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Variable tissue expression of transferrin receptors: relevance to acute respiratory distress syndrome

Depts of ¹ Critical Care and ² Histopathology, Royal Brompton Hospital, Imperial College Faculty of Medicine

CORRESPONDENCE: G.J. Quinlan, Unit of Critical Care, Royal Brompton Hospital, Sydney St, London, SW3 6NP, UK. Fax: 44 2073518524. E-mail: g.quinlan@imperial.ac.uk

Keywords: acute respiratory distress syndrome, iron regulation, oxidative stress, transferrin receptor

Received: August 15, 2002
Accepted March 28, 2003

This study was supported by a British Lung Foundation/BOC Group Programme Grant, the Dunhill Medical Trust and British Heart Foundation. ^¶Joint first authors.

	Abstract

TOP Abstract Materials and methods Results Discussion References

 
Acute respiratory distress syndrome (ARDS) is associated with altered plasma and lung iron chemistry. Iron can promote microbial virulence and catalyse pro-oxidant reactions, thereby contributing to the oxidative stress that characterises the syndrome.

Therefore, the expression of ferritin and transferrin receptors (TfR) were sought in the lungs and hearts of rodents treated with lipopolysaccharide (LPS), and measurements of TfR and ferritin protein expression were taken from lung biopsy specimens from patients with ARDS and appropriate controls.

TfR messenger ribonucleic acid (mRNA) was significantly upregulated in the lungs and significantly downregulated in the hearts of rats 4 h after LPS. Ferritin mRNA levels (light and heavy chains) remained unaltered. Protein TfR levels were significantly upregulated in lungs and downregulated in hearts 4 h post-LPS. Ferritin protein levels were significantly downregulated in lungs compared to baseline values but were unaltered in hearts. Nonhaem iron levels were increased in lungs and decreased in hearts, and iron-regulatory-protein activity increased in hearts but not lungs. TfR protein levels were significantly increased in lung biopsies from patients with ARDS compared to controls.

Transferrin receptors are upregulated in rodent lungs during inflammation but are downregulated in the heart. Transferrin receptor protein levels were significantly increased in the lungs in clinical acute respiratory distress syndrome. These findings have implications for the pathogenesis of acute respiratory distress syndrome, especially in relation to the role of iron as a mediator of oxidative stress.

Acute respiratory distress syndrome (ARDS) in adults is defined clinically by refractory hypoxaemia and bilateral pulmonary infiltrates, attributable to increased permeability of the alveolar capillary membrane. ARDS may complicate a wide variety of infective and noninfective pro-inflammatory insults, not all of which involve the lung directly. Why such provocative factors should render the lung, rather than other organ systems, susceptible to the inflammatory processes implicated in the pathogenesis of the syndrome is, however, unclear 1. Patients with ARDS are also subjected to conditions of severe oxidative stress, which are implicated in the pathogenesis of the inflammatory processes that characterise the condition. Thus, abnormalities in plasma iron chemistry (elevated levels of iron saturation of transferrin) and impaired antioxidant protection (normally afforded by the binding of iron to transferrin and the iron-oxidising, ferroxidase activity of caeruloplasmin) have been described previously 2, and bronchoalveolar lavage fluid from patients with ARDS has been shown to contain increased levels of low molecular mass iron compared to that taken from normal healthy controls 3. Iron is a catalyst at the active centres of numerous oxidase, oxygenase and antioxidant enzymes, and in other proteins for the transport of electrons and oxygen, but when not under such protein structural constraints it is capable of catalysing the formation of numerous aggressive and damaging inorganic and organic reactive oxygen species (ROS) 4, 5.

