Disruption of normal iron homeostasis after bronchial instillation of an iron-containing particle

Andrew J. Ghio1, Jacqueline D. Carter1, Judy H. Richards1, Luisa E. Brighton2, John C. Lay2, and Robert B. Devlin1

1 National Health and Environmental Effects Research Laboratory, Environmental Protection Agency, Research Triangle Park 27711; and 2 Center for Environmental Medicine and Lung Biology, University of North Carolina, Chapel Hill, North Carolina 27599

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The atmosphere constitutes a prime vehicle for the movement and redistribution of metals. Metal exposure can be associated with an oxidative stress. We tested the hypothesis that, in response to an iron-containing particle, the human respiratory tract will demonstrate an increased expression of both lactoferrin and ferritin as the host attempts to transport and store the metal in a chemically less-reactive form and therefore diminish the oxidative stress the particle presents. Subjects (n = 22) were instilled with 20 ml of saline and 20 ml of an iron-containing particle suspended in saline in a right middle lobe bronchus and a lingular bronchus, respectively. At either 1, 2, or 4 days after this exposure, the volunteer was lavaged for a sample of the lower respiratory tract, and concentrations of L-ferritin, transferrin, and lactoferrin were measured by enzyme immunoassay, immunoprecipitin analysis, and enzyme-linked immunosorbent assay (ELISA), respectively. Transferrin receptor was also quantified by ELISA. The concentrations of L-ferritin in the lavage fluid of lung exposed to particles were significantly increased relative to the levels of the protein in the segment exposed to saline. Relative to saline instillation, transferrin was significantly diminished after exposure to the iron-containing particle, whereas both lactoferrin and transferrin receptor concentrations in the segment of the lung exposed to the particle were significantly elevated. We conclude that instillation of an iron-containing particle was associated with a disequilibrium in iron metabolism in the lower respiratory tract. The response included increased ferritin and lactoferrin concentrations, whereas transferrin concentrations diminished. This coordinated series of reactions by the host effects a decrease in the availability of catalytically reactive iron to likely diminish the consequent oxidative stress to the human host.

air pollution; ferritin; transferrin; transferrin receptors; lactoferrin

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

ON A GLOBAL SCALE, the atmosphere constitutes a prime vehicle for the movement and redistribution of metals (35). With the exception of mercury, all metals in the atmosphere are associated with particles. Among particles of anthropogenic origin, such as those originating from combustion emission, metals are enriched in the finest fractions for which control devices are least effective. Natural sources of airborne particles such as terrestrial dusts, sea spray, and volcanic emissions similarly include metals. That metal in greatest abundance in all ambient air pollution particles of rural and urban environments is iron.

Metal exposure can be associated with an oxidative stress whether the specific metal can (e.g., iron, vanadium, and chromium) or cannot (e.g. arsenic, zinc, and cadmium) assume two stable valences and therefore transport electrons to directly catalyze free radical generation (38). It would therefore be of benefit to the exposed tissue to isolate the metal in a chemically less- reactive form. Such sequestration of iron by macrophages diminishes the potential of the metal to catalyze oxidant generation (30). The storage of iron within intracellular ferritin confers an antioxidant function to this protein and, in certain cells, provides cytoprotection in vitro against oxidants (2, 9).

Ferritin expression is regulated by a posttranscriptional mechanism. A specific sequence at the 5'-untranslated end of ferritin mRNA called the iron responsive element (IRE) binds a cubane iron-sulfur cluster, the iron regulatory protein (IRP), when the IRP exists in the apoprotein form (3, 22). Iron included in atmospheric particles could react with IRP, which alters its conformation. This decreases the affinity of the protein to the mRNA, and it is displaced, allowing translation of ferritin to proceed.

Although numerous metal chelates that present an oxidative stress to the cell are extracellular, ferritin is produced intracellularly. Therefore, the sequestration and detoxification of metal by ferritin requires that it be transported across the cell membrane. To transfer iron across a membrane, animal cells most frequently use the glycoprotein transferrin (43). Similar to ferritin, expression of the transferrin receptor is controlled by five IREs, but these are at the 3'-untranslated region (18, 19). When iron levels are high, this posttranscriptional control of protein expression can function to decrease the synthesis of transferrin receptor. Subsequently, specific cells rapidly downregulate transferrin receptor expression with exposure to iron salts, and metal chelation leads to an increase in these surface receptors (6, 33, 46). Transferrin-dependent transport of iron across the membrane is therefore unlikely to contribute to the sequestration of metal by ferritin.

