Induction of ferritin and heme oxygenase-1 by endotoxin in the
lung
M. S.
Carraway1,
A. J.
Ghio2,
J. L.
Taylor1, and
C. A.
Piantadosi1
1 Department of Medicine, Duke
University Medical Center, Durham 27710; and
2 United States Environmental
Protection Agency, Chapel Hill, North Carolina 27599
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ABSTRACT |
Heme oxygenase (HO)-1 expression is increased by
forms of oxidative stress that also induce ferritin. Even though this
could result from release of iron by heme degradation, we hypothesized that ferritin expression in the lung after endotoxin
[lipopolysaccharide (LPS)] would occur independently of
HO-1 because iron sequestration is an important response to infection.
We tested this hypothesis by instilling saline or LPS (1 mg) into lungs
of rats and measuring ferritin expression, HO-1 expression and
activity, and HO-1 and ferritin mRNAs at different times. Lungs were
also inflation fixed for immunohistochemistry for HO-1 and ferritin.
Studies were performed with and without the HO inhibitor tin
protoporphyrin. Ferritin and HO-1 labeling were minimal (macrophages
only) in control lungs. By 4 h after LPS instillation, ferritin
staining was present in bronchial epithelium and macrophages, became
diffuse at 16 h, and was nearly gone by 48-72 h. HO-1 was
detectable in macrophages 4 and 16 h after LPS instillation, increased
in macrophages and bronchial epithelium at 24 h, and diffusely
increased in bronchial epithelium and the alveolar region at 48-72
h. Lung ferritin content increased significantly by 4 h and peaked at
16 h before declining. HO-1 protein was present by Western blot in
control lung, stable at 4 h, and increased by 24 h after LPS
instillation, whereas HO enzyme activity had increased by 4 h after LPS
instillation. After complete inhibition of HO enzyme activity with tin
protoporphyrin, ferritin increased threefold at 4 h and sixfold at 24 h
after LPS instillation. HO-1 mRNA increased by 4 h and was sustained at
24 h, whereas ferritin mRNA did not change after LPS instillation. These results indicate that intratracheal LPS rapidly induces ferritin
protein in the lung independently of its mRNA synthesis or HO enzyme
activity. LPS induces HO-1 mRNA, which is followed by increased
expression of protein.
lipopolysaccharide; oxidants; acute lung injury
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INTRODUCTION |
HEME OXYGENASE (HO) catalyzes the degradation of heme
into biliverdin, an intermediate in the production of bilirubin (22, 23). Iron and carbon monoxide are also products of this reaction. Three
genetic isoforms of HO, designated HO-1, HO-2, and, more recently,
HO-3, have been characterized (16, 17). HO-1 is present constitutively
in small amounts in many organs including the lung, liver, heart, and
spleen, and it is inducible by many stimuli including heme,
hyperthermia, heavy metals, lipopolysaccharide (LPS), hyperoxia, and
hypoxia (7). Oxidant induction of HO-1 is often accompanied by
increased expression of other stress proteins including ferritin (4,
21, 25). Ferritin is regulated posttranscriptionally by an
iron-responsive element (IRE) and is rapidly induced in response to
iron and heme (1).
Because many oxidants increase HO-1 expression, its induction has been
proposed to be a part of the integrated response to oxidative stress
(7). Induction of HO-1 before injury protects against oxidant-mediated
cytotoxicity in cell culture (14) and prolongs survival after LPS
instillation in rats (20). In both cases, this protection is reversed
by treatment with competitive inhibitors of the enzyme (19, 20).
Because LPS and the early-response cytokines, e.g., interleukin-1,
tumor necrosis factor-
, and interferon-
, influence the
intracellular uptake of iron (8, 10, 12), ferritin expression after LPS
instillation also may be an early antioxidant response. Induction of
ferritin in endothelial cells protects against oxidant-mediated
cytotoxicity (2). Several studies (4, 21, 25) have found simultaneous
induction of ferritin and HO-1 during in vivo injuries such as
glycerol-induced renal failure and hyperoxia- and hemoglobin-induced
lung injury in rats.
