Diminished injury in hypotransferrinemic mice after exposure to a metal-rich particle

Andrew J. Ghio1, Jacqueline D. Carter1, Judy H. Richards1, Kay M. Crissman1, Heather H. Bobb2, and Funmei Yang2

1 National Health and Environmental Effects Research Laboratory, Environmental Protection Agency, Research Triangle Park, North Carolina 27711; and 2 Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, Texas 78284


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Using the hypotransferrinemic (Hp) mouse model, we studied the effect of altered iron homeostasis on the defense of the lung against a catalytically active metal. The homozygotic (hpx/hpx) Hp mice had greatly diminished concentrations of both serum and lavage fluid transferrin relative to wild-type mice and heterozygotes. Fifty micrograms of a particle containing abundant concentrations of metals (a residual oil fly ash) was instilled into wild-type mice and heterozygotic and homozygotic Hp animals. There was an oxidative stress associated with particle exposure as manifested by decreased lavage fluid concentrations of ascorbate. However, rather than an increase in lung injury, diminished transferrin concentrations in homozygotic Hp mice were associated with decreased indexes of damage, including concentrations of relevant cytokines, inflammatory cell influx, lavage fluid protein, and lavage fluid lactate dehydrogenase. Comparable to other organs in the homozygotic Hp mouse, siderosis of the lung was evident, with elevated concentrations of lavage fluid and tissue iron. Consequent to these increased concentrations of iron, proteins to store and transport iron, ferritin, and lactoferrin, respectively, were increased when assayed by immunoprecipitation and immunohistochemistry. We conclude that the lack of transferrin in Hp mice did not predispose the animals to lung injury after exposure to a particle abundant in metals. Rather, these mice demonstrated a diminished injury that was associated with an increase in the metal storage and transport proteins.

ferritin; lactoferrin; transferrin; iron; oxidants


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

METALS CONTAINED IN AIR POLLUTION particles can be associated with an inflammatory lung injury (34). The mechanism of tissue damage after particle exposure can include an oxidative stress catalyzed by these metals (Folz RJ, Abushama A, Carter JD, Ghio AJ, and Solomon HB, unpublished observations). In vitro and in vivo generation of oxygen-based free radicals by one emission source particle (a residual oil fly ash) has been associated with the concentrations of catalytically active metals included in the particle (25, 34). Biological end points, such as signal transduction and release of pertinent mediators, after exposure of respiratory epithelial cells to this particle have similarly corresponded to its content of metals and the catalyzed oxidative stress (10, 36). Finally, the in vivo lung injury after exposure to this ash was also associated with its content of metals (14).

As a result of this injury mediated by metal-catalyzed oxidants, it would be of benefit to the host to sequester available metal in response to an exposure. Such sequestration of iron by macrophages restricts the potential of the metal to generate free radicals and can prevent cellular damage resulting from its exposure (31). The storage of iron in a chemically less reactive form within intracellular ferritin confers an antioxidant function to this protein and, in certain cells, provides cytoprotection in vitro against oxidants (4).

To transport the metal associated with the particle into a cell for storage within ferritin, transferrin can potentially be employed. Transferrin assumes all six coordination sites of the metal in transport, and, consequently, the capacity of iron to catalyze production of oxidants is decreased after it is bound to this chelator. This glycoprotein can account for a significant portion of the antioxidant activity of the serum (20). Similarly, this chelator is considered the predominant antioxidant in the lining fluid of the respiratory tract in both health (33) as well as in disease (27). In the lung, apotransferrin has been demonstrated to ameliorate respiratory failure and inhibit lipid peroxidation in the epithelial lining fluid catalyzed by iron (21). However, the expression of human transferrin protein by cells resident in the lung can be diminished after exposure to metal (16). In addition, many cell types, including respiratory epithelium, rapidly downregulate the expression of transferrin receptor after exposure to metal chelates (17, 30). Therefore, transferrin-dependent transport of iron across the cell membrane may not be the only mechanism that contributes to either the sequestration of metal by ferritin or the antioxidative defenses of the lung.

