Resistance of hypotransferrinemic mice to hyperoxia-induced lung injury

Funmei Yang1, Jacqueline J. Coalson2, Heather H. Bobb1, Jacqueline D. Carter3, Jameela Banu1, and Andrew J. Ghio3

Departments of 1 Cellular and Structural Biology and 2 Pathology, The University of Texas Health Science Center, San Antonio, Texas 78229; and 3 National Health and Environmental Effects Research Laboratory, Environmental Protection Agency, Research Triangle Park, North Carolina 27711


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Oxidative stress plays a central role in the pathogenesis of acute and chronic pulmonary diseases. Safe sequestration of iron, which participates in the formation of the hydroxyl radical, is crucial in the lung's defense. We used a mouse line defective in the major iron transport protein transferrin to investigate the effect of aberrant iron metabolism on the lung's defense against oxidative injury. The tolerance to hyperoxic lung injury was greater in the hypotransferrinemic than in wild-type mice as documented by histopathology and biochemical indexes for lung damage. There was no increase in the levels of intracellular antioxidants, inflammatory cytokines, and heme oxygenase-1 in the hypotransferrinemic mouse lung compared with those in wild-type mice. However, there were elevated expressions of ferritin and lactoferrin in the lung of hypotransferrinemic mice, especially in the alveolar macrophages. Our results suggest that pulmonary lactoferrin and ferritin protect animals against oxidative stress, most likely via their capacity to sequester iron, and that alveolar macrophages are the key participants in iron detoxification in the lower respiratory tract.

iron metabolism; lactoferrin; transferrin; ferritin; antioxidants


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

OXYGEN RADICALS and their metabolites appear to be principal mediators of the inflammatory events involved in acute and chronic lung diseases. The association of oxidative injury with bronchopulmonary dysplasia (23), acute respiratory distress syndrome (1), and lung inflammation induced by toxic mineral dusts (27) is well documented. In the presence of transition metals, especially iron, oxygen metabolites can be converted to hydroxyl radicals in a Fenton reaction (15). The highly reactive hydroxyl radical inevitably causes damage such as lipid peroxidation, protein oxidation, and DNA breakage at the site of its formation. An impressive body of evidence supports the role of iron in the pathophysiology of oxidant-mediated tissue damage (21, 22, 37). Patients with acute respiratory distress syndrome were found to have chelatable redox-active iron in their lung fluid (14). Free iron released from iron storage sites appeared to promote tissue oxidation and contributed to ischemia-reperfusion lung injury (37).

Because of its extreme toxicity, iron must be safely transported and sequestered to maintain a normal, healthy state. Ferritin is the major intracellular iron storage protein. Iron sequestered in this protein infrequently participates in electron exchange and oxidant generation. Transferrin, the major iron-transporting protein in vertebrates, regulates iron fluxes between sites of absorption, storage, and utilization via its interaction with the transferrin receptor. Because the expression of transferrin receptor and ferritin is tightly regulated by intracellular iron levels, iron absorbed by the cell via the transferrin-transferrin receptor pathway is safely sequestered in ferritin under normal conditions.

The importance of iron-associated proteins in lung biology and defense has been recognized. Several investigators have shown that transferrin and ceruloplasmin, a ferroxidase that promotes incorporation of iron into its transporting proteins, are important extracellular antioxidants in bronchoalveolar lavage (BAL) fluid (19, 24). We have demonstrated that significant amounts of transferrin and ceruloplasmin are synthesized in specific lung cells in rodents, baboons, and humans (34, 35). Expression of these genes is regulated during development, inflammation, and injury. The degree of iron saturation in transferrin was greatly increased in epithelial lining fluid (from 24 to 80%) but not in serum (remained at 24%) of animals with respiratory failure, suggesting that the iron originated in the lung compartment (16). Several factors, including iron and hyperoxia, have been shown to regulate the synthesis of pulmonary ferritin. In response to hyperoxia, mRNA encoding the ferritin light subunit, which is involved in long-term iron storage, increased severalfold (26), supporting an association between hyperoxic lung injury and iron metabolism. Recent work by several laboratories also suggests an intimate relationship between mechanisms of lung defense and iron equilibrium (6, 8).