Lactoferrin is a monomeric, cationic metal-binding glycoprotein (molecular mass of 76.4 kDa) that, during an inflammatory state, can transport iron across a cell membrane comparable to transferrin (4, 25, 45). After such transport, the metal is deposited in the ferritin. The location of lactoferrin at sites of interaction between a host and its external environment supports a potential role in a detoxification of metal chelates. In the respiratory tract, lactoferrin is synthesized by serous cells of bronchial epithelium (5). We tested the hypothesis that, in response to an iron-containing particle, the human respiratory tract will demonstrate an increased expression of both lactoferrin and ferritin as the host attempts to transport and store the metal in a chemically less-reactive form.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Concentrations of iron-related proteins in normal, unexposed volunteers. Healthy, nonsmoking volunteers 18-35 yr of age underwent fiber-optic bronchoscopy with lavage. Before participation in the study, subjects were informed of the procedures and potential risks, and each signed a statement of informed consent. The protocol and consent form were approved by the University of North Carolina School of Medicine Committee on the Protection of the Rights of Human Subjects. The screening procedures for each subject included a Minnesota Multiphasic Personality Inventory, medical history, physical examination, chest X ray, and routine hematological and biochemical tests. None of the subjects had a history of asthma, allergic rhinitis, chronic respiratory disease, or cardiac disease. Subjects were excluded from the study if they had a recent acute respiratory illness and were asked to avoid exposure to air pollutants such as tobacco smoke and paint fumes. The fiber-optic bronchoscope was wedged into a segmental bronchus of the lingula. Six aliquots of sterile saline were instilled and immediately aspirated. The first was 20 ml, and this fraction was labeled the bronchial sample. The remaining five aliquots were 50 ml each, and the returns from these are considered to reflect the environment of the distal respiratory tract. These were designated the alveolar samples. The procedure was repeated on the right middle lobe, again using 270 ml of saline. Samples were put on ice immediately after aspiration and centrifuged at 300 g for 10 min at 4°C. Total cell counts and differentials were obtained using a hemocytometer. The supernatant was assayed for ferritin, transferrin, transferrin receptor, and lactoferrin concentrations.

Particle generation. Spherical iron-containing particles were generated as previously described (1, 15) from suspensions of colloidal iron oxide (29) using a spinning disk aerosol generator (27) but without radioactive label. The resultant particles had a 2.6-µm-count median diameter with a geometric standard deviation of 1.3 µm. These were collected in sterile saline in an impinger, concentrated by centrifugation, and then washed two times in sterile saline. Although the majority of the product is ferric oxide, some portion of the metal was present as an undefined iron salt and/or iron oxyhydroxides. This was made evident by both a release of iron (240 ng iron/mg particle) by this product when in aqueous solution and a capacity of the particle to support electron transfer and catalyze oxidant generation (unpublished observation).

Initial particle batches were sterilized by autoclaving at 121°C for 3.5 h, which also ensured the destruction of any endotoxin activity that might be present in the particle suspension. Batches of all the particle suspensions were tested for endotoxin activity using a gelatin capillary method (Endotect; ICN Biomedical, Costa Mesa, CA) or using a semiquantitative method (performed by University of North Carolina Tissue Culture Facility). Both assays for endotoxin are based on the Limulus amebocyte lysate (LAL) assay. The capillary LAL method detects endotoxin concentrations as low as 0.06-0.10 ng/ml and provides only a positive or negative indication of the presence of endotoxin. The semiquantitative LAL method is equally as sensitive and provides an indication of the actual concentration of endotoxin present. All particle suspensions tested by the capillary method were negative. Particle suspensions tested by the semiquantitative LAL method were <= 0.06 endotoxin units (=0.1 ng/ml).

Just before instillation, concentrated particles were suspended in 2.0-3.0 ml of sterile saline and placed in an ultrasonic bath for 30 min to disperse clumps of particles. Particles were examined and counted in a hemocytometer to ensure the dispersion of clumps and to quantify particle numbers. Finally, 3.0 × 108 particles were suspended in 10.0 ml of sterile saline and transferred to a sterile syringe for instillation.

Instillation of particles in volunteers. The iron-containing particle was instilled into a segmental bronchus in a small volume of sterile saline via a flexible fiber-optic bronchoscope. The instrument was positioned at, but not wedged into, the lingula. A sterile flexible catheter was inserted through the biopsy channel and extended into the orifice of the bronchial segment selected. Ten milliliters of sterile saline containing 3.0 × 108 (5 mg) microspheres were slowly instilled through the catheter coincident with inspirations to maximize placement of particles in the alveolar region. This was followed by an additional 10.0 ml of saline from a different syringe (for a total of 20 ml), with the intent of washing particles remaining in the airways into the alveoli. As a control, 20.0 ml of saline with no particles were similarly instilled into the medial segment of the right middle lung lobe. To assess the number of particles that were lost to the syringe and catheter, simulated instillations were performed in vitro by injecting particle suspensions through the catheter into a glass vial and counting the particles deposited in the vial. Based on these simulations, almost one-third of the particles (31.4 ± 2.2%) were lost to the syringe and catheter.

Repeat bronchoscopy was done at either 1, 2, or 4 days after particle instillation. Subjects underwent segmental bronchopulmonary lavage of the same segment of the lung in which the particles and the saline (control) had been previously instilled. The instrument was wedged into the segmental bronchus, and six aliquots of sterile saline were instilled and immediately aspirated. The procedure was repeated on the right middle lobe, again using 270 ml of saline. Samples were put on ice immediately after aspiration and centrifuged at 300 g for 10 min at 4°C. Total cell counts and differentials were obtained using a hemocytometer. The supernatant was assayed for L-ferritin, transferrin, transferrin receptor, and lactoferrin concentrations.