We tested the hypothesis that transferrin-independent transport systems exist in the lung that could contribute to the defense of the lung against the oxidative stress caused by catalytically active metal. This was accomplished by employing the hypotransferrinemic (Hp) mouse model. Homozygotic (hpx/hpx) Hp mice have either a point mutation or a small deletion in the transferrin gene, causing defective splicing (24). As a result, the homozygotic Hp mice produce <1% of the normal levels of transferrin.


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INTRODUCTION
METHODS
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Materials. The particle used in this investigation was an oil fly ash acquired from Southern Research Institute (Birmingham, AL). It was collected in Florida with the use of a Teflon-coated fiberglass filter located downstream from the cyclone of a power plant that was burning a low-sulfur number 6 residual oil (collection temperature of 204°C). This emission source air pollution particle has a mass median aerodynamic diameter of 1.95 ± 0.18 µm and is unique in its pronounced quantities of metals (34). However, it contains little to no organics or biological components (34). These characteristics of this oil fly ash make it a useful model particle to use in studies to test the association between metals present in particles, oxidative stress, and various biological end points.

All other materials were obtained from Sigma (St. Louis, MO) unless otherwise specified.

Animals and particle exposure. Homozygous and heterozygous Hp mice (hpx/hpx and hpx/+) were obtained from matings between either hpx/hpx and hpx/+ or hpx/+ and hpx/+ animals. Homozygous Hp pups were small, anemic, and pale at birth. They were maintained by weekly intraperitoneal injection of mouse serum (up to 0.3 ml) (5). Heterozygous and wild-type mice (originally derived from BALB mice) were distinguished by their serum concentrations of transferrin and used as controls. All animals were kept in pathogen-free facilities and routinely monitored for pathogens and viruses.

Mice with a mean age of 105 days (range 90-120 days) were anesthetized with Metafane (Pitman-Moore, Mundelein, IL) and intratracheally instilled with either normal (0.9%) saline (0.05 ml) or 50 µg of oil fly ash in normal saline (0.05 ml; n = 5/dose). Twenty-four hours after exposure, the mice were anesthetized with Metafane, killed by exsanguination, and tracheally lavaged with 1.0 ml of normal saline. This lavage was repeated twice. After centrifugation at 600 g for 10 min to remove cells, the supernatant was stored either on ice or at -80°C.

Serum transferrin concentrations. Blood was collected by heart puncture immediately preceding exsanguination of the animals. After centrifugation, transferrin concentrations in sera were measured by rocket immunoelectrophoresis (39) with purified mouse transferrin (Cappel Laboratories, West Chester, PA) as a standard. Goat anti-mouse transferrin antiserum (Cappel Laboratories) was embedded in 1% agarose gel (in 0.075 M barbital buffer) at a final dilution of 1:200. Each of the 1.75-µl serum samples, diluted at either 1:25 or 1:50, was applied in a circular well (1 mm diameter). Purified mouse transferrin (Cappel Laboratories) was loaded next to the samples at varying concentrations for quantitative comparison. Electrophoresis was performed at 10 V/cm for 16 h. After electrophoresis, the gel was washed with PBS and dried with blotting paper in a vacuum oven at 60°C for 3 h. The dried gel was stained with Coomassie blue, destained, washed, and photographed. With this method, the phenotypes of the mice were easily determined.

Transferrin concentrations and total iron-binding capacity in the lavage fluid. Transferrin concentrations in the lavage fluid supernatants were measured with a commercially available immunoprecipitation analysis kit from INCSTAR (Stillwater, MN). This assay was modified for use in the Cobas Fara II centrifugal spectrophotometer (Hoffman-LaRoche, Branchburg, NJ).

Lavage fluid total iron and unsaturated iron-binding capacity (UIBC) were determined with colorimetric, enzymatic methodology (Sigma Diagnostics). The total iron-binding capacity (TIBC) was calculated as the total iron plus the UIBC. This assay was modified for use in the Cobas Fara II centrifugal spectrophotometer (Hoffman-LaRoche).

Lavage fluid ascorbate, urate, and glutathione concentrations and oxidized products in tissue. Lavage fluid supernatant was acidified (35 µl 60% perchloric acid/ml) and centrifuged at 20,000 g for 30 min at 4°C. The supernatant was stored at -80°C until assayed for ascorbate and urate through the use of HPLC (Waters RCM µBondapak C18 column, Millipore, Marlborough, MA) with electrochemical detection (BAS model LC-4B, Bioanalytical Systems, West Lafayette, IN) (28). Nonprotein sulfhydryls, reflecting total glutathione, were also measured in the supernatant (3).