The antioxidant properties of ferritin are likely mediated by
sequestration and storage of iron, thus preventing ferryl or hydroxyl
radical generation (13). The specific mechanisms for the cytoprotection
by HO-1 have remained uncertain. Biliverdin and bilirubin have been
shown to have antioxidant properties, and their production has been
suggested as a mediator of the protective effects of HO-1 (7). On the
basis of in vivo studies in cultured fibroblasts, another mechanism of
cytoprotection postulated for HO-1 is induction of ferritin by iron
released from heme degradation (9, 24). In rat lung endothelium,
however, methemoglobin induces ferritin even after inhibition of HO-1
activity (3). The interactions between ferritin and HO-1 during
inflammation and their relative contributions to protection against
tissue injury have not yet been defined in vivo.
Intravenous LPS stimulates HO-1 mRNA, protein, and enzyme activity in
the heart, liver, and lungs of rats (20). In the lung, HO-1 is
upregulated diffusely in alveolar and bronchial epithelium and
inflammatory cells after intravenous LPS (5). Intravenous LPS also
increases stainable iron and induces ferritin expression in the spleen
(12) and liver (8). In rats, instillation of LPS into the trachea
produces a gradually progressive lung injury characterized by increased
permeability and influx of inflammatory cells (11, 18). We hypothesized
that lung injury in rats caused by tracheal instillation of LPS would
rapidly induce ferritin independently of HO-1 expression and enzyme
activity. If true, ferritin expression would remain intact after
inhibition of HO-1 activity. We tested this hypothesis by measuring
ferritin and HO-1 protein and mRNA as a function of time after
instilling LPS into the lungs of rats. These experiments were performed
with and without the competitive inhibitor of HO, tin protoporphyrin (SnPP). We specifically sought to determine whether HO-1 activity was
required for ferritin expression to increase in response to LPS.
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METHODS |
Materials. Unless otherwise specified,
all chemicals were obtained from Sigma (St. Louis, MO).
Experimental and animal protocols.
Male Sprague-Dawley (Charles River Laboratories, Research Triangle
Park, NC) rats weighing ~250 g were used for the experiments. All
animals were handled under protocols approved by the Duke University
(Durham, NC) Animal Care and Use Committee in accordance with National
Institutes of Health guidelines for the use of laboratory animals. The
rats were anesthetized with halothane (Ayerst, Philadelphia, PA), and the trachea was cannulated transorally with an 18-gauge catheter. LPS
(Escherichia coli 0111:B4, 1 mg in 0.5 ml of 0.9% NaCl) or NaCl solution alone was rapidly instilled into the
trachea. For control rats, NaCl alone was administered intratracheally.
To inhibit HO-1, SnPP [100 µM/kg (75 mg/kg); Porphyrin
Products, Logan, UT] was given subcutaneously 4 h before LPS
instillation. We studied two experimental groups, LPS and LPS+SnPP, and
two control groups, saline and saline+SnPP. Lungs were taken from animals killed at 0, 4, 16, 24, 48, and 72 h after intratracheal LPS
instillation for immunohistochemistry. At 0, 4, 16, 24, 48, and 96 h
after LPS instillation, lungs from four additional rats in each group
were obtained for quantitation of ferritin protein. At the early time
points (0, 4, and 24 h), lungs from four rats of each group were used
to quantitate lung HO-1 enzyme activity and protein and to measure HO-1
and ferritin mRNAs. From four control animals, the spleens were also
removed as a positive control for HO-1 and the livers were used to
prepare biliverdin reductase for the HO activity assay.
To obtain lungs for immunohistochemistry, the rats were deeply
anesthetized with intraperitoneal ketamine (5.0 mg/kg) and valium (0.5 mg/kg). The trachea was isolated and cannulated with an 18-gauge
catheter. The lungs were flushed with 50 ml of 0.9% NaCl through the
right ventricle while the aorta was transected. The lungs were
inflation fixed en bloc with 4% paraformaldehyde at 20 cmH2O fixative pressure. To obtain
lungs for the remainder of the analyses, the rats were anesthetized
with ketamine and valium (see above). The abdominal and chest cavities
were opened, the lungs were flushed with 0.9% NaCl through the right
ventricle, and the aorta was transected. The lungs were removed, frozen
immediately in liquid nitrogen, and stored at
80°C until
used a few days later.