Iron-related proteins. L-Ferritin and transferrin concentrations were measured using commercially available kits (an enzyme immunoassay and an immunoprecipitin analysis, respectively), controls, and standards from Microgenics (Concord, CA) and Incstar (Stillwater, MN). These assays were modified for use in the Cobas Fara II centrifugal spectrophotometer (Hoffman-LaRoche, Branchburg, NJ). Transferrin receptor and lactoferrin were measured with commercially available enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN, and Calbiochem, La Jolla, CA, respectively).

Iron concentrations in the lavage fluid. After centrifugation to remove cells, iron concentrations in the supernatant were quantified using inductively coupled plasma emission spectroscopy (model P40; Perkin-Elmer, Norwalk, CT).

Statistics. Data are expressed as mean values ± SD. Differences between two groups were compared with t-tests of independent means, whereas those between multiple groups were analyzed employing analysis of variance (8). Linear regression techniques were used to analyze relationships between two continuous variables. Significance was assumed at P < 0.05.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The group of healthy volunteers lavaged to determine normal concentrations of L-ferritin, transferrin, transferrin receptor, and lactoferrin in blood, bronchial samples, and alveolar samples included 12 males and 4 females who were 27 ± 4 yr of age. Among these subjects, there were significant differences in the concentrations of L-ferritin between the plasma, the bronchial sample, and the alveolar sample (Fig. 1). The concentration of this iron storage protein was significantly lower in the plasma relative to both bronchial and alveolar fractions of lavage fluid. This was despite the profound dilution (up to 100-fold) of the lining fluid of the lower respiratory tract by the lavage process (8). There was no difference between the concentrations of L-ferritin in bronchial and alveolar fractions. There was no correlation between the plasma concentration of L-ferritin and the concentration in either the bronchial or alveolar sample (P = 0.94 and 0.16, respectively). Similarly, there was no correlation between the concentrations of L-ferritin in the bronchial and alveolar samples (P = 0.16).


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Fig. 1.   L-Ferritin concentrations in plasma, bronchial samples, and alveolar samples of healthy, unexposed volunteers. Subjects were lavaged with 20 ml of saline for the bronchial sample and 250 ml of saline for the alveolar sample. After centrifugation to remove cells, concentrations of the storage protein were measured in the supernatant using an enzyme immunoassay. Even after dilution, the concentrations of L-ferritin were significantly higher in the lavage samples relative to that in plasma (*).

Among these normal, unexposed individuals, there was a severalfold increase in the concentration of transferrin in the plasma relative to values in the lavage fluid collected from the bronchial and alveolar compartments (Table 1). Concentrations of this transport protein were significantly lower in the bronchial fraction and alveolar fraction relative to plasma. Correlations of the transferrin concentrations in plasma, bronchial sample, and alveolar sample approached significance (plasma to bronchial sample, P = 0.08; plasma to alveolar sample, P = 0.09; and bronchial sample to alveolar sample, P = 0.13).

                              
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Table 1.   Transferrin and transferrin receptor concentrations in healthy volunteers

The concentrations of transferrin receptor in plasma, the bronchial sample, and the alveolar sample were significantly different among healthy volunteers (Table 1). Comparable to transferrin, the concentration of transferrin receptor was significantly higher in plasma relative to both bronchial and alveolar samples. There were no differences between the two lavage fractions (P = 0.72). However, transferrin receptor concentrations in the plasma and bronchial and alveolar samples demonstrated no significant correlations.

Finally, there were differences among concentrations of lactoferrin in the plasma, the bronchial sample, and the alveolar sample among these normal subjects (Fig. 2). Lactoferrin concentrations were significantly higher in the bronchial sample relative to both the plasma (P = 0.04) and alveolar (P = 0.03) samples. There were no significant correlations between the lactoferrin concentrations in the plasma, bronchial sample, and alveolar sample (plasma to bronchial sample, P = 0.70; plasma to alveolar sample, P = 0.98; and bronchial sample to alveolar sample, P = 0.34). Similarly, there was no significant correlation of the total neutrophil count in the blood (2.4 ± 0.6 × 103/µl) with the concentration of lactoferrin in the plasma.


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Fig. 2.   Lactoferrin concentrations in plasma, bronchial samples, and alveolar samples of healthy, unexposed volunteers. Subjects were lavaged with 20 ml of saline for the bronchial sample and 250 ml of saline for the alveolar sample. After centrifugation to remove cells, concentrations of lactoferrin were measured in the supernatant using a commercially available enzyme-linked immunosorbent assay (ELISA) kit. Post hoc testing revealed significant differences (*) in the concentrations of this transport glycoprotein between the bronchial sample and both concentrations in the plasma and alveolar samples.

Correlations between the concentrations of L-ferritin, transferrin receptor, transferrin, and lactoferrin in normal subjects provided disparate results. There was a significant correlation (negative) between L-ferritin and transferrin concentrations (P = 0.0002), whereas there were no associations between either L-ferritin and lactoferrin (P = 0.99) or transferrin and lactoferrin (P = 0.45) concentrations. The concentrations of transferrin receptor were associated with both transferrin (positive correlation; P = 0.001) and ferritin (negative correlation; P = 0.001) but not with lactoferrin (P = 0.59).