Lung tissue (~50 mg wet weight) was homogenized in 1.15% KCl. To 0.2 ml of homogenate, 0.2 ml of SDS and 1.5 ml of 20% acetic acid were added. The pH was adjusted to 3.5, 1.5 ml of 0.8% thiobarbituric acid (TBA) were added, the volume was adjusted to 4.0 ml, and the reaction mixture was heated to 95°C for 60 min. One milliliter of distilled water and 5.0 ml of n-butanol-pyridine (15:1 vol/vol) were added. After centrifugation for 10 min, absorbance of the organic layer was measured at 532 nm.

Concentrations of macrophage inflammatory protein-2 and tumor necrosis factor in lavage fluid. Concentrations of macrophage inflammatory protein-2 (MIP-2) and tumor necrosis factor (TNF) in the supernatants of lavage fluid were measured by ELISA with the use of Quantikine kits purchased from R&D Systems (Minneapolis, MN).

Lavage fluid protein and lactate dehydrogenase concentrations. Lavage fluid protein was determined with the Pierce Coomassie Plus Protein Assay Reagent (Pierce, Rockford, IL). This assay was modified for use in the Cobas Fara II centrifugal spectrophotometer (Hoffman-LaRoche). BSA served as the standard. Lactate dehydrogenase (LDH) concentration in the lavage fluid was also measured with a commercially prepared kit (Sigma Diagnostics) as modified for automated measurement (Cobas Fara II centrifugal analyzer).

Total iron concentrations in lavage fluid. Concentrations of total iron in the lavage fluid supernatant were quantified with inductively coupled plasma emission spectroscopy (ICPES; model P40, Perkin-Elmer, Norwalk, CT) operating at a wavelength (lambda ) of 238.204 nm. Standards included ferric chloride in 1% HCl.

Nonheme iron in lung tissue. Nonheme iron concentrations in mouse lungs were measured after precipitation of hemoglobin (37). Tissue disintegrates during incubation in 3 N HCl and 10% TCA. The separation of heme from nonheme iron is possible because the heme molecule is very resistant to acid hydrolysis. It precipitates and is sequestered in the residue. A clear extract and a small residue result.

Lung tissue (0.10 g wet weight) was hydrolyzed in a solution of 3 N HCl and 10.0% TCA (0.10 g lung/1.0 ml solution) at 70°C for 16 h. Although not usually necessary, the hydrolyzed lung tissue was centrifuged at 1,200 g for 10 min. Iron in the supernatant was measured in duplicate with ICPES (lambda  = 238.204 nm). Standards included ferric chloride in 3 N HCl and 10% TCA.

Slot blots for ferritin and lactoferrin. Aliquots of lavage fluid containing 10 µg of protein were vacuum slot blotted onto 0.45-mm nitrocellulose (Schleicher and Schuell, Keene, NH) in a saline buffer containing 100 mM Tris, pH 8.0. The blot was allowed to air-dry, blocked with 5% powdered milk for 30 min, and then incubated with a 1:2,000 dilution of either rabbit anti-human ferritin antibody (DAKO, Carpinteria, CA) or rabbit anti-human lactoferrin antibody (Sigma) in 5% dry milk overnight at 4°C. The sensitivities of the anti-ferritin and anti-lactoferrin antibodies to rodent proteins have previously been determined (18, 19). The blot was washed in PBS-Tween (0.05%) and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG in 5% dry milk for 1 h. Detection was accomplished on film with enhanced chemiluminescence reagents as per the manufacturer's instructions (Amersham, Arlington Heights, IL). Band optical densities were quantified with a Millipore Digital Bioimaging System (Bedford, MA).

