Published ahead of print on January 18, 2008, doi:10.1165/rcmb.2007-0179OC
© 2008 American Thoracic Society DOI: 10.1165/rcmb.2007-0179OC Carbon Monoxide Reversibly Alters Iron Homeostasis and Respiratory Epithelial Cell Function1 National Health and Environmental Effects Research Laboratory, Office of Research and Development, United States Environmental Protection Agency, Research Triangle Park, North Carolina; 2 Center for Environmental Medicine, Asthma and Lung Biology, University of North Carolina, Chapel Hill, North Carolina; 3 Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, Texas; and 4 Department of Medicine, Duke University Medical Center, Durham, North Carolina Correspondence and requests for reprints should be addressed to Andrew J. Ghio, MD, Campus Box 7315, Human Studies Division, US EPA, 104 Mason Farm Road, Chapel Hill, NC 27599-7315. E-mail: ghio.andy{at}epa.gov
The dissociation of iron from heme is a major factor in iron metabolism and the cellular concentrations of the metal correlate with heme degradation. We tested the hypotheses that (1) exposure to a product of heme catabolism, carbon monoxide (CO), alters iron homeostasis in the lung and in cultured respiratory epithelial cells; (2) this response includes both decreased uptake and increased release of cell metal; and (3) the effects of CO on cell function track changes in metal homeostasis. In rats exposed to 50 ppm CO for 24 hours, non-heme iron concentrations decreased in the lung and increased in the liver. In respiratory epithelial cells cultured at air–liquid interface, CO exposure decreased cell non-heme iron and ferritin concentrations within 2 hours and the effect was fully reversible. CO significantly depressed iron uptake by epithelial cells, despite increased expression of divalent metal transporter-1, while iron release was elevated. The loss of non-heme iron after CO reduced cellular oxidative stress, blocked the release of the proinflammatory mediator (interleukin-8), and interfered with cell cycle protein expression. We conclude that CO reduces the iron content of the lung through both the metal uptake and release mechanisms. This loss of cellular iron after CO is in line with certain biological effects of the gas that have been implicated in the protection of cell viability.
Key Words: oxidants • oxidative stress • inflammation • cell proliferation • ferritin
Iron is an essential micronutrient for normal cell and tissue function but its complex biochemistry leads to intricacy in maintaining cellular metal homeostasis. Under aerobic aqueous conditions, iron forms oxyhydroxides that are often biologically inaccessible. In addition, iron-catalyzed oxidant production creates the potential for cell damage. As a result, cells have acquired strategies to procure adequate iron for homeostasis and function. The dietary requirement for iron (  1 mg daily) is small in relation to the body's iron stores, approximately 4 g, and most of the iron used by mammalian cells (70%) is found within heme moieties (1). Therefore, the dissociation of iron from heme and its release by the cell are major factors in heme–protein homeostasis. The mechanisms for reutilization of iron bound in heme are not fully understood. Heme degradation is the responsibility of the heme oxygenases (HO), which convert it to biliverdin, iron, and carbon monoxide. For instance, the induction of HO-1 alters iron stores; the metal is released in a labile form within minutes to hours, suggesting that its egress is associated with either HO activity or a product of the enzyme (2). Cells transfected with HO demonstrate not only a decrease in iron uptake but also an increase in iron release (3). HO knockout mice develop iron-deficiency anemia, but also accumulate significant amounts of iron in several tissues, including the liver and kidneys (4). Similarly, anemia and elevated tissue iron have been reported in a 6-year-old male with HO deficiency (5). In cells, tissues, and organs, increased HO activity is associated with iron release while loss in HO function causes the metal to accumulate. Carbon monoxide (CO) is one of the three products of HO. This gas has a high affinity for the ferrous ion and acts as a monodentate ligand (forms only one bond with the central atom) to complete the coordination shell of iron. CO binding of iron affects the reactivity of the metal and its homeostasis (6). The physiological and pathogenic effects of numerous biological agents correspond to their capacity to influence iron homeostasis. Moreover, another diatomic gas like CO, nitric oxide (NO), plays an important role in iron metabolism, and certain effector functions depend on its ability to bind this metal (6). Similarly, CO alters iron metabolism by tight coordination of free ferrous iron as the carbonyl or by binding iron-containing macromolecules, such as ferrous heme proteins, thereby interfering with or activating reactions central to cell function. In this work, we tested the hypothesis that (1) CO alters iron homeostasis in the intact lung and in cultured respiratory epithelial cells, (2) this alteration includes both decreased uptake and increased release of the metal from lung cells, and (3) the cell functional effects of CO correspond to changes in iron homeostasis consistent with the apparent antioxidant and antiproliferative actions of the gas.