The group of volunteers instilled with iron-containing particles and saline was predominantly male (18 males and 4 females) and also young (25 ± 5 yr old). These subjects were studied 1 day (n = 10), 2 days (n = 6), and 4 days (n = 6) after instillation of the particle. The concentrations of L-ferritin in the lavage of the lower respiratory tract exposed to particles were significantly different from that of an unexposed segment at 1 day after exposure (Fig. 3). However, ferritin concentrations in lavage fluid returned to normal values at 2 and 4 days after particle instillation. Relative to saline instillation, transferrin was significantly diminished after exposure to the iron-containing particle (Fig. 4). Similar to discrepancies in ferritin after exposure, these changes were significant only at 1 day (P = 0.01). Contrary to these results, transferrin receptor concentrations were elevated after instillation of the particles (Fig. 5). Post hoc testing revealed differences in transferrin receptor levels at 1, 2, and 4 days after instillation of the particles. Finally, lactoferrin concentrations in the segment of the lung exposed to the particle were significantly elevated relative to that segment instilled with saline (Fig. 6). These differences were significant at both 1 day (P = 0.008) and 2 days (P = 0.04) after instillation of the particle. There was a significant influx of inflammatory cells at 1 day after exposure. The total number of neutrophils in the lavage fluid after saline and particle exposures was 3.4 ± 2.9 and 33.9 ± 23.9 × 105/ml, respectively. However, the concentration of lactoferrin did not correlate with the neutrophil count in the lavage fluid (P = 0.71). Finally, the concentrations of iron in lavage fluid actually decreased 1 day after the instillation of particles rather than increasing (Fig. 7). Differences in the concentrations of this metal in the lavage fluid from saline- and particle-exposed lung were not significant at 2 and 4 days after instillation.


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Fig. 3.   L-Ferritin concentrations in the alveolar sample of healthy volunteers exposed to saline and an iron-containing particle. Twenty milliliters of saline and 20 ml of a suspension of the particle (10.0 mg) were instilled into bronchi of the right middle lobe and the lingula, respectively. At either 1, 2, or 4 days after exposure, subjects were lavaged with 250 ml. After centrifugation to remove cells, concentrations of the storage protein in the supernatant were determined using an enzyme immunoassay. There was a significant increase (*) in L-ferritin concentration 1 day after exposure to the iron-containing particle. However, this difference was not apparent at 2 and 4 days after instillation.


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Fig. 4.   Concentrations of transferrin in the alveolar sample of healthy volunteers exposed to saline and an iron-containing particle. Twenty milliliters of saline and 20 ml of a suspension of the particle (10.0 mg) were instilled into bronchi of the right middle lobe and the lingula, respectively. At either 1, 2, or 4 days after exposure, subjects were lavaged with 250 ml. After centrifugation to remove cells, concentrations of the transport protein in the supernatant were determined using an immunoprecipitin analysis. There was a significant decrease (*) in transferrin concentration but only at 1 day after exposure to the particle.


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Fig. 5.   Transferrin receptor concentrations in the alveolar sample of healthy volunteers exposed to saline and an iron-containing particle. Twenty milliliters of saline and 20 ml of a suspension of the particle (10.0 mg) were instilled into bronchi of the right middle lobe and the lingula, respectively. At either 1, 2, or 4 days after exposure, subjects were lavaged with 250 ml. After centrifugation to remove cells, concentrations of the receptor in the supernatant were determined using a commercially available ELISA. There were significant increases (*) in transferrin receptor concentration at 1, 2, and 4 days after instillation of the particle.


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Fig. 6.   Lactoferrin concentrations in the alveolar sample of healthy volunteers exposed to saline and an iron-containing particle. Twenty milliliters of saline and 20 ml of a suspension of the particle (10.0 mg) were instilled into bronchi of the right middle lobe and the lingula, respectively. At either 1, 2, or 4 days after exposure, subjects were lavaged with 250 ml. After centrifugation to remove cells, concentrations of the transport glycoprotein in the supernatant were determined using a commercially available ELISA. There were significant increases (*) in transferrin receptor concentration at 1 and 2 days after exposure to the iron-containing particle.