Immunohistochemistry for ferritin and lactoferrin. Lungs were fixed at inflation with 10% Formalin (35 ml/kg body wt; Fisher, Pittsburgh, PA). Tissue sections were cut at 4 µm, floated on a protein-free water bath, mounted on silane-treated slides (Fisher, Raleigh, NC), and air-dried overnight. The slides were heat fixed at 60°C in a slide dryer (Shandon Lipshaw, Pittsburgh, PA) for 10 min and cooled to room temperature. Sections were then deparaffinized and hydrated to 95% alcohol (xylene for 10 min, absolute alcohol for 5 min, and 95% alcohol for 5 min). Endogenous peroxidase activity was blocked with H2O2 in absolute methanol (30% hydrogen peroxide in 30 ml of 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 the primary antibody diluted in 1% BSA for 45 min at 37°C in PBS at the following dilutions: anti-ferritin, 1:100 and anti-lactoferrin, 1:100. Slides were incubated with biotinylated linking antibody from Stat-Q Staining System (Innovex Biosciences) for 10 min at room temperature and washed with PBS, and peroxidase enzyme label from Stat-Q Staining System (Innovex Biosciences) was then applied. After incubation for 10 min at room temperature and washes with PBS, tissue sections were developed with 3,3'-diaminobenzidine tetrahydrochloride for 3 min at room temperature. Sections were counterstained with hematoxylin, dehydrated through alcohols, cleared in xylene, and covered with a coverslip with a permanent mounting medium.

Statistics. Data are expressed as means ± SE. Differences between multiple groups were analyzed with ANOVA (11). The post hoc test employed was Scheffe's test. Two-tailed tests of significance were employed. Significance was assumed at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Rocket electrophoresis confirmed that, among those animals instilled with saline only, homozygotic Hp mice had serum concentrations of transferrin that were barely detectable, whereas those values of the wild-type mice (4 mg/ml) were approximately twice those of the heterozygotic animals. Twenty-four hours after exposure, there was no effect of particle instillation on serum transferrin concentrations. The concentrations of transferrin in lavage fluid after saline instillation were also decreased in the homozygotic Hp mice relative to wild-type and heterozygotic mice (Fig. 1). However, lavage fluid transferrin increased after exposure to the particle in both the wild-type and heterozygotic Hp mice but not in the homozygotes (Fig. 1). TIBC in the lavage fluids were 10.8 ± 1.8, 19.9 ± 1.8, and 15.3 ± 1.6 µg/dl in homozygotic, heterozygotic, and wild-type animals, respectively, instilled with saline only. In the wild-type and heterozygotic animals, only a small fraction of the TIBC was complexed with metal (5 and 7%, respectively), whereas a larger fraction (18%) of the TIBC contained the metal in the homozygotic mice. After instillation of the particle, the TIBC did not change significantly in any group of animals, with values of 17.0 ± 2.8, 15.8 ± 1.8, and 13.3 ± 1.4 µg/dl in wild-type, heterozygotic, and homozygotic mice, respectively. However, the percentage of lavage fluid TIBC from the wild-type and heterozygotic mice complexed with iron was significantly elevated to 11 and 18%, respectively, whereas that of the homozygotic Hp mice remained unchanged at 18%.


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Fig. 1.   Concentration of transferrin in lavage fluid of wild-type and hypotransferrinemic (Hp) mice 24 h after instillation of either saline (0.05 ml) or oil fly ash (50 µg in 0.05 ml of saline). Transferrin was measured with a commercially available immunoprecipitation analysis kit. Among mice instilled with saline, concentration of transferrin in lavage fluid was significantly lower in homozygotic Hp mice. After particle exposure, transferrin concentrations significantly increased in wild-type and heterozygotic mice (n = 5 /group). * Significantly decreased relative to saline-instilled wild-type mice. ** Significantly increased relative to saline-instilled wild-type mice.

Lavage fluid concentrations of low-molecular-weight antioxidants were measured to assess the oxidative stress in the lower respiratory tract after exposure to the metal-rich ash. After instillation of saline, there were no differences between the three groups of animals in the lavage fluid concentrations of ascorbate, glutathione, and urate (Fig. 2, A-C, respectively). Instillation of the particle was associated with decreased ascorbate concentrations in all three groups of mice (Fig. 2A). There were no differences in either glutathione or urate concentration between the saline- and particle-exposed mice (Fig. 2, B and C).