To avoid the deleterious consequences of iron misuse, elaborate physiological mechanisms have evolved to ensure its efficient recycling, turnover and control 6. These include the expression of transferrin receptors (TfRs) on cells to facilitate the uptake of iron-loaded transferrin and the expression of cellular apoferritin (both heavy and light chains) in which to store iron intracellularly. The expression of these two proteins is regulated at the post-transcriptional level by the iron-regulatory proteins (IRP)-1 and -2. Activated IRP-1 and nondegraded IRP-2 bind to iron-responsive elements (IRE) in the noncoding regions of ferritin and TfR messenger ribonucleic acid (mRNA). Such responses are controlled by the prevailing levels of iron (albeit by different mechanisms for IRP-1 and -2). When cellular iron levels are low, inhibition of ferritin synthesis occurs together with a stabilisation of TfR mRNA leading to increased TfR synthesis. By contrast, when iron levels are high the reverse process occurs 7, 8. Mechanisms of IRP activation independent of iron signalling have also been reported to contribute to iron regulation. These include activation by ROS 9 and inflammatory mediators 10.

Therefore, the presence of iron and its regulation has a potentially crucial part to play in the pathogenesis of ARDS. However, the extent to which dysfunctional iron regulation might render the lung particularly susceptible to injury following pro-inflammatory insults has not been explored. The aims of this study were, therefore, three-fold. First, usinga rodent model, the effects of a pro-inflammatory insult (lipopolysaccharide (LPS)) on the mobilisation of iron (cellular uptake and storage) were investigated through the expression of TfRs and ferritin. Secondly, the hypothesis thatthe expression of these iron uptake and storage proteinswas organ-, and by inference, cell-specific was explored, since differences may have important implications for the pathogenesis of ARDS. Finally, evidence of TfR and ferritinexpression was sought in archived lung biopsy specimens taken from patients with ARDS and appropriate controls.

	Materials and methods

TOP Abstract Materials and methods Results Discussion References

 
Animal studies
All procedures and protocols were performed in accordance with the Animal (Scientific Procedures) Act, 1986, and approved by the Home Office (UK) Inspectorate. Male Wistar rats (n=4 per group except where otherwise stated, 275–300 g) received LPS (20 mg·kg^–1 i.p.) or an equivalent volume of saline. Either 120 or 240 min post-LPS, the animal was anaesthetised with pentobarbitone (60 mg·kg^–1 i.p.), sacrificed by cervical dislocation, and the heart and lungs were harvested for reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting.

Clinical studies
Archived lung tissue biopsies (stored at –70°C) taken for diagnostic purposes from patients with ARDS (n=7) and defined by America-European Consensus Conference criteria (for patient demographics see table 1), and normal tissue from seven control patients (six male and one female) undergoing lung resection for bronchogenic or metatastic carcinoma (mean age 58 yrs, range 45–75 yrs), were homogenised and processed. TfR and ferritin protein levels were determined using electrophoresis and protein blotting. The methodology employed was as for the animal studies described below.

Western blotting
Rodent hearts and lungs, and human lung tissue were homogenised with a pH 7.0 cell lysis buffer containing proteinase inhibitor Complete^TM (Roche Diagnostics, Lewes, UK), and centrifuged at 10,000xg for 10 min. Supernatants were divided into aliquots and total protein determined by the Lowry method. Volume adjustments weremade with loading buffer to ensure that samples containing equal amounts of protein were used for electrophoresis. The protein concentration used for a given analysiswas established by trial and error, and differed according to the type and tissue. For TfR studies in hearts,wells were loaded with 25 µL of 1.0 µg·µL^–1, and for those in lungs, 2.0 µg·µL^–1. For ferritin studies in hearts, wellswere loaded with 25 µL of 0.2 µg·µL^–1, and in lungs,0.4 µg·µL^–1. In heart-derived samples, TfR gel electrophoresis was performed under nondenaturing conditions (7.5% weight/volume (w/v) gel), which facilitated the movement and determination of the parent TfR homodimer. Inlung supernatants, it was decided for technical reasons to perform TfR electrophoresis using standard denaturing (inthe presence of sodium dodecylsulphate (SDS) and after heating) conditions (15% w/v gel). In this way the subunit corresponding to the TfR monomer was separated and determined. Ferritin electrophoresis was also performed under standard denaturing conditions because of the largesize of the parent multi-subunit protein and again with 15% w/v SDS-PAGE gels.