A mutant mouse line (hpx) defective in the transferrin gene (3) has been used as an animal model to study human disorders of iron metabolism. Homozygous hypotransferrinemic mice (hpx/hpx) are characterized by a 50-fold decrease in the level of circulatory transferrin, anemia, an increased storage of iron in the liver, and an elevated ferritin level in the serum (3, 29). Using this mouse line, we have investigated the effect of aberrant iron metabolism on the lung's defense against oxidative stress. Unexpectedly, we have found that the hpx/hpx mice are more resistant to hyperoxic lung injury than the wild-type animals. We present the results of our investigation on the factors that may contribute to the resistance of these mice to hyperoxia. Our analysis indicates that the elevated expression of two iron-mobilizing proteins, lactoferrin and ferritin, may contribute to the resistance of hpx/hpx mice to oxidative injury.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and hyperoxic exposure. Homozygous and heterozygous transferrin-deficient mice (hpx/hpx and hpx/+) were obtained from matings between hpx/hpx and hpx/+ or between hpx/+ and hpx/+ animals. The hypotransferrinemic (hpx/hpx) pups were anemic and pale in appearance at birth. They were maintained by a weekly intraperitoneal injection of mouse serum (up to 0.3 ml) as described by Bernstein (3). Heterozygous and wild-type (BALB/c) mice were distinguished by their serum concentrations of transferrin. All animals were kept in pathogen-free facilities and routinely monitored for possible infection. Young adult mice (2.5-5 mo old) were used in all experiments. Mice were exposed to hyperoxia in a well-equipped stainless steel-Plexiglas chamber. The chamber was flushed continuously with oxygen at a controlled flow rate and maintained at 1 atm pressure. This provided sufficient flow to maintain measured oxygen concentration consistently at >99%. Food and water were provided ad libitum. Female mice, which do not need to be housed individually in the chamber, were used for most of the studies.

Collection of lavage fluid and lung tissues. At 72-96 h after exposure, the mice were anesthetized, euthanized, and tracheally lavaged with 1.0 ml of normal saline. The lavage was repeated twice. The combined lavage fluid was centrifuged at 600 g for 10 min to remove cells. The supernatant was stored on ice or at -80°C for biochemical assays. In some experiments, mouse lungs were intratracheally instilled with 4% paraformaldehyde at a constant pressure of 20 cmH2O, immersed in the same fixative for 16 h, and blocked and embedded in paraffin by the standard histological procedure. In other experiments, lung tissue was immediately stored in liquid nitrogen for the preparation of tissue homogenates or RNA.

Serum transferrin concentrations. Blood was collected from the tail vein of the mice or by heart puncture immediately before exsanguination of the animals. After centrifugation, transferrin concentrations in sera were measured by rocket immunoelectrophoresis with use of goat anti-mouse transferrin antiserum (Cappel Laboratories) embedded in 1% agarose gel. Purified mouse transferrin (Cappel Laboratories) was loaded next to the samples at various concentrations for quantitative comparison. By use of this method, the genotypes of the mice in regard to the transferrin allele can be determined.

Concentrations of total protein, lactate dehydrogenase, and tumor necrosis factor in lavage fluids. Lavage protein levels were determined using the Coomassie Plus Protein Assay Reagent (Pierce, Rockford, IL). This assay was modified for use in the Cobas Fara II centrifugal spectrophotometer (Hoffmann-LaRoche). BSA served as the standard. The lactate dehydrogenase (LDH) concentration in the lavage fluid was measured using a commercially prepared kit (Sigma Diagnostics) modified for automated measurement. Tumor necrosis factor-alpha (TNF-alpha ) in the lavage fluid was measured by ELISA with Quantikine kits (R&D Systems, Minneapolis, MN).

Intracellular antioxidant activities. Lung tissues were homogenized in 50 mM Tris · HCl and 1.15% KCl, pH 7.4, with a lung weight-to-buffer volume ratio of 1:3. The homogenate was centrifuged at 20,000 g for 20 min at 4°C, and the supernatant was used to measure the intracellular antioxidant enzyme activities as described previously (11).

Lavage concentrations of transferrin, ferritin, and lactoferrin. Transferrin was measured using a commercially available immunoprecipitation analysis kit (INCSTAR, Stillwater, MN). This assay was modified for use in the Cobas Fara II centrifugal spectrophotometer. Ferritin and lactoferrin were measured by the slot blot method. Aliquots of lavage fluid were vacuum slot blotted onto 0.45-mm nitrocellulose filters (Schleicher and Schuell, Keene, NH) in a saline buffer containing 100 mM Tris, pH 8.0. The blot was air-dried, blocked with 5% powdered milk for 30 min, and then incubated with a 1:2,000 dilution of rabbit anti-human ferritin antibody (DAKO, Carpinteria, CA) or rabbit anti-human lactoferrin antibody (Sigma Chemical) in 5% dry milk overnight at 4°C. The sensitivities of the anti-ferritin and the anti-lactoferrin antibodies to rodent proteins have previously been determined (12). The blot was washed in PBS containing 0.05% Tween and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG in 5% dry milk for 1 h. Reaction signals were detected using enhanced chemiluminescence reagents according to the manufacturer's instructions (Amersham, Arlington Heights, IL). Band optical densities were quantified using the Digital Bioimaging System (Millipore, Bedford, MA).