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Fig. 2.   Ascorbate (A), glutathione (B), and urate (C) concentrations in lavage fluid of wild-type and Hp mice 24 h after instillation of either saline (0.05 ml) or oil fly ash (50 µg in 0.05 ml of saline). Ascorbate and urate were measured with HPLC with electrochemical detection, whereas glutathione was quantified as nonprotein sulfhydryls. Ascorbate concentrations were significantly diminished in all groups of mice exposed to the particle, whereas glutathione and urate remained unchanged. * Significantly decreased relative to saline-instilled wild-type mice.

The expression of mediators associated with lung injury can be influenced by oxidants catalyzed by metals in this ash (10). Lavage fluid demonstrated no differences in MIP-2 and TNF concentrations among wild-type, heterozygotic, and homozygotic mice after instillation of saline (Fig. 3). After exposure to the particle, the quantities of both cytokines increased in the wild-type and heterozygotic Hp animals but not in the homozygotic Hp mice (Fig. 3).



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Fig. 3.   Concentrations of macrophage inflammatory protein-2 (MIP-2; A) and tumor necrosis factor (TNF; B) in lavage fluid of wild-type and Hp mice 24 h after instillation of either saline (0.05 ml) or oil fly ash (50 µg in 0.05 ml of saline). Cytokine concentrations were determined with commercially available ELISA kits. Concentrations of MIP-2 were significantly elevated after particle exposure in both wild-type and heterozygotic mice but not in homozygotes. Lavage fluid concentrations of TNF were similarly elevated after particle exposure in both wild-type and heterozygotic mice but not in homozygotes. * Significantly increased relative to saline-instilled wild-type mice.

Tissue injury was evaluated directly with histology and also was assessed biochemically (i.e., lavage fluid protein and LDH concentrations). There was no evidence of either an inflammatory cell incursion or an injury in the lung in any group of animals after saline instillation (Fig. 4A). However, corresponding to the disparities in cytokine concentrations in the lavage fluid among the animals, there was a notable neutrophilic influx into the large airways and peribronchial regions of both wild-type and heterozygotic Hp mice after particle exposure but not in the homozygotic Hp animals (Fig. 4, B and C). Lavage fluid protein and LDH similarly exhibited no disparities among the three groups of mice after saline instillation (Fig. 5). The wild-type and heterozygotic Hp mice both had elevated concentrations of protein and LDH in the lavage fluid after exposure to the oil fly ash (Fig. 5). These two indexes were not increased in the homozygotic mice, supporting a diminished injury after particle exposure.


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Fig. 4.   Hematoxylin and eosin staining of lung tissue from wild-type and Hp mice 24 h after instillation of either saline (0.05 ml) or oil fly ash (50 µg in 0.05 ml of saline). After saline instillation, there were no differences among the 3 groups of mice by examination with light microscopy. However, both wild-type and heterozygotic mice demonstrated a neutrophilic inflammatory injury focused around the airways. Relative to wild-type animals instilled with saline (A), wild-type mice exposed to particle (B) exhibited an incursion of neutrophils, perivascular edema, and proliferation of respiratory epithelium. However, homozygotic Hp mice exposed to particle (C) demonstrated a diminished injury, with a decreased incursion of neutrophils and a smaller accumulation of perivascular edema. Original magnification, approximately ×200.




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Fig. 5.   Lavage fluid protein (A) and lactate dehydrogenase (LDH; B) concentrations in wild-type and Hp mice 24 h after instillation of either saline (0.05 ml) or oil fly ash (50 µg in 0.05 ml of saline). Lavage fluid protein was determined with Coomassie Plus protein assay reagent, whereas LDH was measured with a commercially prepared kit. Concentrations of both protein and LDH were significantly elevated after exposure to particle. However, these indexes of injury were diminished in homozygotic mice after instillation of ash relative to those in wild-type and heterozygotic Hp mice. * Significantly increased relative to saline-instilled wild-type mice.

Hp animals can demonstrate a siderosis of peripheral organs (26). Such an imbalance in metal metabolism could affect the expression of iron-associated protein genes. ICPES measurements of total iron in the lavage fluid supernatant demonstrated greater concentrations of iron in the homozygotic Hp mice among the saline-exposed animals (Fig. 6A). After instillation of the particle, iron concentrations in the lavage fluid in all groups of animals were elevated, but the homozygotic Hp mice again were significantly increased relative to wild-type and heterozygotic mice (Fig. 6A). Vanadium and nickel concentrations were not measurable in any sample of lavage fluid. Similarly, nonheme iron in lung tissue was elevated in homozygotic Hp mice after instillation of saline only (Fig. 6B). After exposure to the particle, values of nonheme iron were unchanged relative to those after saline instillation (Fig. 6B).



