining

In the third trimester placenta, the STB was a continuous uninterrupted multinucleated layer without cell borders (Fig 1A–E). In the STB, nuclei could be found bunched together, associated with structures called syncytial knots (‘k’ in Fig 1A, B and E). In contrast, in the first trimester placenta, the STB nuclei were more evenly distributed and the underlying villous cytotrophoblast was more apparent (Fig 1F).

image

Figure 1.  Localisation of transferrin receptor-1 (TfR1) to the apical (maternal facing) membrane and of ferroportin (FPN) to the basal (fetal facing) membrane of the placental syncytiotrophoblast (STB). (A) Peroxidase staining of term placenta with anti-TfR1 antibody shows staining of apical STB facing into the maternal spaces (arrows). Nuclei are blue (haematoxylin stain). (B) Peroxidase staining of term placenta with anti-FPN antibody shows staining of basal STB facing the fetal tissue (arrows). (C–E) Double staining of term placenta with anti-TfR1 (red) and anti-FPN (green) antibodies shows clear polarisation of the two proteins to different faces of the STB. Nuclei are blue (DAPI stain). (F) Staining of first trimester placenta reveals the same pattern of TfR1 and FPN localisation on the developing trophoblast. m, maternal circulation; f, fetal compartment; k, syncytial knot. Bars in B, C = 10 μm.

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Peroxidase staining of term placenta confirmed that TfR1 was present on the apical (facing the maternal circulation) side of the STB (Fig 1A, arrows) whereas FPN was present on the basolateral (fetal) side (Fig 1B, arrows). This was shown more clearly by the fluorescence double labelling of three different term placentas (Fig 1C–E), where the polarisation of TfR1 to apical and FPN to the basal side of the STB was distinctive. This localisation of TfR1 and FPN to different sides of the STB was also apparent on the trophoblast in the first trimester sample (Fig 1F).

Earlier work suggested widespread strong expression of HFE on the apical side of the STB in term placenta (Parkkila et al, 1997a). However, using the monoclonal antibody JB1 we found much weaker HFE staining on STB (Bastin et al, 1998). To try and resolve this issue we examined the staining of three different anti-HFE antibodies (the monoclonal antibody 8C10 and two polyclonal sera against the C-terminus and alpha-3 domain of HFE) on five third trimester placentas. The antibodies all stained COS cells transfected with HFE (data not shown). Immunofluorescence detected some HFE on STB, but it was relatively sparse and distributed discontinuously (Fig 2A and B). In addition, where HFE was found on the STB, it was localised to the basolateral, and not the apical side. Figure 2A and B shows HFE (green) staining localised to the opposite side of the STB compared to TfR1 staining (red). This localisation of HFE was the same whether using anti-C-terminus or anti-alpha-3 sera. Figure 2C and D shows staining of term placenta with the anti-HFE monoclonal 8C10 (red) and polyclonal anti-FPN sera (green). HFE and FPN colocalised (yellow/orange in Fig 2E and F) on the basal (fetal) side of the STB, as indicated by the position of STB nuclei projecting in front of the HFE/FPN staining towards the maternal circulation (arrows in Fig 2E and F). However, it must be stressed that when the whole section was assessed, the levels of HFE expressed in STB were minimal compared to FPN and TfR1, which were both abundant and continuous over the STB.

image

Figure 2.  HFE, when present on the syncytiotrophoblast (STB), localises to the basal (fetal) not the apical (maternal) membrane. (A, B) Co-staining of term placenta with anti-transferrin receptor-1 (TfR1) (red) and anti-HFE (green, anti-alpha-3 domain polyclonal in A and anti-C-terminus polyclonal in B) shows that HFE is not present on all STB and does not colocalise with TfR1 on the apical membrane. (C–F) HFE (red, D–F, 8C10 monoclonal) is present on the basal STB membrane and colocalises with ferroportin (FPN) (green, C, E, F). C, D and E show single staining of FPN and HFE and a merge, F shows another example of the merged double staining. Arrows in E, F show nuclei (blue) of the STB that are on the maternal side of the FPN/HFE staining. m, maternal circulation; f, fetal compartment. Bar in C = 10 μm.