Animal Exposures The experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee at Duke University. Adult male Sprague-Dawley rats (300–400 g body weight) from Charles River Laboratories (Wilmington, MA) were used for the experiments. For the exposure, 1% CO in air was bled into Plexiglas exposure cages to achieve the desired concentrations and steady state. The CO concentration was monitored continuously using a calibrated infrared CO detector (Snifit Model 50; Bacharach, Pittsburgh, PA). The CO concentration was maintained within ± 5 ppm of the desired level. The rats were exposed to either filtered air or 50 ppm CO for 24 hours. The rats were anesthetized with halothane and an aliquot of blood collected by cardiac puncture. The animal was killed by transecting the abdominal aorta and the trachea was cannulated for lung lavage with 0.9% NaCl (saline). The volume of saline instilled was equal to 90% of the total lung capacity (35 ml/kg of body weight) based on published allometric equations. Saline was withdrawn after a 3-second pause, re-instilled twice more in identical fashion, and centrifuged (600 x g for 10 min) to remove cells. The lungs and liver were resected en bloc.
Measures of Inflammation and Injury in the Animal Model
Non-Heme Iron Concentrations in the Serum, Lavage Fluid, and Tissues
Cultured Cell Exposures
Non-Heme Iron Concentrations in Cell Lysates
Ferritin and Transferrin Concentrations in Cell Lysates
Western Blot Analysis for DMT1 Protein samples (50 µg) were separated by electrophoresis on a 4 to 15% SDS acrylamide gel and transferred to nitrocellulose membranes (Bio-Rad), blocked with 3% nonfat milk in PBS, and incubated with an antibody against either DMT1 or metal transport protein 1 (MTP1). The membrane was stained with a horseradish peroxidase–conjugated goat anti-rabbit IgG antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA) and developed using enhanced chemiluminescence (ECL kit; Amersham Pharmacia Biotech, Piscataway, NJ).
Oxidative Stress in BEAS-2B Cells and Proinflammatory Mediator Release
Cell Proliferation
Quantitative RT-PCR for cdk2, cdk4, and p21CIP1/WAF1
Western Blotting for p21
Statistics
Animal Exposures to CO The rats exposed to 50 ppm CO for 24 hours significantly increased their COHb concentrations to 6.9 ± 0.6% (relative to 0.6 ± 0.2% after filtered air). CO inhalation was associated with mild neutrophilic influx and slight elevations of lavage protein and LDH. Lavage neutrophils increased significantly after CO exposures, but were still less than 10% of total cells (Figure 1A). CO exposure increased the lavage concentrations of the proinflammatory mediator MIP-2 (Figure 1B). There were small but significant elevations of both total protein (Figure 1C), and LDH (Figure 1D) in the lavage fluid of rats exposed to CO for 24 hours. However, lavage albumin concentrations were not significantly increased by CO inhalation (39.3 ± 5.7 micrograms/ml) compared with filtered air (35.8 ± 4.8 micrograms/ml).
Iron status in the lower respiratory tract was altered significantly by exposure of rats to 50 ppm CO for 24 hours. Lavage iron concentrations increased more than 4-fold (Figure 2A). Levels of the metal storage protein ferritin in lavage also increased significantly (Figure 2B); however, lavage transferrin concentrations did not change. In contrast to lavage iron, serum iron concentration decreased after CO inhalation (695 ± 69 and 552 ± 22 µg/dl for filtered air and CO, respectively). No significant differences in either serum ferritin or transferrin were observed after CO exposure. Fresh lung tissue was used for the analysis of non-heme iron, which was found to be significantly decreased after CO inhalation (Figure 2C). In comparison, the non-heme iron concentration of liver tissue was increased (Figure 2D).