Immunohistochemistry for ferritin and lactoferrin. Lung paraffin sections were cut at 4 µm, mounted on silane-treated slides (Fisher, Raleigh, NC), and air-dried overnight. The slides were heat fixed at 60°C for 10 min and cooled to room temperature. Sections were then deparaffinized and hydrated. Endogenous peroxidase activity was blocked with H2O2 in absolute methanol for 8 min. Slides were rinsed in 95% alcohol for 2 min, placed in deionized H2O, and washed in PBS. After treatment with Cyto Q Background Buster (Innovex Biosciences, Richmond, CA) for 10 min, slides were incubated with a 1:100 dilution of primary antibody in 1% BSA and PBS for 45 min at 37°C. Slides were incubated with biotinylated linking antibody from the Stat-Q Staining System (Innovex Biosciences) for 10 min at room temperature and washed with PBS. A peroxidase enzyme label from the Stat-Q Staining System was then applied. After tissue sections were incubated for 10 min at room temperature and washed with PBS, they were developed with 3,3'-diaminobenzidine tetrahydrochloride for 3 min at room temperature. Sections were then counterstained with hematoxylin.

Isolation of RNA and Northern blot analysis. Total RNA was extracted from the lung tissue using TRIzol Reagent (Life Technologies) according to the protocol provided by the vendor. Northern blotting was performed by formaldehyde-agarose gel electrophoretic separation of RNA. Rat HO-1 cDNA insert (28) (kindly provided by A. M. Choi, Yale University) was 32P labeled by a random-priming procedure and used as a hybridization probe. Quantitative analysis of hybridization signals was conducted using a PhosphorImager analyzer (Molecular Dynamics, Sunnyvale, CA). Relative amounts of rRNA in each gel slot were used as gel loading controls.

Tissue in situ hybridization. Mouse lung paraffin sections of 5 µm thickness were prepared. Processing and hybridization of lung sections were performed as previously described by Zeller and Rogers (36). 35S-labeled single-stranded RNA probes were synthesized by using the Riboprobe System (Promega) according to the procedure supplied by the vendor. The plasmid DNA template used for riboprobe synthesis contains a 2.2-kb mouse lactoferrin cDNA insert (25) (a gift from C. T. Teng, National Institute of Environmental Health Sciences). After hybridization, sections were treated with 20 µg/ml RNase A at 37°C for 30 min and washed at high stringency (50% formamide, 2× saline-sodium citrate, and 0.1% beta -mercaptoethanol at 55°C). For autoradiography, slides were coated with film emulsion (Kodak NTB-2) and exposed at 4-8°C for 3-5 days. Slides were then developed with Kodak D19 developer and stained with hematoxylin and eosin.

Statistics. Data are expressed as means ± SE. Differences between multiple groups were analyzed by ANOVA. Two-tailed tests of significance were employed. Significance was assumed at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To study the effects of aberrant iron metabolism in the lung's defense against oxidant-induced injury, age-matched homozygous (hpx/hpx) and heterozygous transferrin-deficient mice, together with wild-type mice, were subjected to hyperoxia (>99% oxygen) as described in MATERIALS AND METHODS. After 72-96 h of hyperoxic exposure, lung tissues and lavage fluids were collected for histological and biochemical studies. Tissues from the air-breathing control mice were included in all studies.