Gels were transferred onto nitrocellulose membranes. Afterblocking, blots were probed with a polyclonal antibodyagainst TfR (Bioquote Ltd, York, UK) or ferritin(Sigma-Aldrich Ltd). The samples were then incubated with a second anti-immunoglobulin G horseradish peroxidase conjugate. Membranes were treated with enhanced chemiluminescence reagent to reveal immunoreactive proteins. A TfR or ferritin standard was run on each gel as an added control. Results are expressed as mean densitometric readings.

Total nonhaem iron measurement
These were measured in tissue homogenates prepared in the absence of iron chelators using iron-free reagents. A kit method was employed (Total Iron Assay Kit; Sigma Chemical Company, Dorset, UK).

Bleomycin-detectable iron measurement
Free iron can be chelated by bleomycin, which is the basis of this assay. The protocol is fully described in 16.

Iron-regulatory protein activity
IRP activity was determined by use of an RNA gel shift assay technique, as described previously 17. A vector plasmid (pTZ19-IRE) containing the IRE for heavy-chain ferritin was obtained from Fermentas AB (Vilnius LT, Lithuania). The plasmid was linearised and ³²P labelled RNA was transcribed to produce the radiolabelled IRE probe. Protein extracts (2 µg) from hearts and lungs were mixed with saturating amounts of the IRE probe (1.3x10⁵), and incubated at ambient temperature for 10 min. Heparin displaced nonspecific interactions. ß-Mercaptoethanol (2%) was added to one set of samples for total IRP activation; for endogenous activity ß-mercaptoethanol was omitted. RNA-protein complexes were resolved on native 6% polyacrylamide gels. Gels were developed by autoradiography. In work-up experiments with smooth muscle cells, IRP-1 and -2 activity could be differentiated. However, IRP-2 activity could not be consistently demonstrated in samples derived from the in vivo model. This is probably due to the known labile nature of IRP-2, the activity of which may be lost on processing tissue samples to release protein.

Statistical analysis
Results are expressed throughout as mean±sem. Where two groups were compared, differences were assessed using the Mann-Whitney U-test. One way analysis of variance (ANOVA) and Dunnett's multiple comparisons test were used for comparisons of multiple groups. A p-value of <0.05 was considered significant.

	Results

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Rats treated with LPS expressed significantly decreased levels of TfR mRNA in the heart over time. Thus, in the heart, differences between groups were significant overall (p=0.036). Post-tests revealed a significant downregulation of TfR mRNA expression 4 h after LPS, as compared to time 0 (fig. 1a). Decreased levels of TfR protein were also observed. Although these differences were not significantly different overall, the trend was for a decrease (fig. 1b). However, one data point in the control group was skewed; with this point removed, differences reached overall significance (p=0.016). Furthermore, post-tests showed a significant decrease for TfR protein 4 h after LPS, as compared to time 0.

View larger version (33K): [in this window] [in a new window]   Fig. 1.— Levels of transferrin receptor (TfR) in the heart of rats. a) Levels of expression of TfR messenger ribonucleic acid (mRNA), measured as densitometric ratios relative to the ß-actin housekeeping gene (n=4 for all groups) and a representative blot for TfR and ß-actin mRNA after reverse transcriptase polymerase chain reaction (RT-PCR) amplification. b) Levels of expression of TfR protein measured as denisitometric values (n=4 for all groups) and a representative Western blot of TfR protein from the heart. Levels are shown both pre- (; 0 h) and post- (; 2 and 4 h) lipopolysaccharide treatment. *: p<0.05.