Immunohistochemistry. Inflation-fixed
lungs were paraffin embedded and cut into 10-µm sections. Before the
tissue sections were labeled, they were deparaffinized in xylene and
then rehydrated in graded alcohol solutions. The sections were blocked
in a solution of 5% nonfat dry milk, 1% BSA, 5% goat serum in 0.01 M
PBS, and 0.1% Triton X-100 before incubation overnight at 4°C with
antibodies to ferritin (Dako, Carpenteria, CA) or HO-1 (Stress-Gen,
Vancouver, BC) in 1% milk and 1% BSA in 0.01 M PBS and 0.1% Triton
X-100 at dilutions of 1:100 and 1:500, respectively. The sections were washed three times with PBS with 0.1% Triton X-100 (5 min each) and
incubated with the secondary antibody, biotinylated goat anti-rabbit IgG (Jackson Laboratories), at a dilution of 1:1,000 in 1% milk in
0.01 M PBS and 0.1% Triton X-100 at room temperature for 1 h. The
signal was detected with peroxidase-conjugated avidin and diaminobenzidine. The slides were counterstained with hematoxylin. For
negative controls, sections were processed as above except that the
primary incubation was performed with nonimmune rabbit serum (Jackson
Laboratories) instead of the primary antibodies.
Western blot. Samples of lungs and
spleen were homogenized on ice in cold lysis buffer [150 mM NaCl,
50 mM Tris, pH 7.6, 1% SDS (Bio-Rad), 3% Nonidet P-40, 5 mM EDTA, 1 mM MgCl2, 2 mM
1,3-dichloroisocoumarin, 2 mM 1,10-phenanthroline, and 0.5 mM
trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64)]. The homogenate was centrifuged at 10,000 g for 10 min. The supernatant was
decanted, and an aliquot was stored at
20°C for measurement
of protein concentration. The remaining supernatant was mixed with an
equal volume of double-strength Laemmli sample buffer [250 mM
Tris · HCl (Bio-Rad), pH 6.8, 4% SDS, 10% glycerol, 0.006% bromphenol blue, and 2%
-mercaptoethanol], divided
into aliquots, and stored at
80°C.
Electrophoresis was performed on 12% polyacrylamide gels under
reducing conditions with a minigel system (Hoefer Scientific Instruments, San Francisco, CA). All lanes were loaded with 15 µg of
protein, and electrophoresis was performed over 1.5 h under a constant
current of 30 mA. The proteins were electrotransferred on a TE series
Transphor unit at 100 V (Hoefer Scientific) to a polyvinylidene
fluoride membrane (Millipore, Amersham Life Sciences, Cleveland, OH)
and blocked overnight at 4°C in Tris-buffered saline (TBS) with 1%
polyoxyethylenesorbitan monolaureate (Tween 20; TBS-T) containing 5%
nonfat dry milk. The following day, the membranes were washed six times
over 30 min in TBS-T at room temperature. Western blots were performed
with rabbit polyclonal antibodies against rat HO-1 (Stress-Gen).
Incubation with the primary antibody was performed for 1 h at room
temperature in TBS-T with 5% milk at a dilution of 1:1,000. After
multiple washes in TBS-T, the membranes were incubated with horseradish
peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Jackson
Laboratories) at a 1:15,000 dilution in TBS-T with 5% milk. The
membranes were then washed six times in TBS-T, and the signal was
detected on Biomax film (Eastman Kodak, Rochester, NY) with the
enhanced chemiluminescence kit (Amersham, Arlington Heights, IL).