Lung histopathology. To assess the degree of lung injury after hyperoxia, stained paraffin sections of lung specimens were evaluated without prior knowledge of the mouse genotypes. The histopathological assessment was repeated four times with 8-10 hyperoxia-treated animals each time. Lung injury became evident in wild-type and heterozygous mice after 84 h of hyperoxic exposure. After 96 h, there was a striking, reproducible difference in the histopathology of the lung between hpx/hpx and wild-type (or heterozygous) mice. A typical hpx/hpx mouse lung after exposure to hyperoxia is shown in Fig. 1, A and B. Fifteen of the sixteen hpx/hpx mice showed this pattern of well-expanded lung parenchyma and minimal pathological changes. The well-expanded alveoli seen in Fig. 1B showed congestion only within the capillaries of the alveolar walls. One hpx/hpx mouse lung had fibrin deposits over the alveolar ducts. The wild-type mouse lung (Fig. 1, C and D), on the other hand, showed significant pathological abnormalities after hyperoxia. Thick hyaline membranes lined many of the air spaces, most of which are respiratory bronchioles and alveolar ducts. Sites of microatelectasis (collapsed alveolar walls) were situated between the dilated air spaces that were lined by hyaline membranes. The findings are consistent with diffuse alveolar damage and exudative phase and differ greatly from those in the lung of the mutant mouse (Fig. 1, A and B). Although mild perivascular edema could be observed in some hpx/hpx mice after hyperoxia, no sign of severe lung injury, such as hyaline membrane deposition, was detected in these mice. There was little difference in the lung pathology between wild-type and heterozygous animals after they were exposed to hyperoxia for 96 h. Consistent results were obtained with all four experiments.


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Fig. 1.   Homozygous mutant (hpx/hpx) and wild-type mouse lungs after animals were exposed to 100% oxygen for 96 h. In A and B (hpx/hpx mouse), well-expanded lung parenchyma with airway (A), vascular, and alveolar compartments are identified; there was minimal pathological change. In C and D (wild-type mouse), lung shows significant pathological abnormalities. Thick hyaline membranes (arrowheads) line some air spaces, most of which are respiratory bronchioles and alveolar ducts. Sites of microatelectasis (collapsed alveolar walls, double-headed arrow in C) are situated between dilated air spaces that are lined by hyaline membranes. Magnification: ×230 (A and C) and ×460 (B and D).

The hpx/hpx mice are anemic at birth and require a transferrin supplement, which is administered by injecting a small amount of transferrin or normal mouse serum. We maintained the hpx/hpx mice by a weekly injection containing 0.25 ml of normal mouse serum. To rule out the possibility that serum injections could affect the resistance of the lung to hyperoxic injury, two different experiments were performed. In one, the wild-type mice were injected with the same serum once; in the other, the wild-type, like the hpx/hpx, mice were injected with serum weekly for 3 mo starting 4 days after birth. These mice were exposed to hyperoxia together with noninjected mice. In neither of the two experiments did we observe a significant difference in lung histopathology between serum-treated and noninjected animals (data not shown).

Biochemical evidence of tissue injury and oxidative stress. To biochemically assess oxidative injury, BAL fluids were collected from mice after the animals were exposed to hyperoxia for 72 h, which is before severe lung damage in wild-type mice occurred. BAL fluid was analyzed for indexes of tissue injury and inflammation. Although there was no significant difference in the level of total BAL protein among all genotypes in air-breathing animals (Fig. 2A), the BAL protein level of wild-type mice was 2.7-fold of that detected in the hpx/hpx mice after exposure. In the hyperoxia-treated group, the concentration of BAL protein was higher in the heterozygous than in the hpx/hpx mice. Figure 2 represents a typical profile of each of the three repeated experiments using a total of 76 mice. This result indicates significantly less lung damage and permeability leak in hpx/hpx than in wild-type and heterozygous mice in hyperoxia-induced lung injury. This conclusion was further supported by the LDH activities detected in BAL fluid (Fig. 2B). In air-breathing mice, similar LDH levels were detected in all three genotypes. After hyperoxic exposure, the LDH activities in BAL fluid were 50-70% higher in wild-type and heterozygous than in hpx/hpx mice. We have also measured the lavage fluid concentration of TNF-alpha , which is one of the important cytokines elevated during lung inflammation. As shown in Fig. 3, exposure to hyperoxia greatly increased the concentrations of lavage fluid TNF-alpha in wild-type and heterozygous, but not in hpx/hpx, mice.



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Fig. 2.   Comparison of total lavage protein (A) and lactate dehydrogenase (LDH) activity (B) in air-breathing mice and mice exposed to hyperoxia. * Significantly increased relative to air-breathing mice. There is no difference in protein or LDH level among 3 groups of mice exposed to air. After hyperoxia, protein level is significantly lower in homozygotes than in heterozygotes, and levels are significantly less in homozygotes and heterozygotes than in wild-type mice; LDH is significantly lower in homozygotes than in heterozygotes, and LDH in heterozygotes is similar to that in wild-type mice.