View larger version (39K): [in this window] [in a new window]   Fig. 2.— Levels of transferrin receptor (TfR) in the lungs of rats. a) Levels of expression of TfR messenger ribonucleic acid (mRNA), measured as densitometric ratios relative to the ß-actin housekeeping gene (n=4 for all groups) and a representative blot for TfR and ß-actin mRNA after reverse transcriptase polymerase chain reaction (RT-PCR) amplification. b) Levels of expression of TfR protein, measured as denisitometric values (n=4 for all groups) and a representative Western blot of TfR protein in the lungs. Levels are shown both pre- (; 0 h) and post- (; 2 and 4 h) lipopolysaccharide treatment. *: p<0.05.

Ferritin protein levels in the heart remained unaltered at all time points (fig. 3a). In the lungs, differences between groups were significant overall (p=0.031). Post-tests showed a significant downregulation of ferritin protein expression in thelungs 4 h after LPS, as compared to baseline (fig. 3b andc). Changes in mRNA levels do not exclude a post-transcriptional control mechanism.

View larger version (24K): [in this window] [in a new window]   Fig. 3.— Levels of expression of ferritin protein, measured as densitometric ratios relative to a ferritin protein standard, in a) the heart and b) the lungs of rats (n=4 in all cases). c) A representative Western blot showing ferritin protein (heavy chain) in the lung. Levels are shown both pre- (; 0 h) and post- (; 2 and 4 h) lipopolysaccharide treatment. *: p<0.05.

Levels of total nonhaem iron were measured at baseline and at 4 h in control (n=6) and LPS-treated (n=3) rats. An increase was seen in the lungs (fig. 4a), but decreases in total nonhaem iron 4 h post-LPS, as compared to baseline values, were seen in the heart (fig. 4b). Interestingly, levels of low molecular mass/loosely bound (bleomycin-detectable) iron were significantly (p<0.05) decreased in the lungs 4 h after LPS, as compared to baseline (fig. 5a). By contrast, in the heart, the pattern mirrored those seen for total nonhaem iron (albeit at lower iron concentrations) (fig. 5b).

View larger version (28K): [in this window] [in a new window]   Fig. 4.— Changes in total nonhaem iron in a) lung and b) heart homogenates from control animals (; n=6) and 4 h post-administration of lipopolysaccharide (; n=3).

View larger version (28K): [in this window] [in a new window]   Fig. 5.— Changes in bleomycin-detectable iron (low molecular mass iron) in a) lung and b) heart homogenates from control animals (; n=6) and 4 h post-administration of lipopolysaccharide (; n=3). *: p<0.05.

View larger version (31K): [in this window] [in a new window]   Fig. 6.— Levels of iron-regulatory protein (IRP) activity (n=3 for both groups), measured as densitometric ratios relative to total activity in a) the heart and b) lungs of control () and lipopolysaccharide-treated () rats. *: p<0.05.

View larger version (13K): [in this window] [in a new window]   Fig. 7.— Transferrin receptor (TfR) protein levels in lung tissue homogenates from patients with acute respiratory distress syndrome (ARDS) (n=7) and appropriate controls (n=7). Equivalent concentrations of total protein were used throughout. Levels of TfR were significantly increased (#: p=0.02) in patients with ARDS.

	Discussion

TOP Abstract Materials and methods Results Discussion References

In endotoxaemia, increased levels of TfR mRNA and protein were observed in lung tissue when compared to controls, suggesting a degree of transcriptional regulation of the expression of this protein, although increased mRNA stability cannot be conclusively excluded as contributing to this finding. However, the four-fold increase in lung TfR mRNA at the early (2 h) time point strengthens the argument for LPS-mediated transcriptional effects. Ferritin was significantly decreased in lung tissue, whilst the level of mRNA for both subunits remained unaltered. This suggests that regulatory control at the translational level occurred. However, there was no apparent change in IRP activity in the lungs. This discrepancy is difficult to explain, but if correction for total activity is not applied, apparent changes in activity can be seen (fig. 8a, lanes 1 and 2).