HO-1 activity assay. Lungs were
homogenized on ice in one volume of 100 mM phosphate buffer with 2 mM
MgCl2 (HO activity buffer). The
homogenates were sonicated and centrifuged for 15 min at 18,800 g. The
supernatant was used to quantitate HO-1 activity. The reaction mixture
consisted of 200 µl of sample homogenate, 100 µl of liver cytosol
(source of biliverdin reductase), 20 mM hemin, 0.8 mM NADPH, 2 mM
glucose 6-phosphate, and 0.0016 U/µl of glucose-6-phosphate dehydrogenase, and the reaction was performed in duplicate in the dark.
The samples were incubated in a 37°C water bath for 1 h. To extract
bilirubin, an equal volume of chloroform was added to each tube, and
the tubes were mixed thoroughly and centrifuged for 5 min at 15,000 g. The samples were scanned on a
spectrophotometer from 464 to 530 nm. Bilirubin concentration was
calculated based on the change in optical density at 464 nm from that at 530 nm, with an extinction coefficient of 40 mM/cm. The
values are expressed as picomoles of bilirubin formed per hour per
milligram of protein. The spleen from a control animal was processed
identically to the lung samples to provide a positive control; an
NADPH-free reaction mixture provided a baseline against which the
measured concentrations were determined.
Slot blots for ferritin. Lung tissue
was homogenized (1.0 g/5 ml) in lysis buffer. Fifteen micrograms of
protein in the homogenate were vacuum slot blotted onto 0.45-mm
nitrocellulose (Schleicher and Schuell, Keene, NH) in saline buffer
containing 100 mM Tris, pH 8.0. The blot was air-dried, blocked with
5% milk for 30 min, and incubated with a 1:2,000 dilution of rabbit
anti-human ferritin antibody (Dako) in 5% dry milk for 2 h. The blot
was washed in PBS-0.05% Tween and incubated with horseradish
peroxidase-conjugated goat anti-rabbit IgG in 5% dry milk for 1 h.
Signals were obtained in a linear portion of the detection curve on
film with enhanced chemiluminescence reagents. Band optical densities
were quantified with a Millipore digital bioimaging system (Bedford,
MA).
RT-PCR. Lung tissue was homogenized (1 g/5 ml) with 4 M guanidine thiocyanate (Boehringer Mannheim,
Indianapolis, IN), 50 mM sodium citrate, 0.5% sarkosyl, and 0.01 M
dithiothreitol. RNA was pelleted by ultracentrifugation through cesium
chloride (Boehringer Mannheim) and 0.1 M EDTA. One hundred nanograms of
total RNA were reverse transcribed (M-MLV Reverse Transcriptase, Life
Technologies), and the resultant cDNA was amplified for 24, 25, and 24 cycles for glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), ferritin, and HO, respectively, in separate reactions with
gene-specific primers. Oligonucleotide sequences were synthesized with
an Applied Biosystems 391 DNA synthesizer (Foster City, CA) based on
sequences published in the GenBank DNA database. The following sense
and antisense sequences, respectively, were employed: GAPDH,
5'-CCATGGAGAAGGCTGGGG-3' and
5'-CAAATTGTCATGGATGACC-3'; ferritin,:
5'-TCGCAGGTGCGCCAGAACTA-3' and
5'AAGGAAGATTCGGCCACCTC-3'; and HO, 5-'ATTGGAGGCTGGAGCTATTCTG-3' and
5-'CCTTCGGTGCAGCTCCTCAG-5'. Amplification
products were separated on 2% denaturing agarose gel, stained with
ethidium bromide, and photographed under ultraviolet light. The
resulting negative (type 55 film, Polaroid, Cambridge, MA) was
quantitated with a BioImage densitometer (BioImage, Ann Arbor, MI). For
each experimental condition, the integrated optical densities of the
ferritin and HO DNA bands were divided by that of the GAPDH DNA band
(as a housekeeping gene) to correct for variation in the amount of
amplifiable cDNA in each sample.