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Fig. 7.   Concentrations of total iron in the alveolar sample of healthy volunteers exposed to saline and an iron-containing particle. Twenty milliliters of saline and 20 ml of a suspension of the particle (10.0 mg) were instilled into bronchi of the right middle lobe and the lingula, respectively. At either 1, 2, or 4 days after exposure, subjects were lavaged with 250 ml. After centrifugation to remove cells, iron concentrations in the supernatant were measured using inductively coupled plasma emission spectroscopy. Concentrations of the metal were unexpectedly decreased (*) 1 day after exposure to the particle. There were no significant differences in iron concentrations at 2 and 4 days after instillation.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The lack of correlations in L-ferritin between the plasma, bronchial sample, and alveolar sample in healthy, unexposed volunteers supports an influence of local factors in the control of expression of this storage protein. The correlation of L-ferritin with transferrin concentrations is likely to reflect a dependence of both on tissue iron concentrations. The concentration of L-ferritin that we measured in lavage samples is included within the range of values for total ferritin (9.9-5,600 ng/ml) previously reported in normal, unexposed volunteers (26, 40, 49). The actual concentrations of constituents in respiratory epithelial lining fluid are difficult to determine with assurance as a result of uncertainties in the methods used to measure this volume (31). Considering that unconcentrated lavage fluid represents an ~100-fold dilution of the lining layer of the respiratory tract (31), these values of ferritin concentrations appear to be extremely high. The concentrations of this storage protein in blood can range up to ~200 ng/ml in healthy individuals. The higher concentrations in the respiratory tract lining fluid may reflect the direct interaction of the lung with the external environment. The lungs are subsequently exposed on a continuous basis to metals included in air- pollution particles. An increased expression of ferritin is predicted to be included in the response of cells resident in the respiratory tract in their attempt to diminish the oxidative stress associated with exposure to the metal.

With the instillation of an iron-containing particle, L-ferritin concentrations in the lavage fluid significantly increased at 1 day after exposure but rapidly returned to normal values by 2 days after instillation. This supports an increased expression of ferritin by the host in response to elevated concentrations of available iron. The cell responsible for this increased production of this iron storage protein was not determined but is likely to include alveolar macrophages and respiratory epithelial cells (12 and unpublished observation). The presence of L-ferritin in extracellular fluid (i.e., bronchoalveolar lavage) suggests the existence of a cycle of this protein in the milieu of the lung (36). The protein is produced intracellularly but appears to be released into the external environment. An alternative reason for the appearance of ferritin in lavage fluid is its liberation after cell necrosis. However, the high concentrations of this protein in the bronchoalveolar lavage of unexposed healthy volunteers does not support this speculation.

Correlations between transferrin concentrations in plasma, the bronchial sample, and the alveolar sample approached significance, supporting a possible translocation of this glycoprotein from the plasma to the lower respiratory tract as a source in lavage samples. Relative to levels of transferrin in the plasma of normal healthy volunteers, concentrations of this iron transport protein were low in unconcentrated lavage. These values approximate levels of transferrin in the lavage of normal volunteers previously reported (24, 37, 40-42, 48, 49). When the dilutions by the procedure are accounted for, it appears that transferrin concentrations in the lining fluid of the respiratory tract are approximately equivalent to that in plasma. This further supports the plasma concentrations of transferrin functioning as the source of the glycoprotein in lavage fluid specimens.

To transport the metal associated with the particle into a cell and effect an increased expression of ferritin, transferrin and lactoferrin can be employed. Similar to previous investigation, decrements in transferrin concentrations in lavage fluid after instillation of particles demonstrate that this transport protein can function as an "anti-acute phase protein" (17). As a result of complexation of iron by transferrin and the resultant decrease in the ability of the metal to catalyze radical generation, transferrin has been described as a major antioxidant in the lining fluid of the lower respiratory tract (31). However, decrements in the concentration of this protein in the lavage in response to the instilled particle were contrary to the anticipated host response, which is to protect itself against the oxidative stress associated with the particle. One possible mechanism to explain the diminished concentrations of transferrin in the lavage fluid after exposure to iron-containing particles is that transferrin receptor expression by macrophages can be increased after exposure to iron. This receptor mediates the endocytosis of iron-transferrin, and the transport protein is internalized by the cell. However, if transferrin is to participate in decreasing iron available to catalyze radical generation, oxidative stress to the host, and injury after exposure to the metal, this glycoprotein would also have to have an increase in concentration that corresponded to transferrin receptor changes. This was not observed, and transferrin appears unlikely to participate in the transport and detoxification of metal in the lower respiratory tract after exposure to the particle.

Transferrin receptor is a transmembrane, disulfide-linked glycoprotein dimer of two identical 95-kDa subunits expressed on a variety of cell types that either require iron to fulfill specific functions (20, 23) or facilitate acquisition of the metal for purposes of storage (10). Although erythroblasts exemplify cells with a high requirement for iron (16), alveolar macrophages and hepatocytes are included in those cells with transferrin receptor for purposes of metal sequestration (14, 20). The mechanism of formation of extracellular transferrin receptor is not known, but it involves, at least in part, proteolysis at a site ~100 amino acid residues from the NH2 terminus, beyond the disulfide bond between subunits, and beyond the transmembrane portion of the receptor. The resulting extracellular transferrin receptor is a monomer of ~85 kDa. Concentrations of transferrin receptor in lavage samples of healthy volunteers were greatly decreased relative to that in plasma. The measurement of this receptor in lavage specimens has not been reported previously, and comparisons to levels in other investigations cannot be made. In healthy, unexposed volunteers, there were no correlations between concentrations of transferrin receptor in plasma, bronchial samples, and alveolar samples. This may reflect local factors in the control of expression of this receptor. However, concentrations of transferrin receptor correlated positively with transferrin and negatively with ferritin levels. This suggests a relationship of transferrin receptor with total body iron similar to that of ferritin and transferrin.

