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
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ABSTRACT |
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
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INTRODUCTION |
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.
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MATERIALS AND METHODS |
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.
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RESULTS |
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 (*).
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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).
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.
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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.
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DISCUSSION |
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.
 |
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