Biochemical measurements. Protein
concentrations were measured in all samples with the bicinchoninic acid
assay with BSA as a standard (15). Iron concentrations in LPS and SnPP
were measured to exclude iron contamination as a source of error in the
experiments. LPS and SnPP were solubilized in 1 N HCl (1.0 mg/1.0 ml).
After centrifugation, iron concentrations were determined by
inductively coupled plasma emission spectroscopy (model P40,
Perkin-Elmer, Norwalk, CT).
Statistical analysis. Group data are
expressed as means ± SD. Statistical comparisons were made by
two-way analysis of variance, and post hoc comparisons were made with
Fisher's exact test with commercially available software (Statview
4.01, Calabasas, CA) on a Macintosh computer (7600/120).
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RESULTS |
Pathology and immunohistochemistry. At
the dose we administered, intratracheal LPS resulted in the development
of an inflammatory lung injury over 72 h. This injury was characterized
early by increased cellularity in the alveolar capillary region
(16-24 h), followed by an accumulation in the alveolar spaces of
erythrocytes and inflammatory cells including polymorphonuclear and
mononuclear leukocytes. These changes are consistent with the injury
reported in earlier studies (11, 18) of intratracheal LPS in rats.
Figure 1 compares representative
immunohistochemistry for ferritin
(A-D)
with HO-1
(E-H)
in the bronchial regions of the rat lung 0, 4, 16, and 24 h after LPS
exposure. In the control lung, no airway staining of ferritin was
detected, and scattered staining for ferritin was found in macrophages
(Fig. 1A). Four hours after LPS
instillation (Fig. 1B), ferritin
staining increased in the bronchial epithelium and macrophages. At 16 h, similar staining for ferritin was present in the bronchial
epithelium, macrophages, and alveolar region (Fig.
1C). By 24 h, ferritin staining was no longer present in the bronchial epithelium but remained detectable in the macrophages and alveolar region (Fig.
1D). Negative controls in the lung
16 h after LPS instillation (rabbit serum in place of primary antibody)
showed minimal nonspecific staining (data not shown). The contrasting
time course of HO-1 induction after intratracheal LPS is shown in Fig.
1,
E-H.
The control lung [time (t) = 0] showed minimal light staining of macrophages for HO-1, and
HO-1 was not present in the airway (Fig.
1E). Four and sixteen hours after
LPS instillation, the lung was progressively more cellular, and
staining for HO-1 remained limited primarily to the inflammatory cells
(Fig. 1, F and
G). At 16 h, some staining was also
present in bronchial epithelium. By 24 h, there was intense staining of
the bronchial epithelium, macrophages, and alveolar region for HO-1
(Fig. 1H). The negative control lung
at 72 h after LPS instillation showed minimal nonspecific staining
(data not shown).

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Fig. 1.
Immunohistochemistry of bronchial region for ferritin
(A-D)
and heme oxygenase (HO)-1
(E-H)
after intratracheal lipopolysaccharide (LPS) instillation.
A: ferritin was not found in bronchial
epithelium in control lungs [time
(t) = 0]. It was present only
in macrophages. B: 4 h after LPS
instillation, ferritin staining was present in bronchial epithelium.
C: 16 h after LPS instillation,
ferritin was still present in bronchial epithelium.
D: 24 h after LPS instillation,
ferritin staining was almost absent in bronchial epithelium. Influx of
mononuclear inflammatory cells, however, stained positively for
ferritin. E: control lung
(t = 0).
F: 4 h after LPS instillation. HO-1
staining was not present in airway epithelium.
G: at 16 h, airway staining for HO-1
was barely detectable, whereas macrophages stained intensely.
H: at 24 h, definite staining for HO-1
was present in airway. Macrophages stained intensely for HO-1. Original
magnification, ×220.
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Figure 2,
A-C,
shows more detailed immunohistochemistry of the alveolar region of the
lung for ferritin 0, 4, and 24 h, respectively, after LPS exposure. In
the control lung (t = 0), scattered
staining for ferritin was found in macrophages (Fig.