After instillation of the iron-containing particle, the concentration of transferrin receptor increased. Although many cell types respond to iron exposure with decrements in transferrin receptor expression, monocytes and macrophages increase the production of this receptor (39). The increment in transferrin receptor concentrations after instillation of the particle likely reflects an effect on alveolar macrophages, since respiratory epithelial cells, similar to fibroblasts (47) and lymphocytes (32), decrease expression of transferrin receptor after in vitro exposure to iron (unpublished observation). After instillation of the iron-containing particle, elevations in transferrin receptor concentration in lavage fluid could contribute to a rapid decrement in diferric transferrin. This could subsequently effect decrements in iron concentrations in the lavage fluid.

Lactoferrin provides an alternative glycoprotein for the transport of iron across a cell membrane. Our concentrations of lactoferrin in lavage fluid are similar to others (11, 41, 42). The concentrations of lactoferrin in lavage fluid are high relative to those in plasma. The lack of correlations between concentrations in plasma, bronchial samples, and alveolar samples suggests that the production of lactoferrin in these three compartments is independent of each other. Contrary to decrements in transferrin, lactoferrin concentrations increased 1 day after instillation of an iron-containing particle. Similar to other investigations demonstrating lactoferrin in lavage fluid, there was a lack of correlation between the neutrophil count and lactoferrin after particle instillation, suggesting sources of lactoferrin other than these cells (41, 44). This glycoprotein is assumed to participate in the transport of catalytically active iron across a cell membrane and assists in its storage as a less-reactive form of the metal within ferritin. At 1 day after instillation, lactoferrin and ferritin concentrations correlated positively, supporting a common function (i.e., the detoxification of chemically reactive iron). Lactoferrin concentrations in the lavage fluid returned to normal by 4 days.

The concentrations of iron measured in lavage fluid are included within a range of normal values (50-190 µg/l) previously reported (34). One day after exposure to an iron-containing particle, concentrations of the metal were not increased but were actually diminished relative to saline instillation. This could be the product of several reactions that include the hypoferric effects of increased concentrations of lactoferrin. Increased concentrations of transferrin receptor react with diferric transferrin to diminish lavage iron. In addition, lactoferrin receptors have been demonstrated for several cells resident in the lower respiratory tract, including macrophages (4, 25, 45). Diferric lactoferrin will react with these receptors, therefore decreasing the concentrations of iron in the respiratory tract. Finally, a neutrophilic influx can be associated with decreased iron concentrations (28).

In conclusion, instillation of an iron-containing particle was associated with a disequilibrium in iron metabolism in the lower respiratory tract. The host responds with an increase in ferritin, transferrin receptor, and lactoferrin, whereas transferrin decreases. These changes are apparent within 1 day of exposure. This coordinated series of reactions by the host effects a decrease in the availability of catalytically reactive iron to likely diminish the consequent oxidative stress. Pertinent cell types were not identified, but alveolar macrophages are likely to participate because they have previously been demonstrated to maintain iron homeostasis (7). The hypoferric retort by the host is not permanent, and normal iron homeostasis returns within 4 days of the iron challenge.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

Address for reprint requests: A. J. Ghio, MD-58D, HSD, Environmental Protection Agency, 104 Mason Farm Rd., Chapel Hill, NC 27599.

Received 2 September 1997; accepted in final form 15 December 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Albert, R. E., H. G. Petrow, A. S. Salam, and J. R. Spiegelman. Fabrication of monodisperse lucite and iron oxide particles with a spinning disk generator. Health Phys. 10: 933-940, 1964.

2.   Balla, J., D. A. Nath, G. Balla, M. B. Juckett, H. S. Jacob, and G. M. Vercellotti. Endothelial cell heme oxygenase and ferritin induction in rat lung by hemoglobin in vivo. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L321-L327, 1995[Abstract/Free Full Text].

3.   Beinert, H., and M. C. Kennedy. Aconitase, a two-faced protein: enzyme and iron regulatory factor. FASEB J. 7: 1442-1449, 1993[Abstract/Free Full Text].

4.   Birgens, H. S., L. O. Kristensen, N. Borregaard, H. Karle, and N. E. Hansen. Lactoferrin-mediated transfer of iron to intracellular ferritin in human monocytes. Eur. J. Haematol. 41: 52-57, 1988[Medline].

5.   Bowes, D., A. E. Clark, and B. Corin. Ultrastructural localization of lactoferrin and glycoprotein in human bronchial glands. Thorax 36: 108-115, 1981[Abstract].

6.   Bridges, K. R., and A. Cudkowicz. Effect of iron chelators on the transferrin receptor in K562 cells. J. Biol. Chem. 259: 12970-12977, 1984[Abstract/Free Full Text].

7.   Byrd, T. F., and M. A. Horwitz. Regulation of transferrin receptor expression and ferritin content in human mononuclear phagocytes. Coordinate upregulation by iron transferrin and downregulation by interferon gamma. J. Clin. Invest. 91: 969-976, 1993[Medline].