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Blood vessels in the fetal compartment were localised using a mouse monoclonal to CD31 protein (Fig 3, red). In Fig 3A and B FPN is stained green in term placenta and in Fig 3C FPN and CD31 staining on first trimester placenta is shown. Figure 3D shows co-staining of HFE and CD31 on term placenta. FPN and HFE on the basal STB were situated very close to, but still distinct from, fetal blood vessels (arrows in Fig 3A and D). Ferritin was strongly expressed in the placental stroma, but absent from the maternal spaces and the lumen of the fetal capillaries (Fig 3E).

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Figure 3.  Positions of ferroportin (FPN), HFE and ferritin in the placenta relative to CD31+ blood vessel endothelium. (A,B) Anti-CD31 antibody (red) stains blood vessels (v) in the stroma of term placenta, some of which (arrow in A) are adjacent to, but not overlapping, the FPN-positive basal membrane of the syncytiotrophoblast (STB) (green). (C) Same staining pattern as A and B, seen in first trimester developing trophoblast. (D) Similarly HFE (green) on the basal STB is close to but not overlapping (arrow) some CD31+ endothelium (red). A larger blood vessel is also present in the stroma (v). (E) The iron storage protein ferritin (green) is present in the fetal stroma and is expressed by some CD31+ endothelial cells (red/orange). m, maternal circulation; f, fetal compartment; v, blood vessel lumen. Bar in A = 10 μm.

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The locations of proteins of iron metabolism in the STB and the placental stroma were further investigated. Anti-cytokeratin-7 monoclonal stains trophoblast (red in Fig 4A and B). Figure 4A indicates the iron-deficient state of the STB as ferritin (green) was mostly confined to the stroma of the placenta with very little ferritin staining of the STB. Figure 4B shows first trimester trophoblast cytokeratin-7 (red) and FPN on the basal side (green overlapping red giving orange, arrows). The function of Tf is to bind iron and transport it safely. Figure 4C shows that Tf (green) was present on the apical (maternal facing) STB (similar to TfR1 in Figs 1 and 2) and also in the stroma. Ferritin (red) is mainly confined to the stroma. DMT1 mediates iron transport through endosomal membranes into the cytoplasm. Figure 4D shows discontinuous staining of DMT1 (green) on both STB and stromal cells, and TfR1 (red) shows continuous staining of STB. There was some overlap of DMT1 staining and TfR1 staining on the STB (red/orange).

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Figure 4.  Locations of iron transport and storage proteins relative to the trophoblast and the placental stroma. (A) Term placenta stained with anti-cytokeratin-7 (red) and anti-ferritin (green) antibodies shows that ferritin is largely absent from the syncytiotrophoblast (STB). (B) First trimester placenta showing overlap of cytokeratin-7 (red) and ferroportin (FPN) staining on the basal side of the developing trophoblast (orange colour, shown by white arrows). (C) Transferrin (green) localises to the maternal-facing apical membrane of the term STB while, as in A, ferritin (red) is found in stromal cells. (D) Transferrin receptor-1 (TfR1) (red) is on the maternal-facing apical membrane of the term STB. The divalent metal transporter 1 (DMT1, green) is present discontinuously on STB and stromal cells. m, maternal circulation; f, fetal compartment. Bar in A = 10 μm.

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The placental villous stroma is constructed of fixed connective tissue cells that form a network. Stromal cells include fetal blood vessels (CD31+), Hofbauer cells (CD68+) and fibroblasts. CD68 is present in the cytoplasmic granules and on the surface of macrophages, monocytes, neutrophils and basophils. Hofbauer cells are agreed to be the fetal tissue macrophages of human placenta. Figure 5A shows that stromal CD68 cells (red) co-expressed FPN (green, cells indicated by arrows). Note also FPN staining of the basal STB that is CD68 (arrowhead). Figure 5B shows term STB stained with CD68 (red) and anti-alpha-3 HFE (green). Hofbauer cells co-expressing CD68 and HFE are yellow and indicated by arrows; HFE staining on the basal STB is indicated by an arrowhead. Figure 5C–E shows double staining of FPN and HFE (using 8C10) on a stromal cell. This cell is presumed to be a Hofbauer cell from Fig 5A and B.