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Fig. 3.   Analysis of lavage fluid tumor necrosis factor-alpha (TNF-alpha ) in air-breathing mice and mice subjected to hyperoxia. * Significantly increased relative to air-breathing mice. There is no difference among 3 groups exposed to air. After hyperoxia, TNF-alpha is significantly lower in homozygotes than in heterozygotes and wild-type mice. Although TNF-alpha is lower in heterozygotes than in wild-type mice, there is no significant difference between these groups after hyperoxia.

Intracellular antioxidant activities in the lung. Inasmuch as several factors have been reported to affect the lung's defense against hyperoxia, we determined whether tolerance to oxidative injury observed in hpx/hpx mice was due to increased levels of intracellular antioxidants. Lung homogenates were prepared from air-breathing and hyperoxia-treated (72 h) animals and assayed for total superoxide dismutase, glutathione peroxidase, and catalase activities. As shown in Fig. 4, there were no significant differences in total superoxide dismutase activities among all the animal groups (Fig. 4A). Glutathione peroxidase activities in the lung extracts increased in all three mouse genotypes after the animals were exposed to hyperoxia (Fig. 4B). However, similar levels of glutathione peroxidase activity were detected in all three mouse types in air-breathing or hyperoxia-treated groups. The level of catalase activity was lower in lungs of hpx/hpx mice than in lungs of heterozygous or wild-type mice, but similar levels of this enzyme activity were detected in all mouse groups after hyperoxia (Fig. 4C). These results demonstrated that resistance to oxidative injury developed in hpx/hpx mice was not due to increased intracellular antioxidant activities.




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Fig. 4.   Comparison of total superoxide dismutase (SOD, A), glutathione peroxidase (B), and catalase (C) activities in lung extracts of air-breathing mice and mice subjected to hyperoxia. In A, there is no difference among all 6 groups of animals. In B, activities in all hyperoxia-treated groups (*) are significantly increased relative to air-breathing mice, but there is no difference among 3 air-breathing groups or 3 groups subjected to hyperoxia. In C, catalase level in air-breathing homozygotes (*) is lower than in all other groups, among which there is no significant difference.

Lung heme oxygenase-1 activity. Previous studies on heme oxygenase-1 (HO-1), a stress-responsive protein, have shown that HO-1 may mediate protection against oxidant insults. Because HO-1 activity is controlled mainly at the transcriptional level, we analyzed the mRNA levels of HO-1 to determine whether expression of the HO-1 gene in the lung was different among different genotypes of hpx mice. Northern blot analysis (Fig. 5) was performed with the total lung RNA isolated from air-breathing and hyperoxia-treated mice. No significant differences were observed in HO-1 mRNA levels among the three air-breathing mouse groups. After the animals were exposed to hyperoxia, the mean level of HO-1 mRNA was lower in hpx/hpx than in wild-type or heterozygous mice. However, the difference in the HO-1 mRNA level was not statistically significant among the three hyperoxia-treated mouse groups (P > 0.05).



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Fig. 5.   Comparison of lung heme oxygenase (HO-1) mRNA levels in air-breathing mice and mice subjected to hyperoxia. A: Northern blot analysis was performed with 10 µg of total lung RNA from each animal and hybridized with radioactive rat HO-1 cDNA insert. B: quantitative analysis was conducted by using a PhosphorImager. There is no difference among 3 air-breathing groups. After hyperoxia, all mouse types (*) showed a significant increase relative to air-breathing groups. Although means are different among 3 groups subjected to hyperoxia, there is no significant difference among these groups.

Comparison of transferrin, ferritin, and lactoferrin levels in lavage fluids. Because of a deficiency in the major iron-transporting protein transferrin, the hpx/hpx mice have an aberrant iron metabolism. These mice have nonregulated iron absorption in the intestine, increased storage of iron in the liver, and elevated ferritin in the serum. Ghio et al. (12) reported that the levels of pulmonary ferritin and lactoferrin were increased in rats treated with metal chelates. To determine whether abnormal iron metabolism in hpx/hpx mice could affect the expression of the major iron-transporting and iron storage proteins in the lung, we have studied the levels of transferrin, ferritin, and lactoferrin proteins in the lung fluids of these mice (Fig. 6). As predicted, transferrin levels in the air-breathing hpx/hpx mice approached the limit for detection. In the hyperoxia-treated group, the level of transferrin in hpx/hpx mice was the lowest among all three mouse types (Fig. 6A). On the other hand, the concentrations of ferritin and lactoferrin were much higher in hpx/hpx than in wild-type and herterozygous mice (Fig. 6, B and C). After hyperoxia, the concentrations of ferritin and lactoferrin were elevated in all three mouse types, but the levels of these two proteins were still significantly higher in the hpx/hpx mice than in the other two mouse types. The sources of these iron-binding proteins in the lavage fluid are discussed below.