8.   Colton, T. Statistics In Medicine. Boston, MA: Little, Brown, 1974.

9.   Cozzi, A., P. Santambrogio, S. Levi, and P. Arosio. Iron detoxifying activity of ferritin. Effects of H and L human apoferritins on lipid peroxidation in vitro. FEBS Lett. 277: 119-122, 1990[Medline].

10.   Deiss, A. Iron metabolism in reticulendothelial cells. Semin. Hematol. 20: 81-90, 1983[Medline].

11.   Eklund, A., O. Eriksson, L. Hakansson, K. Larsson, K. Ohlsson, P. Venge, H. Bergstrand, A. Bjornson, R. Brattsand, C. Glennow, M. Linden, and E. Wieslander. Oral N-acetylcysteine reduces selected humoral markers of inflammatory cell activity in BAL fluid from healthy smokers: correlation to effects on cellular variables. Eur. Respir. J. 1: 832-838, 1988[Abstract].

12.   Ghio, A. J., J. D. Carter, J. M. Samet, J. Quay, I. A. Wortman, J. H. Richards, T. P. Kennedy, and R. B. Devlin. Post-transcriptional regulation of ferritin expression after in vitro exposures of human alveolar macrophages to silica is iron-dependent. Am. J. Respir. Cell Mol. Biol. 17: 533-540, 1997[Abstract/Free Full Text].

14.   Hamilton, T. A., J. E. Weiel, and D. O. Adams. Expression of the transferrin receptor in murine peritoneal macrophages is modulated in the different stages of activation. J. Immunol. 132: 2285-2290, 1984[Abstract/Free Full Text].

15.   Hass, F. J., P. S. Lee, and R. V. Lourenco. Tagging of iron oxide particles with 99mTc for use in the study of deposition and clearance of inhaled particles. J. Nucl. Med. 17: 122-125, 1976[Abstract].

16.   Iturralde, M., J. K. Vass, R. Oria, and J. H. Brock. Effect of iron and retinoic acid on the control of transferrin receptor and ferritin in the human promonocytic cell line U937. Biochim. Biophys. Acta 1133: 241-246, 1992[Medline].

17.   Krsek-Staples, J. A., R. R. Kew, and R. O. Webster. Ceruloplasmin and transferrin levels are altered in serum and bronchoalveolar lavage fluid of patients with the adult respiratory distress syndrome. Am. Rev. Respir. Dis. 145: 1009-1015, 1992[Medline].

18.   Kuhn, L. C., and M. W. Hentze. Coordination of cellular iron metabolism by post-transcriptional gene regulation. J. Inorg. Biochem. 47: 183-195, 1992[Medline].

19.   Lash, A., and A. Saleem. Iron metabolism and its regulation. Ann. Clin. Lab. Sci. 25: 20-30, 1995[Abstract].

20.   Laskey, J., I. Webb, H. M. Schulman, and P. Ponka. Evidence that transferrin supports cell proliferation by supplying iron for DNA synthesis. Exp. Cell Res. 176: 87-95, 1988[Medline].

22.   Leibold, E. A., and B. Guo. Iron-dependent regulation of ferritin and transferrin receptor expression by the iron-responsive element binding protein. Annu. Rev. Nutr. 12: 345-368, 1992[Medline].

23.   Lu, J., C. Kaur, and E.-A. Ling. Expression and upregulation of transferrin receptors and iron uptake in the epiplexus cells of different aged rats injected with lipopolysaccharide and interferon-gamma. J. Anat. 187: 603-611, 1995[Medline].

24.   Lykens, M. G., W. B. Davis, and E. R. Pacht. Antioxidant activity of bronchoalveolar lavage fluid in the adult respiratory distress syndrome. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L169-L175, 1992[Abstract/Free Full Text].

25.   Markowetz, B., J. L. Van Snick, and P. L. Masson. Binding and ingestion of human lactoferrin by mouse alveolar macrophages. Thorax 34: 209-212, 1979[Abstract].

26.   McGowan, S. E., and S. A. Henley. Iron and ferritin contents and distribution in human alveolar macrophages. J. Lab. Clin. Med. 111: 611-617, 1988[Medline].

27.   Mitchell, J. P. The production of aerosols from aqueous solutions using the spinning top generator. J. Aerosol Sci. 15: 33-45, 1984.

28.   Molloy, A. L., and C. C. Winterbourn. Release of iron from phagocytosed Escherichia coli and uptake by neutrophil lactoferrin. Blood 75: 984-989, 1990[Abstract].

29.   Neidle, M., and J. Barab. Studies in dialysis II. The hot dialysis of the chlorides of ferric iron, chromium, and aluminum, and the rapid preparation of their colloidal hydrous oxides (Abstract). J. Am. Chem. Soc. 39: 79, 1917.

30.   Olakanmi, O., S. E. McGowan, M. B. Hayek, and B. E. Britigan. Iron sequestration by macrophages decreases the potential for extracellular hydroxyl radical formation. J. Clin. Invest. 91: 889-899, 1993[Medline].