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Figure 5.  HFE and ferroportin (FPN) are co-expressed on CD68+ stromal Hofbauer cells. (A) CD68+ (red) Hofbauer cells in the stroma of term placenta co-express FPN (green), indicated by white arrows. Note also FPN expression on the basal syncytiotrophoblast (STB) (arrowhead). (B) CD68+ (red) Hofbauer cells of the placental stroma co-express HFE (green), indicated by white arrows. Note also HFE expression on the basal STB (arrowhead). (C–E) FPN (C, E, green) and HFE (D, E, red) colocalise on a stromal cell, indicated by white arrow. m, maternal circulation; f, fetal compartment. Bar in A = 10 μm.

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Liver staining

The liver consists of plates of hepatocytes that are interspersed with sinusoids through which blood percolates. The sinusoids have endothelial lining cells and Kupffer cells (the resident liver macrophages) that project into the sinusoidal space and contact the blood passing through. Figure 6A shows APAAP staining of the liver with the W6/32 antibody, which recognises major histocompatibility complex (MHC) I molecules and reveals the liver architecture; MHC I was strongly expressed by the endothelial cells that line the sinusoids (indicated by asterisks in Fig 6B) and also by the Kupffer cells (arrows). The hepatocytes that lie between the sinusoids (marked by the letter ‘h’) were also MHC I positive but less so than the sinusoid endothelium and the macrophages. The Kupffer cells were specifically stained by the anti-CD68 antibody as shown in Fig 6B (arrows).

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Figure 6.  Staining of liver section for human leucocyte antigens (HLA) and CD68 reveals location of hepatocytes, sinusoid lining cells and Kupffer cells. (A) Alkaline phosphatase anti-alkaline phosphatase (APAAP) staining of liver with W6/32 antibody (revealed by red stain), which reacts with HLA. Sinusoids are lined with endothelial cells that strongly express HLA. Plates of hepatocytes (marked with ‘h’) express HLA weakly but are clearly positive (pink). Liver macrophage Kupffer cells (arrows) face into sinusoids and are strongly positive. (B) APAAP staining of liver for the macrophage marker CD68 picks out Kupffer cells (arrows). Liver sinusoids are marked with an asterisk; plates of hepatocytes (‘h’) are not stained. Bar in A = 10 μm.

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Whether Kupffer cells or hepatocytes express human HFE is currently under debate. Frozen liver sections were stained with four anti-HFE mouse monoclonal antibodies JB1 (Fig 7A), 8C10 (Fig 7B), 10G4 (Fig 7C) and 2F5 (Fig 7D) from three different laboratories (Bastin et al, 1998; Salter-Cid et al, 1999; Ben-Arieh et al, 2001). Liver tissue was also stained with polyclonal rabbit antisera against two different regions of the HFE protein, the alpha-3 domain (Fig 7E) and the C-terminus (Fig 7F). All six stains clearly showed that the pattern of HFE expression (HFE-positive cells indicated by arrows in Fig 7A and B) mirrored the staining of CD68+ Kupffer cells shown in Fig 6B. The hepatocytes (marked by the letter ‘h’ in Fig 7A and B) were not detectably HFE positive with any of the six antibody preparations, in contrast to the MHC I staining detected on hepatocytes in Fig 6A.

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Figure 7.  Alkaline phosphatase anti-alkaline phosphatase (APAAP) staining of liver with four different anti-HFE mouse monoclonal antibodies and two different anti-HFE rabbit polyclonal antibodies reveal that HFE is expressed strongly by Kupffer cells and not detectably by hepatocytes. (A–D) Four mouse monoclonals (JB1, 8C10, 10G4 and 2F5) all react with cells in a Kupffer cell distribution (examples are shown by arrows in A, B) and do not detectably stain hepatocytes (‘h’ in A, B). (E, F) Two polyclonal sera raised against different regions of HFE (anti-alpha-3 domain in E, anti-C-terminus in F) also show HFE staining of Kupffer cells. Bar in A = 10 μm.