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Fig. 6.   Levels of lavage fluid transferrin, ferritin, and lactoferrin in air-breathing mice and mice subjected to hyperoxia. Among air-breathing mice, level of transferrin (A) is higher in wild-type mice than in heterozygotes and homozygotes. After hyperoxia, transferrin is higher in wild-type mice than in heterozygotes and higher in heterozygotes than in homozygotes. * Significantly increased relative to air-breathing wild-type mice. In air-breathing and hyperoxia-treated groups, ferritin (B) and lactoferrin (C) levels were higher in homozygotes than in wild-type mice and heterozygotes. In B and C, there is no significant difference between heterozygotes and wild-type mice. * Significantly increased relative to air-breathing wild-type mice. ** Significantly higher in homozygotes than wild-type mice or heterozygotes after hyperoxia.

Detection of ferritin and lactoferrin in lung sections. The localization of transferrin, ferritin, and lactoferrin in air-breathing hpx/hpx and wild-type mouse lungs by the method of immunohistochemistry was examined in a separate study (10). Transferrin was localized in several lung cells but mainly in airway epithelial cells in wild-type mouse lung. It was present at a very low level in the hpx/hpx mouse lung. Ferritin was present in alveolar epithelium, interstitial tissues, and alveolar macrophages, with the highest concentration in macrophages. Lactoferrin was detected mainly in airway epithelium, interstitial tissues, and alveolar epithelium. The immunostaining of ferritin and lactoferrin in the lung section was higher in hpx/hpx than in heterozygous or wild-type mice (10). In this study, we have examined the tissue levels of ferritin and lactoferrin in the lung after the animals were exposed to hyperoxia for 96 h. The immunostaining for ferritin again appeared to be higher in hpx/hpx than in wild-type mice (Fig. 7). In both mouse types, alveolar macrophages, among all lung cells, contained the highest amount of ferritin. To show the immunostaining of alveolar macrophages in the wild-type mouse lung, Fig. 7B was obtained from one of the better-expanded and less-damaged parts of an injured lung. The severity of tissue injury in this lung sample was still evident by the presence of abundant hyaline membranes. In the hpx/hpx mouse lung, immunostaining for lactoferrin was detected in macrophages in addition to airway and alveolar epithelia after hyperoxia (Fig. 8A). In contrast, very little immunostaining was seen in macrophages and epithelial cells in the wild-type mouse lung (Fig. 8B), although some staining was detected in the interstitial tissue.


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Fig. 7.   Immunohistochemistry of ferritin. Hpx/hpx (A) and wild-type (B) mouse lungs exposed to hyperoxia were stained with antibody against ferritin. Immunostaining can be seen in macrophages (arrowheads) in hpx/hpx and wild-type mouse lungs but is stronger in hpx/hpx mouse. Stain was also detected in alveolar wall cells in hpx/hpx (A), but not in wild-type (B), mice. Arrows in B, hyaline membranes in injured lung of wild-type mouse. Magnification: ×460.



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Fig. 8.   Immunohistochemistry of lactoferrin. Lung sections from hyperoxia-exposed hpx/hpx (A) and wild-type (B) mice were stained with antibody against lactoferrin. Immunostaining was more intense in macrophages (arrowheads) of hpx/hpx than wild-type mice. Arrows in B, hyaline membranes in injured wild-type mouse lung. Magnification: ×460.

Activation of lactoferrin gene expression in lung alveolar cells. Although ferritin gene expression is regulated largely at the translational level, lactoferrin gene expression appears to be controlled at the transcriptional level. The in situ hybridization technique was used to investigate the regulation of lactoferrin mRNA production in lung cells. Lung sections derived from air-breathing and hyperoxia-treated mice were hybridized with antisense mouse lactoferrin cRNA. In all groups of mice, strong hybridization signals were detected in submucosal glandular cells in the trachea (Fig. 9A). Hybridization signals were also found in the epithelial cells of the upper airways (trachea and bronchi) but not of the lower airways. A high level of lactoferrin mRNA was found in some cells in the alveolar space of hpx/hpx but not of wild-type mice. Furthermore, the number of lactoferrin-synthesizing cells (Fig. 9, B and C) in the alveolar space increased >20-fold in hpx/hpx mice after hyperoxia. Because of extensive processing of the lung sections during the experimental procedure, the identity of these lactoferrin-synthesizing cells was not clear, but they could be a subset of alveolar macrophages or infiltrating immune cells.