31.   Pacht, E. R., and W. B. Davis. Role of transferrin and ceruloplasmin in antioxidant activity of lung epithelial lining fluid. J. Appl. Physiol. 64: 2092-2099, 1988[Abstract/Free Full Text].

32.   Pelosi, E., U. Testa, F. Louache, P. Thomopoulos, G. Salvo, P. Samoggia, and C. Peschle. Expression of transferrin receptors in phytohemagglutinin-stimulated human T-lymphocytes. Evidence for a three-step model. J. Biol. Chem. 261: 3036-3042, 1986[Abstract/Free Full Text].

33.   Rao, K. K., D. Schapiro, E. Mattia, K. Bridges, and R. D. Klausner. Effects of alterations in cellular iron on biosynthesis of the transferrin receptor in K562 cells. Mol. Cell. Biol. 5: 595-600, 1985[Medline].

34.   Romeo, L., G. Maranelli, F. Malesani, I. Tommasi, A. Cazzadori, and M. S. Graziani. Tentative reference values for some elements in broncho-alveolar lavage fluid. Sci. Total Environ. 120: 103-110, 1992[Medline].

35.   Schroeder, W. H., M. Dobson, D. M. Kane, and N. D. Johnson. Toxic trace elements associated with airborne particulate matter: a review. J. Air Pollution Control Assoc. 37: 1287-1285, 1987.

36.   Sibille, J.-C., H. Kondo, and P. Aisen. Interactions between isolated hepatocytes and Kupffer cells in iron metabolism: a possible role for ferritin as an iron carrier protein. Hepatology 8: 296-301, 1988[Medline].

37.   Stites, S. W., M. E. Nelson, and L. J. Wesselius. Transferrin concentrations in serum and lower respiratory tract fluid of mechanically ventilated patients with COPD or ARDS. Chest 107: 1681-1685, 1995[Abstract/Free Full Text].

38.   Stohs, S. J., and D. Bagchi. Oxidative mechanisms in the toxicity of metal ions. Free Radic. Biol. Med. 18: 321-336, 1995[Medline].

39.   Testa, U., M. Petrini, M. T. Quaranta, E. Pelosi-Testa, G. Mastroberardino, A. Camagna, G. Boccoli, M. Sargiacomo, G. Isacchi, A. Cozzi, P. Arosio, and C. Peschle. Iron up-modulates the expression of transferrin receptors during monocyte-macrophage maturation. J. Biol. Chem. 264: 13181-13187, 1989[Abstract/Free Full Text].

40.   Thompson, A. B., T. Bohling, A. Heires, J. Linder, and S. I. Rennard. Lower respiratory tract iron burden is increased in association with cigarette smoking. J. Lab. Clin. Med. 117: 493-499, 1991[Medline].

41.   Thompson, A. B., T. Bohling, F. Payvandi, and S. I. Rennard. Lower respiratory tract lactoferrin and lysozyme arise primarily in the airways and are elevated in association with chronic bronchitis. J. Lab. Clin. Med. 115: 148-158, 1990[Medline].

42.   Thompson, A. B., M. B. Mueller, A. J. Heires, T. L. Bohling, D. Daughton, S. W. Yancey, R. S. Sykes, and S. I. Rennard. Aerosolized beclomethasone in chronic bronchitis. Improved pulmonary function and diminished airway inflammation. Am. Rev. Respir. Dis. 146: 389-395, 1992[Medline].

43.   Thorstensen, K., and I. Romslo. The role of transferrin in the mechanism of cellular iron uptake. Biochem. J. 271: 1-9, 1990[Medline].

44.   Van De Graaf, E. A., T. A. Out, A. Kobesen, and H. M. Jansen. Lactoferrin and secretory IgA in the bronchoalveolar lavage fluid from patients with a stable asthma. Lung 169: 275-283, 1991[Medline].

45.   Van Snick, J. L., B. Markowetz, and P. L. Masson. The ingestion and digestion of human lactoferrin by mouse peritoneal macrophages and the transfer of its iron into ferritin. J. Exp. Med. 146: 817-827, 1977[Abstract].

46.   Ward, J. H., I. Jordan, J. P. Kushner, and J. Kaplan. Heme regulation of HeLa cell transferrin receptor number. J. Biol. Chem. 259: 13235-13240, 1984[Abstract/Free Full Text].

47.   Ward, J. H, J. P. Kushner, and J. Kaplan. Transferrin receptors of human fibroblasts. Analysis of receptor properties and regulation. Biochem. J. 28: 19-26, 1982.

48.   Wesselius, L. J., C. H. Flowers, and B. S. Skikne. Alveolar macrophage content of isoferritins and transferrin. Comparison of nonsmokers and smokers with and without chronic airflow obstruction. Am. Rev. Respir. Dis. 145: 311-316, 1992[Medline].

49.   Wesselius, L. J., M. E. Nelson, and B. S. Skikne. Increased release of ferritin and iron by iron-loaded alveolar macrophages in cigarette smokers. Am. J. Respir. Crit. Care Med. 150: 690-695, 1994[Abstract].


AJP Lung Cell Mol Physiol 274(3):L396-L403
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