2A). Four hours after LPS
instillation (Fig. 2B), there were
increased inflammatory cells that stained for ferritin, and there was a
modest increase in ferritin labeling in the alveolar region. At 16 h,
similar staining was present in the macrophages and alveolar region
(data not shown). By 24 h, ferritin staining was strong in both the cells and the alveolar region (Fig.
2C).

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Fig. 2.
Immunohistochemistry of alveolar region for ferritin after
intratracheal LPS instillation. A:
ferritin was present mostly in macrophages (arrowhead) in control lungs
(t = 0).
B: 4 h after LPS instillation,
ferritin stained prominently in macrophages and was present in alveolar
region (arrowhead). C: 24 h after LPS
instillation, there were increased mononuclear inflammatory cells that
stained intensely for ferritin, and staining persisted in alveolar
region. Original magnification, ×440.
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Figure 3,
A-C,
shows representative immunohistochemistry for HO-1 0, 4, and 24 h,
respectively, after LPS exposure. The control lung
(t = 0) showed minimal light staining
of macrophages for HO-1 (Fig. 3A).
Four hours after LPS instillation, macrophages progressively increased,
and these cells stained for HO-1 (Fig. 3B). At 24 h, there was increased
staining of the alveolar capillary region for HO-1 and more
inflammatory cells, including macrophages, that labeled for HO-1 (Fig.
3C).

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Fig. 3.
Immunohistochemistry of alveolar region for HO-1 after intratracheal
LPS instillation. A: HO-1 was barely
detectable in macrophages in control lung
(t = 0).
B: 4 h after LPS instillation,
macrophages stained lightly for HO-1 (arrowhead).
C: at 24 h, inflammatory cells were
increased and macrophages stained intensely for HO-1. HO-1 staining was
prominent in alveolar region (arrowhead). Original magnification,
×440.
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By 48 h, HO-1 was strongly present in bronchial epithelium, alveolar
regions, and inflammatory cells. Ferritin was not present in alveolar
regions or bronchial epithelium and was only visible in macrophages,
similar to control lungs (data not shown). Figure 4 shows representative immunohistochemistry
sections for HO-1 and ferritin 72 h after LPS instillation. Similar to
48 h, the lung showed diffuse infiltration of inflammatory cells and
thickened interstitium. Ferritin staining was present only in
macrophages, similar to control lungs (Fig.
4A). In contrast, at 72 h, heavy HO-1 staining was present diffusely in the bronchial epithelium, alveolar wall regions, and inflammatory cells (Fig.
4B).

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Fig. 4.
Immunohistochemistry for ferritin
(A) and HO-1
(B) 72 h after intratracheal LPS. By
72 h after instillation of LPS, lung showed diffuse infiltration of
inflammatory cells. Ferritin staining was limited to macrophages,
similar to control rat lungs (t = 0;
see Fig. 1A). At 72 h after
intratracheal LPS instillation, HO-1 was diffusely increased in
alveolar wall region and inflammatory cells. Original magnification,
×220.
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Lung HO-1 and ferritin content. The
immunohistochemistry findings indicated that, after LPS instillation,
ferritin expression increased before that of HO-1. To quantitate these
differences, HO-1 and ferritin were measured in the lung at different
times after LPS instillation. Figure 5
shows the results of quantitation of ferritin protein from immunoblots
of lung samples taken 4, 16, 24, 48, and 96 h after intratracheal LPS
instillation compared with the control
(t = 0) values. At 4 h, ferritin
content in the lung was increased 2.5-fold above the control value. By
16 h, ferritin had peaked eightfold above control value. At 24 and 48 h, ferritin had fallen to four times the control value and by 96 h had
returned to baseline.

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Fig. 5.
Ferritin expression in lung after LPS instillation. Values were
obtained by densitometry of slot blots, with normalization of post-LPS
values to control values (t = 0). In
rat lung, ferritin content was increased by 4 h after intratracheal
LPS. Ferritin content peaked at 16 h and remained increased over
control value at 24 and 48 h. By 96 h, ferritin values were similar to
control value.