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Fig. 6.   Lavage fluid iron (A) and nonheme lung tissue iron (B) in wild-type and Hp mice 24 h after instillation of either saline (0.05 ml) or oil fly ash (50 µg in 0.05 ml of saline). Both lavage fluid and tissue iron concentrations were measured with inductively coupled plasma emission spectroscopy (wavelength = 238.204 nm). Tissue was hydrolyzed in a solution of 3 N HCl and 10.0% TCA (0.10 g lung/ml) at 70°C for 16 h before measurement of metal. After saline instillation, homozygotic Hp mice exhibited higher concentrations of both lavage fluid and tissue iron. After particle exposure, lavage fluid concentrations of iron were elevated in all animals but were significantly greater in homozygotic Hp mice. There was no significant change in tissue iron after exposure to particle. ppb, Parts/billion; ppm, parts/million. * Significantly increased relative to wild-type and heterozygotic Hp mice instilled with saline. ** Significantly increased relative to wild-type and heterozygotic Hp mice instilled with particle.

To better define the dysequilibrium in metal metabolism in the lungs of unexposed homozygotic Hp mice and to delineate changes after instillation of the metal-rich particle, slot blots for ferritin were performed. Analogous to the total iron concentrations, there were greater quantities of ferritin protein in the lavage fluid of unexposed homozygotic mice relative to those in wild-type and heterozygotic animals (Fig. 7). After instillation of the particle, ferritin protein in the lavage fluid supernatant increased significantly in all groups of animals but was highest in the homozygotes (Fig. 7). Immunohistochemistry confirmed that there were elevations in tissue ferritin among the homozygotic Hp mice after saline instillation only (Fig. 8). After the exposure to the oil fly ash, the lung tissue in all groups stained positively for ferritin but appeared greatest in the homozygotes. In the homozygotic Hp animals only, there were sideromacrophages in large numbers in the alveolar region after instillation of the oil fly ash. The respiratory epithelial cells of the bronchi also stained positively for iron in this group of animals after particle instillation.


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Fig. 7.   Slot blots for lavage fluid ferritin in wild-type and Hp mice 24 h after instillation of either saline (0.05 ml) or oil fly ash (50 µg in 0.05 ml of saline). Staining for ferritin was accomplished with a 1:2,000 dilution of rabbit anti-human ferritin antibody. Band optical densities were quantified with a digital bioimaging system. Results are expressed as multiple of increase relative to that in wild-type mice instilled with saline. After exposure to saline only, homozygotic Hp mice had significantly higher concentrations of ferritin in lavage fluid. Instillation of particle was associated with an elevation in ferritin concentrations in all animals. * Significantly increased relative to wild-type mice instilled with saline. ** Significantly increased relative to wild-type mice instilled with particle.



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Fig. 8.   Immunohistochemistry for ferritin 24 h after instillation of either saline (0.05 ml) or oil fly ash (50 µg in 0.05 ml of saline). Staining for ferritin was accomplished with a 1:100 dilution of rabbit anti-human ferritin antibody. After instillation of mice with saline, there was minimal uptake in wild-type (A) and heterozygotic Hp mice, which was centered in airway epithelium. However, there was more uptake of antibody in homozygotic Hp animals exposed to saline only (B). This was predominantly in airway epithelium but did include some staining of structures in the alveolar region, including macrophages. Instillation of particle was associated with an elevation in ferritin staining in all animals.

Finally, for ferritin to function in decreasing the oxidative stress in Hp mice presented by the particle, a chelator must be present to transport the metal across the membrane to the storage protein. However, transferrin was demonstrated to be diminished in that group of animals with the least injury (i.e., homozygotic Hp mice). Slot blots for lactoferrin revealed that, with exposure to saline alone, homozygotic mice had greater concentrations of this transport protein (Fig. 9). After instillation of the particle, lactoferrin concentrations increased in all groups but was greatest in the homozygotic animals (Fig. 9). Immunohistochemistry verified that after instillation of saline only, lactoferrin expression was significantly greater among homozygotes (Fig. 10). After exposure to the particle, lactoferrin expression was increased in all groups of animals after saline instillation but appeared highest in the homozygotic mice.