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Work in mouse liver has shown that FPN was expressed on Kupffer cells that were positive for the mouse macrophage marker F4/80 (Abboud & Haile, 2000; Yang et al, 2002). In humans, FPN expression has been reported on Kupffer cells and more weakly on hepatocytes (Donovan et al, 2000). Human liver sections were stained with anti-FPN (Fig 8, green) and either anti-hepatocyte (Fig 8B and C, red) or an antibody against the human macrophage marker CD68 (Fig 8E and F, red). Figure 8 shows that most cells that strongly express FPN were CD68+ Kupffer cells (arrows in Fig 8F). In contrast, the hepatocytes expressed only very low levels of FPN.

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Figure 8.  Immunofluorescence staining of liver shows strong ferroportin (FPN) staining on CD68+ Kupffer cells. (A–C) Staining of liver with anti-FPN (green in A, C) and anti-hepatocyte (red, B, C) antibodies shows that strong FPN expression does not occur on hepatocytes. (D–F) Staining of liver with anti-FPN (green in D, F) and anti-CD68 (red, E, F) shows that many CD68+ cells also strongly express FPN (white arrows). Bar in A = 10 μm.

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Discussion

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgements
  8. References

Localisation of TfR1, FPN and HFE in human placenta

TfR1 was strongly expressed on the entire apical surface of the STB and rarely, minute quantities were found on basal surface STB. Also, TfR1 was found on some stromal cells. FPN was abundant on all basal STB and some stromal Hofbauer cells. The expression of the two proteins suggests that TfR1 captures Tf-bound iron from the maternal circulation, and then the iron is passed through the STB (by unknown means) before being released into the fetal compartment by FPN.

HFE was found in large amounts on some parts of the basal STB (Fig 2), some Hofbauer cells (Fig 5) and on non-CD68 stromal cells. HFE and FPN colocalised on basal STB (Fig 2) and on Hofbauer cells (Fig 5). The amount of STB HFE varied between samples, from none detected to large discontinuous amounts on the basal STB. This variation could be due to the differing iron status of each mother or to other factors. Stromal HFE was found in all placental samples studied. The basal STB location of HFE, away from TfR1, was in contrast to previous findings (Parkkila et al, 1997a). Three different antibodies were used to identify HFE in our studies. The location of TfR1 and HFE was compared using two different rabbit anti-HFE antibodies with two different mouse monoclonal anti-TfR1 antibodies and the location of HFE and FPN was compared using one rabbit anti-FPN and one mouse monoclonal to HFE. This enabled us to state with confidence that HFE, when present, colocalises with FPN and is separate from TfR1. This staining pattern implies that any association between TfR1 and HFE in the STB is minimal.

Earlier work showed that HFE and TfR1 could be co-immunoprecipitated from placenta (Parkkila et al, 1997a). The technique of immunoprecipitation employed by Parkkila et al (1997a) was as follows: extraction of placental membranes, chemical cross-linking of membrane proteins, immunoprecipitation with anti-TfR antibody and then Western blotting of the immunoprecipitate with anti-HFE. The high sensitivity of the technique employed suggests that the HFE/TfR1 complex was at a low concentration in the placental samples used, consistent with the staining pattern of HFE and TfR1 observed in our study. In non-polarised cell lines engineered to co-express TfR1 and HFE, co-immunoprecipitation of the two molecules is observed with a simpler direct technique (Gross et al, 1998).

The function of HFE is still unclear. HFE is known to bind to TfR1 in competition with Tf (Lebron et al, 1999), but there is a growing consensus that HFE may have other functions besides this activity. The localisation of HFE away from TfR1 in the STB shown in Fig 2 is consistent with this idea.

Placental architecture and proteins of iron metabolism

The CD31 staining on the endothelium of stromal blood vessels was distinct from HFE and FPN (Fig 3). This indicated that FPN and HFE were present on the STB but not on the fetal blood vessels. It is not known exactly how iron passes through the fetal endothelium to enter the fetal circulation from the STB but iron has to be oxidised from Fe(II) to Fe(III) for it to be incorporated into fetal Tf.