View larger version (43K): [in this window] [in a new window]   Fig. 8.— Representative ribonucleic acid (RNA) gel shift assay showing iron regulatory protein (IRP)-1 activity in a) the lungs and b) theheart pre-treatment (1 and 3) and 4 h post-lipopolysaccharide treatment (2 and 4). The total activities were obtained by adding 2-mecaptoethanol (3 and 4).

Several studies have demonstrated decreased expression of TfR in response to pro-inflammatory stimuli such as LPS in combination with -interferon, tumour necrosis factor, interleukin-1 and combinations of chemokines applied to isolated epithelial cells and or macrophages 19–21. Indeed, nitric oxide-mediated degradation of IRP-2 is a key regulatory component under such circumstances 22, 23. As macrophages and epithelial cells are found in the lungs, these studies may be of relevance, however, the present in vivo studies oppose those above in that an increase in TfR levels was seen in the lungs. Hypoxia inducible factor-1-mediated transcriptional regulation of TfR expression in response to hypoxia has also been demonstrated 18. Hypoxia may be highly relevant to this in vivo study as a defining criterion for ARDS. The current authors', as yet, unpublished studies using the same in vivo model (data not shown) are investigating the relationship between haemoxygenase-mediated iron release and the modulation of IRP activity in vivo. Their preliminary findings suggest that iron is a key regulatory component for the modulation of measurable IRP activity in the lungs, but that other mechanisms, possibly related to oxidative stress, are more important in the heart.

In functional terms, the current data indicate that following LPS, iron uptake and storage mechanisms are regulated in such a way as to favour decreased cellular uptake of iron by the rodent heart. In the lungs, the opposite appears to occur (increased TfR and decreased ferritin expression), a situation that would favour iron accumulation. Indeed, preliminary studies suggest that 4 h after LPS, tissue nonhaem iron levels are decreased in the heart but increased in the lungs. Although the lungs may therefore accumulate nonhaem iron in sepsis, mechanisms appear to exist to limit any adverse effects associated with the cellular accumulation of low molecular mass forms of iron, since levels of bleomycin-detectable iron decreased. The storage of iron by ferritin could, in part, account for these findings, although this protein was found to be decreased in the lung in response to LPS in the current study. Other iron-binding mechanisms may account for the sequestration observed, including that of the intracellular iron transport protein Nramp1.

The current authors suggest that the opposing responses demonstrated here, as summarised in figure 9, are related to organ/cell-type specific adaptations that increase antioxidant and antimicrobial protection against iron. Specifically, they propose that the heart, by decreasing TfR expression, may protect itself against the potential pro-oxidant effects of iron. This could be particularly important during acute inflammatory conditions, such as those encountered in this model, since this is a time of increased ROS production 24. The high respiratory demand of the heart may also make it more susceptible to oxidative damage in the presence of redox active iron. Moreover, iron is known to be acutely cardiotoxic 25. Such a response is consistent with an antioxidant response evolved to protect the heart from oxidative damage.

View larger version (20K): [in this window] [in a new window]   Fig. 9.— Schematic diagram summarising the changes in iron-regulatory responses observed in the heart and lungs in response to alipopolysaccharide (LPS) challenge. +–: represents no change; ––: decrease; ++: increase. IRP: iron regulatory protein; mRNA: messenger ribonucleic acid.

In conclusion, the responses observed in this study are likely to represent adaptations to increase antioxidant and antimicrobial protection against iron. The presence of extracellular iron in normal healthy lungs 31 makes it particularly vulnerable to oxidative damage and to infection when such control is lost.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (9) Citing Articles via Google Scholar Google Scholar Articles by Upton, R.L. Articles by Quinlan, G.J. Search for Related Content PubMed PubMed Citation Articles by Upton, R.L. Articles by Quinlan, G.J.