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Fig. 9.   Slot blots for lavage fluid lactoferrin in wild-type and Hp mice 24 h after instillation of either saline (0.05 ml) or oil fly ash (50 µg in 0.05 ml of saline). Staining for lactoferrin was accomplished with a 1:2,000 dilution of rabbit anti-human lactoferrin antibody. Band optical densities were quantified with a digital bioimaging system. Results are expressed as multiple of increase relative to that in wild-type mice instilled with saline. After exposure to saline only, homozygotic Hp mice had significantly higher concentrations of lavage fluid lactoferrin. Instillation of particle was associated with an elevation in lactoferrin concentrations in all animals. * Significantly increased relative to wild-type mice instilled with saline. ** Significantly increased relative to wild-type mice instilled with particle.



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Fig. 10.   Immunohistochemistry for lactoferrin 24 h after instillation of either saline (0.05 ml) or oil fly ash (50 µg in 0.05 ml of saline). Staining for lactoferrin was accomplished with a 1:100 dilution of rabbit anti-human lactoferrin antibody. After instillation of mice with saline, there was minimal uptake in wild-type (A) and heterozygotic Hp mice in airway epithelium. Homozygotic Hp animals exposed to saline demonstrated increased staining relative to wild-type and heterozygotic animals (B). Instillation of particle was associated with an elevation in expression of lactoferrin protein in all animals.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Lavage fluid transferrin concentrations in the homozygous mice were modestly reduced relative to decrements in serum values. This disparity between changes in the lavage fluid and serum concentrations of this glycoprotein may reflect differences in either the collection of the samples or specific methods of analysis. The TIBC in the lavage fluid after exposure of the animals to the metal-containing particle is similar in the three groups despite significant elevations in iron concentrations. This may be the result of decrements in transferrin with exposures to metal (16). Concomitantly, the transport and storage of the metal by lactoferrin and ferritin in the animals increase. In addition, differences in the percent TIBC bound with metal can indicate a transudation of serum transferrin across the alveolar-capillary membrane in the wild-type and heterozygotic animals because injury is greater in these groups of mice.

Despite serum and lavage fluid transferrin concentrations that were significantly lower relative to wild-type and heterozygotic Hp animals, the homozygotic Hp mice did not demonstrate an increased lung injury after exposure to the metal-rich particle but rather had diminished tissue damage. A possible reason for this is that, comparable to other peripheral organs, the homozygotic Hp mice demonstrated an accumulation of iron in the lungs before any exposures to metal. Increased concentrations of iron can elevate the expression of both metal storage and transport proteins, and, indeed, the homozygous Hp mice had increased lung ferritin and lactoferrin concentrations. Consequently, exposure of these mice to the metal-rich particle is likely to have resulted in a diminished oxidative stress because the metal was sequestered by ferritin and lactoferrin. A decreased tissue injury in the homozygotic Hp mice could result.

After the exposure to the oil fly ash, ascorbate decreased equally among all mice, whereas glutathione and urate did not change. Ascorbate is unstable in the presence of transition metal cations (23), and decrements in lavage fluid after exposure to the metal contained in the particle occur with consumption of the antioxidant molecule. The lack of any dissimilarities between the three groups of mice in the concentrations of this low-molecular-weight antioxidant possibly reflects the compartment sampled (i.e., the extracellular alveolar compartment). Radicals catalyzed intracellularly probably contribute to the development of tissue injury also. Alterations in the antioxidative capacity of this compartment were not measured in any of the three groups of mice.

Despite of a lack of clear differences in the oxidative stress in the lower respiratory tract among the three groups of mice, mediators of injury were significantly diminished in the homozygotic Hp mice relative to those in the wild-type mice and heterozygotes after exposure to the particle. The release of such inflammatory mediators has been associated with exposure to oxygen-based free radicals (2, 12). Similarly, mediator expression and release after in vitro exposure of cells to this metal-rich particle can be the result of catalyzed oxidants (10). The increase in MIP-2 and TNF after instillation of particles in mice can subsequently result from the exposure of the lower respiratory tract to metal-catalyzed oxidants because cellular expression of these mediators can be controlled by oxidant-sensitive promoters including nuclear factor-kappa B (22, 35).