The control of the storage and transport of iron in the placenta and fetal tissue is important because of the potential damage to rapidly dividing cells in the formation of new organs. The STB takes up iron bound to Tf in the maternal circulation. Transferrin was found predominantly on the TfR1-expressing apical STB (Fig 4C), but some Tf (presumably of fetal origin) was also found in the stroma, where it may be used for transport between fetal cells. Any iron that is not immediately used or transported is stored in cytosolic ferritin. This study found the STB to be relatively free of ferritin, consistent with the STB relaying iron from the mother to the fetus. Ferritin was instead strongly expressed by the stroma (Figs 3E, 4A and C). Some CD31 and ferritin double staining was observed (orange in Fig 3E); the vascular endothelial cells may have some iron storage capacity (Dumartin & Canivenc, 1992; Burdo et al, 2004). The iron transporter DMT1 was found to be close to some parts of the basal STB (Fig 4D) as found previously (Georgieff et al, 2000). There is little overlap in localisation between TfR1 and DMT1.

The stromal location of ferritin in this study, using one polyclonal antiserum, seems very clear but is in disagreement with earlier studies stating a trophoblast location (Brown et al, 1979; Dumartin & Canivenc, 1992). Placental immunomodulatory ferritin (PLIF), a protein identical to the ferritin heavy chain for the first 118 amino acids but derived from a different gene, is expressed by placental trophoblasts (Moroz et al, 2002). PLIF is not known to be involved in iron storage but has roles in immune regulation. It is possible that the earlier findings showing ferritin in trophoblasts may have identified PLIF rather than conventional ferritin. Further studies using a range of anti-ferritin antibodies are needed.

Staining of HFE and FPN in human liver

We investigated the location of HFE in human liver. Four monoclonal antibodies and two polyclonal sera all showed strong HFE expression on Kupffer cells projecting into liver sinusoids (Fig 7); precisely the same staining pattern revealed by antibodies specific for the macrophage marker CD68 (Fig 6B). This pattern of HFE expression agrees with earlier work on human livers (Bastin et al, 1998; Griffiths et al, 2000; Cardoso et al, 2004).

In rats, Hfe mRNA was shown to be highly expressed in the liver (Holmstrom et al, 2003) and was more highly expressed in hepatocytes compared to Kupffer cells and endothelial cells. Zhang et al (2004) also fractionated rat liver and used quantitative reverse transcription polymerase chain reaction to show that hepatocytes had 10-fold more Hfe mRNA than Kupffer cells. In situ hybridisation and Western blotting confirmed that Hfe mRNA and Hfe protein were expressed mainly in the hepatocytes in rats (Zhang et al, 2004). Therefore, there appears to be a species difference in the liver cell distribution of Hfe between humans (Kupffer cells) and rats (hepatocytes). The precise tissue and cellular distribution of Hfe protein in mouse livers has not, to our knowledge, been thoroughly investigated, although Makui et al (2005) reported Hfe mRNA in both whole liver (predominantly hepatocytes) and purified liver macrophages.

In agreement with previous studies in both humans and mice (Abboud & Haile, 2000; Donovan et al, 2000; Yang et al, 2002), we found that FPN was strongly expressed by liver macrophages (Fig 8F). There may be some FPN expression by hepatocytes but it was much weaker. Thus in contrast to the situation with HFE, there appears to be no species difference in the cellular distribution of FPN.

Conclusions

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgements
  8. References

In the human placenta, TfR1 and FPN occupy opposing membranes of the STB, consistent with their respective roles in iron uptake and iron export. In contrast to previous findings, HFE expression was variable and mostly restricted to the basal STB and to stromal Hofbauer cells. In the human liver, we confirm that HFE and FPN proteins are strongly expressed by Kupffer macrophage cells.

Acknowledgements

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgements
  8. References

We thank R. Ehrlich and Y. Yang for the generous provision of reagents, M. Jones for reagents, liver tissue samples and technical advice, and the Wellcome Trust for funding.

References

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  5. Discussion
  6. Conclusions
  7. Acknowledgements
  8. References
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