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Fig. 9.   Localization of lung cells expressing lactoferrin mRNA. In situ hybridization was performed with radioactive antisense mouse lactoferrin RNA. In all mice, as exemplified by an untreated wild-type mouse (A), lactoferrin mRNA was detected in airway epithelial cells (arrowhead in A), in trachea and bronchi, and in tracheal glands (arrow in A). Open arrow in A, cartilage in trachea. After hyperoxia, lactoferrin mRNA was also detected in cells in alveolar space (arrowheads in B and C) of hpx/hpx, but not of wild-type, mice. B: dark-field picture taken at low magnification. Magnification: ×280 (A), ×77 (B), and ×460 (C).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Several investigators have shown that transferrin is a major extracellular antioxidant in the lung (19, 24). In an in vivo model of respiratory failure, iron-free transferrin was shown to ameliorate respiratory failure and improve surfactant activity in BAL fluid (16). Recently, we showed that transferrin was produced and secreted at a high level in the lung (34). In baboons and humans, transferrin gene expression displayed distinct temporal patterns in different lung cells. The biological significance of the regulated synthesis of transferrin in specific lung cells is not clear. At the cellular level, iron metabolism is tightly regulated by controlling the synthesis of ferritin and transferrin receptor via interaction between iron-regulatory proteins and iron-responsive elements (reviewed in Ref. 17). In a normal healthy state, transferrin is the major iron-transporting protein in vertebrates. Therefore, the function of transferrin might be to regulate the availability of iron to cells by binding the metal and forcing the delivery mechanism for iron exclusively toward the transferrin receptor (31). However, with an increased level of intracellular iron, cells rapidly downregulate the expression of transferrin receptor and upregulate the synthesis of ferritin to sequester the iron that has already entered the cells. Under this condition, cellular uptake of iron via the transferrin-dependent pathway is blocked. Activation of a transferrin-independent iron-transporting system(s) during stressful conditions may provide an alternative mechanism for iron detoxification via specific cells such as macrophages to minimize the potential for oxidative stress and cytotoxicity caused by the presence of redox-active iron in the extracellular space.

The hpx mouse line, which is defective in transferrin gene, has a very low level of transferrin synthesis in the liver and extrahepatic tissues. However, we have found that the homozygous hypotransferrinemic mice (hpx/hpx) were resistant to hyperoxia-induced lung injury in repeated experiments. This finding was supported by pathological and biochemical evidence. Several factors that may contribute to the developed resistance of these mice to oxidative damage were studied. Our results demonstrated that levels of HO-1, intracellular antioxidant activities, and inflammatory cytokines were not higher in lungs of hpx/hpx than in wild-type or heterozygous mice. In fact, the level of catalase activity is slightly lower before hyperoxic exposure and the level of TNF-alpha activity is lower after hyperoxia in hpx/hpx than in wild-type or heterozygous mice.

Because the hpx/hpx mice were maintained by a weekly injection of normal mouse serum, we conducted experiments and demonstrated that tolerance to oxidative injury in these mice was not due to serum injection or endotoxin contamination in the supplemental serum. No induction of tolerance was observed in wild-type mice injected with the same serum used to inject hpx/hpx mice. Our studies on HO-1, intracellular antioxidants, and the inflammatory cytokine also clearly indicated the absence of endotoxin-induced biochemical changes in hpx/hpx mice. We used young adult hpx/hpx mice (3 mo old) for our study. Although they have increased iron storage in the liver, these mice do not show any iron overload-induced abnormality as observed in the older mice (30). These collective data support the theory that tolerance to hyperoxic lung injury in hpx/hpx mice is unlikely related to inflammation-induced factors.

Ferritin was reported to be cytoprotective mainly because of its ability to sequester iron (2). Hyperoxic exposure was associated with an elevated expression of pulmonary ferritin L subunit (26). Instillation of metal-containing particles into airways resulted in an increased production of ferritin and lactoferrin in the lung (12). These observations suggest an induction of host defense mechanisms to control iron-mediated oxidative stress in the lung. The hpx/hpx mice are known to have an elevated level of ferritin in the serum (29). We have found higher amounts of ferritin and lactoferrin in the lavage fluids from these mice than from wild-type or heterozygous mice. After hyperoxia, ferritin, lactoferrin, and transferrin in BAL fluid were increased in all three mouse types. Although these iron-associated proteins are known to be upregulated during inflammation in mice, the increase detected in BAL fluid of wild-type mice may be augmented by damaged lung cells. Immunohistochemically, the tissue levels of ferritin and lactoferrin were higher in the lung cells, especially alveolar macrophages, in hpx/hpx than in wild-type mice. The elevated levels of ferritin and lactoferrin in hpx/hpx mice likely enhance the capacity for iron sequestration in the lung.