Cell Exposures to CO After 24 hours of exposure to as much as 500 ppm CO, there was no change in LDH release or cellular trypan blue exclusion by respiratory epithelial cells, indicating a lack of cytotoxicity in the exposures. However, CO caused BEAS-2B cells to lose non-heme iron, in amounts that depended on both time and dose (Figures 3A and 3B). After CO, cell non-heme iron concentrations could be diminished to approximately 20% of initial values. BEAS-2B cells exposed to 50 ppm CO for 2 hours showed non-heme iron values approximating 50% of those of cells in incubated in filtered air; however, removal of the cells from CO to filtered air at 2 hours for another 22 hours corrected the metal content (Figure 3C). This demonstrates reversibility of the changes in iron homeostasis associated with CO exposure. Comparable to non-heme iron concentrations, cell levels of ferritin decreased with 24 hours of exposure to CO (Figure 4A). Ferritin concentrations were increased in the media of the basolateral compartment after exposure to 50 ppm CO for 24 hours, consistent with release of this storage protein (Figure 4B). Cell concentrations of the transferrin transport protein showed no significant change with CO exposure.
Because respiratory epithelial cells cycle iron, a decrease in metal concentration can reflect changes in uptake, release, or both (10). Therefore, BEAS-2B cells were removed from media and placed in buffer at known concentrations of iron without transferrin. When these cells were exposed to CO, iron uptake decreased relative to control cells in filtered air. After 24 hours of exposure to 50 ppm CO, cells demonstrated less iron uptake compared with those in filtered air (Figure 5A). One protein that regulates intracellular iron importation in BEAS-2B cells is DMT1 (11). Western blots for DMT1 revealed increased protein expression after 24 hours of exposure to 50 ppm CO (Figure 5B). Removal of the cells from the CO to filtered air after 2 hours actually led to an increased uptake of iron at 24 hours (Figure 5C).
To quantify iron release, BEAS-2B cells were removed from media and placed in HBSS without iron. Cells incubated in filtered air showed slight iron release into the apical chamber after 2 hours (Figure 6A). However, cells exposed to 50 ppm CO for 2 hours released a large amount of iron into the apical chamber (Figure 6A). Iron release by respiratory epithelial cells is not well understood but, MTP1 (ferroportin 1) is involved (12). Western blots for MTP1 showed no differences in protein expression after 24 hours of exposure to 50 ppm CO (Figure 6B).
The relationship between biological responses to CO and altered iron homeostasis was evaluated with assays for oxidative stress, mediator release, and cell proliferation. Using aldehyde formation as an index of oxidative stress, we found that 50 ppm CO for 24 hours decreased acetaldehyde in cell extracts relative to cells incubated in filtered air (Figure 7A). In the same BEAS-2B cells exposed to 50 ppm CO for 24 hours, the levels of IL-8 were diminished in the cell supernatant (Figure 7B).
Cell proliferation was evaluated by following 5-bromo-2'-deoxyuridine (BrdU) incorporation in BEAS-2B cells exposed to either 50 ppm CO or filtered air. CO exposure decreased cell proliferation compared with cells incubated in filtered air (Figure 7C). Removing the cells from CO after 2 hours to filtered air eliminated the differences in oxidative stress, supernatant IL-8 concentrations, and cell proliferation at 24 hours (Figures 7A, 7B, and 7C). This functional inhibition after CO exposure corresponded to decrements in cell iron that corrected at 24 hours. An important mechanism of cell cycle arrest is inhibition of cyclin-dependent kinases (cdks). In mammalian cells, cyclin–CDK complexes are negatively regulated by CDK inhibitors, including p21. Levels of cdk2, cdk4, and p21 mRNA after air and CO exposures and iron depletion with 50 µM deferoxamine were measured using RT-PCR. We found small but significant decrements in RNA for cdk2 and cdk4 after 4 hours of exposure to 50 ppm CO (Figures 8A and 8B). These changes in cdk2 and cdk4 were similar to those following 4 h incubation of the cells in 50 µM deferoxamine to effect iron depletion (Figures 8A and 8B). RNA for p21 also decreased after 4 hours of exposure to CO and iron depletion using 50 µM deferoxamine (Figure 8C). Finally, Western blotting confirmed loss of protein expression for p21 after both CO exposure and 50 µM deferoxamine for 24 hours (Figure 9A). After 4 hours, CO and deferoxamine exposure and 20 hours air, there were no differences in p21 expression (Figure 9B). This indicates the reversibility of the biological effect by CO corresponding to the change in iron homeostasis.