Disparities between the animals in the release of cytokines that are chemotactic for inflammatory cells predicted a diminished injury in the homozygotic Hp mice because some portion of tissue damage after exposure to oxidants can be mediated by a release of products by these cells (e.g., proteases). Histology confirmed a decreased neutrophilic incursion and edema in homozygotes while lavage fluid protein and LDH concentrations also were diminished at 24 h after exposure.

In the homozygotic Hp mice, iron is abnormally deposited in parenchymal organs including the liver, pancreas, and heart, and these tissues can become siderotic (13, 26). Through a posttranscriptional mechanism involving the iron-responsive element, elevations in iron can effect an increased production of ferritin, which functions as an antioxidant (4). An accumulation of iron was evident in the lung of homozygotic Hp mice before exposure to the particle with increased concentrations of this metal in both the lavage fluid and the tissue. Some quantity of this metal appeared to be biologically available because concentrations of ferritin protein in both the lavage fluid and lung tissue were increased. Comparable to ferritin, the expression of lactoferrin can be influenced by concentrations of available metal (17). Concentrations of lactoferrin protein were also elevated in the lavage fluid of the homozygotic Hp mice before instillation of the particle. The lower respiratory tract of the homozygotic Hp mice are therefore primed to protect against a metal-catalyzed oxidative stress with increased concentrations of proteins to both transport and then store metals.

Differences between metal transport by transferrin and lactoferrin may reflect dissimilarities in cell handling of the two iron-binding proteins (32). In addition, disparities in the expression of the two chelators could reflect contrasting functions. Transferrin releases the metal to a low-molecular-weight pool of chelates in the cell, which is accessible to meet the needs for metabolism and growth (7). This glycoprotein is subsequently used to meet nutritional demands of the cell but there is a potential of the metal transported by transferrin to participate in a catalysis of oxidants (8). This capacity is greatest while the metal is in transit between the endosomes and cytosolic ligands. In contrast, lactoferrin participates in the transport of metal across a cell membrane but assists in its storage as a less reactive form within ferritin (38). These two metal chelators appear to respond in a diametrical fashion to metal availability. Transferrin-receptor mRNA and transferrin protein concentrations increase with the depletion of iron in the environment (16, 17, 30). With exposure to metal-rich particles, the reverse situation can be evident, with transferrin-receptor mRNA decreasing. However, in response to catalytically active metal, the in vitro and in vivo production of lactoferrin can increase in order to transport metal in a redox-inactive form (17).

Such a balance of the two metal transport systems assists in interpreting previous investigations demonstrating that metal depletion can increase transferrin-mediated transport, whereas iron excess can increase lactoferrin-mediated transport. In exposures to microbial agents, the replication of which is dependent on a mobilization of host iron, the two systems of binding iron respond in a dissimilar fashion. Serum transferrin concentrations diminish after bacterial infection, whereas lactoferrin concentrations in the immediate environment were elevated (6). Exposure of the human lung to aerosolized microbials similarly decrease lavage fluid transferrin or increase lactoferrin, again supporting disparate functions of the two systems of transport (29). With a direct challenge (ingestion) of humans to iron, transferrin concentrations in the serum do not increase to meet an in vivo oxidative stress, whereas lactoferrin levels do (9). Finally, in human injury associated with elevations in catalytically active iron, such as rheumatoid arthritis, the local concentrations of lactoferrin in the synovial fluid, but not of transferrin, increase in concentration (1).

In conclusion, the lack of transferrin in Hp mice did not predispose the animals to lung injury after exposure to a particle abundant in metals. Rather, these mice demonstrated a diminished injury that was associated with an increase in metal transport and storage proteins.


    ACKNOWLEDGEMENTS

This work was supported by a grant from the Morrison Trust.


    FOOTNOTES

This report has been reviewed by the National Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

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: A. J. Ghio, MD58D, HSD, Environmental Protection Agency, 104 Mason Farm Rd., Chapel Hill, NC 27599 (E-mail: ghio.andy{at}epa.gov).

Received 12 May 1999; accepted in final form 9 December 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Lung Cell Mol Physiol 278(5):L1051-L1061