Synthesis of ferritin is controlled mainly at the translational level. Expression of lactoferrin gene, on the other hand, appears to be regulated at the transcriptional level. In the normal mouse lung, lactoferrin mRNA was detected mainly in the secretory glands and epithelial cells of the upper airways. Because these two types of cells represent only a small subset of lung cells, quantitative analysis of the level of lactoferrin mRNA by use of RNA prepared from the whole lung was not informative. As noted above, lactoferrin protein was seen in alveolar macrophages of hpx/hpx mice. In hpx/hpx, but not in wild-type, mice, lactoferrin mRNA was detected in cells in the alveolar space. Although we have not confirmed the identity of these cells, activation of lactoferrin expression at the alveolar level could provide an additional supply of lactoferrin in the lower respiratory tract, a site vulnerable to oxidative injury. Lactoferrin is structurally and functionally closely related to transferrin. Its concentration increases in plasma during inflammation, and its major functions are presumably associated with host defense (reviewed in Ref. 18). Lactoferrin-mediated transfer of iron has been recognized in monocytes and macrophages during inflammatory states (5, 20). It is believed that iron-saturated lactoferrin delivers its iron into intracellular ferritin in mouse macrophages by a receptor-mediated mechanism. Human monocyte/macrophage cells have been shown to possess high-affinity receptors for lactoferrin (4). The affinity of Fe-lactoferrin for the membrane of peritoneal macrophages is indeed higher than that of apolactoferrin (32). These observations further support the role of lactoferrin/lactoferrin receptor in the iron uptake of macrophages.

Results from this study indicate that activation of pulmonary lactoferrin and ferritin gene expression may contribute to the resistance of the hpx/hpx mice against acute oxidative injury via their capacity to sequester iron. It is likely that alveolar macrophages are the key participants in iron detoxification in the lower respiratory tract. This conclusion is further supported by results obtained in a separate study (10) in which hpx/hpx mice exposed to an iron-rich pollution particle, oil fly ash, were found to be resistant to particle-induced lung injury compared with similarly treated wild-type mice. Because injury induced by this particle was shown to be metal dependent (9), the hpx/hpx mice may have developed an efficient iron sequestration system(s) independent of transferrin in the lung.

The mechanism(s) underlying the activation of ferritin and lactoferrin gene expression in the hpx/hpx mouse lung is not clear but could involve the increased level of tissue iron and/or non-transferrin-bound iron species detected in hpx/hpx mice (29). Non-transferrin-bound iron can be transported to cells via transferrin-independent pathways and results in the activation of iron-responsive proteins including ferritin and lactoferrin. Interestingly, a recent study has shown that transgenic tobacco plants overexpressing ferritin are resistant to pathogens and oxidative damage induced by several types of insults (7). In another study, it was found that overexpression of ferritin in transgenic plants increased the iron absorption activity in these plants (33). Apparently, these plants behave as iron deficient and activate iron transport system(s). These findings appear to be analogous to observations we reported in this article. It is not known whether the levels of other proteins possibly involved in transferrin-independent iron transport, such as Nramp 2 (13), are also upregulated in the hpx/hpx mouse lung. These possibilities need exploration to further define the complex role of iron metabolism in lung injury.


    ACKNOWLEDGEMENTS

We thank Dr. Ronald Crawley and Renee Judkins (Laboratory of Animal Resources, The University of Texas Health Science Center at San Antonio) for providing sera from pathogen-free mice. The Laboratory of Animal Resources, Department of Veterans Affairs Medical Center (San Antonio, TX) provided the chamber for the hyperoxia experiments. We appreciate the assistance of Rheanna Urrabaz, Linda Buchanan, Barbara Wadwell, and Katrine Krugger.


    FOOTNOTES

This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-36536; National Institute on Aging Grants AG-06872 and AG-06650; and a grant from the Morrison Trust.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: F. Yang, Dept. of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78229 (E-mail: YangF{at}UTHSCSA.edu).

Received 17 March 1999; accepted in final form 17 August 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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