CO is a respiratory poison at one extreme and an endogenous regulatory molecule at the other largely through its binding effects on ferrous heme. In this study, low-level CO exposure altered lung iron homeostasis both in vivo in rats and in vitro in respiratory epithelial cells grown at air–liquid interface. In the rat, iron was mobilized from the lung accompanied by decrements in non-heme concentration. Correspondingly, elevations in non-heme iron concentrations were found in the alveolar and airway compartments by bronchoalveolar lavage. A portion of this increased iron in the lavage fluid was associated with the storage protein ferritin because extracellular ferritin concentrations were also elevated after CO exposure. Levels of non-heme iron changed in the opposite direction in the liver from those in the lungs, implying that iron homeostasis is regulated systemically during the CO exposure. Although serum iron and ferritin concentrations were not measurably affected by CO in this study, this finding probably reflects late timing and unitary sampling. In mammals, the liver is the preferred site for storing iron in a controlled state to minimize the catalytic capacity of the metal. Thus, physiologically, iron moved from the lungs, where the CO exposure was most intense, to the liver, its main storage site. Exposure of BEAS-2B respiratory epithelial cells to CO led to a rapid, significant, decrease in non-heme iron concentrations in cells grown at an air–liquid interface. Cells exposed to CO at 50 ppm for only 2 hours lost almost half of their non-heme iron compared with those exposed to filtered air. This loss of iron could result from an increased egress, a decreased uptake, or both. It has been proposed that CO can alter iron homeostasis by coordination of Fe2+ and release of metal from transferrin in the endosome (13); this would subsequently inhibit metal entry into the cell. Our data in BEAS-2B cells showed significant decrements in iron uptake during CO exposure in agreement with that earlier study (13). However, removal of the BEAS-2B cells from CO revealed that its effect on intracellular iron was rapidly reversible. Moreover, the DMT1 protein, which participates in the intracellular uptake of Fe2+, actually increased with CO exposure—possibly reflecting a state of intracellular iron depletion. The removal of respiratory epithelial cells from CO to filtered air not only eliminated the decrements in iron uptake but significantly increased intracellular metal transport over 24 hours. This increased cellular iron transport is consistent with the increased expression of DMT1 and perhaps other transporters. The cellular iron egress after CO exposure found here contrasts with an earlier investigation that detected no significant effect of CO on iron release (14). In filtered air, human bronchial epithelial cells normally lose a small amount of iron, which (as in the previous investigation) reflects continuous iron uptake and release by these cells (12). Although CO greatly accelerated the iron loss, expression of the major protein responsible for iron release in these cells, MTP1, was unchanged by the CO exposures. Consequently, the cause of decreased uptake and increased release of iron after CO exposure in respiratory epithelia likely involves another mechanism(s). For instance, after CO exposure, ferritin concentration in the media was elevated, consistent with its release by the airway cells. Investigation in other systems suggests that such ferritin release may be a defense against high intracellular iron concentrations (15). An important factor in the cellular iron depletion by CO could be that the iron carbonyl has limited utility to the cell, in which case, the cell may be forced to release the iron-carbonyl nonspecifically. Alternatively, if CO increases heme release (e.g., from mitochondrial sources) and stimulates HO activity in the cell, cytosolic iron concentration will rise unless the cell exports it (16). Under such circumstances, a reduction in the uptake and/or increase in the release of the metal would reduce iron and oxidative stress in the cell. Thus, CO could signal the need to adjust iron homeostasis to avoid iron accumulation and oxidative stress. Exposure to CO for 24 hours decreased acetaldehyde levels in respiratory epithelial cells consistent with such an antioxidant effect. By coordinating iron, CO can oppose oxidative stress by decreasing or abolishing iron's potential to support electron transfer to active intermediates, especially reactive oxygen species. In addition, the loss of cellular non-heme iron after CO would block oxidant generation by Fenton-based reactions. These results are consistent with previous studies showing that CO can protect against oxidative stress (17–19); however, removing the cells from CO after 2 hours returned non-heme iron concentrations and acetaldehyde production to control levels within 24 hours. CO-dependent changes in non-heme iron concentration also corresponded to lower production of the proinflammatory mediator IL-8. Once again, removing the epithelial cells from CO after 2 hours returned IL-8 levels to those of control cells in filtered air. This could be interpreted as an anti-inflammatory effect, similar to that reported for HO in various models of inflammatory injury including hyperoxia, sepsis, ischemia, and so on (20–22); however, dosing and timing of CO administration is a variable that could confound the interpretation of such effects (23, 24). The data also demonstrate that changes in iron homeostasis by CO are congruent with apparent antiproliferative effects of CO reported in other cell types (25). Adequate cellular iron is vital for cell cycle progression, and the evidence so far indicates that most of this iron is required for DNA synthesis (26), especially during late G1 and S phase. It is also now known that iron depletion results in a compound response that affects ribonucleotide reductase and multiple cell cycle control points (27). Low iron concentrations block cellular proliferation by impinging on pathways that control cell division (e.g., levels of cyclin D, cyclin-dependent kinases) (28). Iron chelation arrests cell cycle progression in late G1, before the G1/S border. Similarly, CO inhibited cell proliferation in accordance with its capacity to deplete iron. Because the cyclins coordinate cell proliferation and their activities are modulated by both cdks and cdk inhibitors, we checked mRNA levels for cdk2 and cdk4 after CO and found them to be decreased relative to air controls. The response was comparable to that of the BEAS-2B cells after iron depletion with deferoxamine, both here and in other investigations (26, 29). Similarly, the mRNA for p21 was decreased by CO exposure. The effect of iron depletion on p21 mRNA appears to be cell specific: it increases in some (30) and decreases in other cell types (31). In BEAS-2B cells, the effect of CO was identical to that of deferoxamine (both decreased the mRNA equally) and fits with the known transcriptional and post-transcriptional influence of iron on p21 protein expression (27). Diminished p21 protein expression after CO or deferoxamine exposure was confirmed by Western blot analysis, and for both protein and message, the effect of CO on BEAS-2B cells was equivalent to that of deferoxamine and consistent with the influence of iron homeostasis on the cell cycle. The results of this study also reinforce the idea that physiological CO and NO share certain cell functions (e.g., as reported for guanylate cyclase)—as both molecules demonstrate common coordination chemistry, a high affinity for iron (6), and now changes in iron homeostasis. However, NO reacts with both Fe (III) and Fe (II), while CO binds only the latter. Comparable to CO, NO also mobilizes cell metal and inhibits iron uptake from transferrin and its incorporation into ferritin (32, 33). Although our findings contrast with those of a previous study (14) by demonstrating significant iron efflux from BEAS-2B cells after CO exposure, iron loss is consistent with observations that cells transfected with HO show less iron uptake from transferrin and elevated iron efflux and that HO knockout mice show not only iron deficiency anemia but iron accumulation in liver and kidneys (4). Differences in iron efflux rates among studies may relate to the cell type, HO expression, culture conditions, and so on, but exposure of the lungs or airway epithelial cells to CO occurs without cell hypoxia, and the use of an air–liquid interface is relevant to pulmonary gas exchange. That CO clearly alters lung iron homeostasis possibly reflects interactions with the control of the oxidative potential of the metal by the host. CO is produced endogenously by heme protein degradation (e.g., hemoglobin, cytochromes, etc.) by the heme oxygenases. To limit the propensity of free iron to generate oxidative stress, the cell can increase the release of the iron and decrease its uptake, and our data support the idea that CO, as a product of enzymatic heme breakdown, participates in just such a response. Moreover, the loss of cellular iron is rapidly reversible and reflected in a reversible change in the growth characteristics of respiratory epithelial cells, which could have positive or negative effects on the lung. In conclusion, exogenous CO alters iron homeostasis in the intact lung and in lung epithelial cells by enhancing net iron release. Some of the biological effects reported for CO correspond to these effects on cellular iron status and are comparable to those of the potent metal chelator, deferrioxamine. Analogous to use of chelators, CO as a deliberate intervention will be complicated by a lack of focused delivery to specific cell targets and a lack of capacity of the gas to distinguish between excess/inappropriate iron which catalyzes harmful oxidative stress and metal required for homeostasis. Perhaps the most interesting new finding reported here, however, is the rapidly reversible but poorly understood effect of CO on iron handling by human respiratory epithelial cells that tracks with changes in cell cycle protein expression.
Originally Published in Press as DOI: 10.1165/rcmb.2007-0179OC on January 18, 2008 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form May 18, 2007 Accepted in final form November 20, 2007
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Abstract
CLINICAL RELEVANCE