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Because ferritin induction was highest in the first 24 h after LPS
instillation, we focused on these first 24 h for quantitative HO-1
protein and activity measurements and experiments with inhibition of
HO-1 enzyme activity. Figure 6 is a Western
blot of lung samples with antibody to HO-1. The HO-1 protein is
identified by Western blot as a strong band at 32 kDa in the spleen,
which is rich in HO-1. HO-1 was present in the control lungs and was
not increased 4 h after intratracheal LPS. By 24 h after intratracheal
LPS instillation, HO-1 protein increased substantially.

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Fig. 6.
HO-1 expression in lung after LPS instillation. Western blot showed a
small amount of HO-1 present in rat lungs at baseline (control;
t = 0). Four hours after intratracheal
LPS instillation, there was no change from baseline. After 24 h, HO-1
protein increased in lung. After pretreatment with tin protoporphyrin
(SnPP), HO-1 increased 4 h after LPS exposure and increased further at
24 h. HO-1 also increased at 24 h after SnPP alone. Rat spleen was a
positive control for HO-1. All lanes were loaded with 15 µg of
protein.
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Figure 7 shows HO-1 activity
in lung homogenates as measured by bilirubin production and expressed
as picomoles per milligram of protein per hour. The control lung
generated ~5 pmol bilirubin · mg
protein
1 · h
1.
Four hours after intratracheal LPS, HO activity was 25 pmol · mg
protein
1 · h
1,
which was significantly different from the control value
(P
0.05). After 24 h, there was a
further increase in HO-1 activity, to 35 pmol · mg
protein
1 · h
1,
which was significantly different from the control samples
(P
0.05) but not different from the
samples from the 4-h group.

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Fig. 7.
HO enzyme activity in rat lung. HO enzyme activity was present at
baseline in lung samples and increased 5-fold at 4 h after
intratracheal LPS instillation (P 0.05). Further increases in enzyme activity were detected after 24 h
(P 0.08 for LPS at 4 vs. 24 h).
After pretreatment with SnPP, HO activity did not increase after
intratracheal LPS at 4 or 24 h.
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Effect of SnPP on ferritin and HO-1.
Figure 7 also shows the effect on HO-1 activity of subcutaneous
administration of SnPP 4 h before intratracheal LPS. HO-1 activity in
the LPS-treated rats at 4 and 24 h was totally inhibited, indicating a
long-lasting effect on enzyme activity by the competitive inhibitor.
Pretreatment with SnPP, however, did not prevent induction of HO-1
protein by LPS as shown by the Western blot in Fig. 6. There was more total HO-1 per microgram of total protein present by Western blot after
4 and 24 h in the SnPP+LPS-treated rats than in the LPS-alone group at
both 4 and 24 h.
Figure 8 summarizes the effect of LPS and
LPS+SnPP on the ferritin content of the lung at 4 and 24 h compared
with the ferritin content of control lungs. Even after total blockade
of HO-1 activity by SnPP pretreatment (see Fig. 7), the ferritin
content in SnPP+LPS lungs was increased threefold over the control
value 4 h after intratracheal LPS. The SnPP-treated animals also showed
a sixfold increase in ferritin over the control value 24 h after
intratracheal LPS. At both time points, the increase in ferritin was
slightly but not significantly higher than in the LPS-treated lungs.
After SnPP+LPS, ferritin was present at 4 and 24 h in amounts similar to those found after LPS alone. After SnPP+saline, ferritin staining was also present but to a lesser degree.

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Fig. 8.
Ferritin content of rat lungs after LPS and LPS+SnPP instillation. By
slot blot, ferritin content increased 2.5-fold over control value 4 h
after LPS instillation. Four hours after LPS+SnPP instillation,
ferritin content increased 3-fold over control value. At 24 h, ferritin
increased 4-fold after LPS instillation and 6-fold after LPS+SnPP
instillation.
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To rule out iron contamination of LPS and saline vehicle as the
mechanism by which ferritin and HO-1 protein expression increased, the
iron content of the LPS and SnPP solutions was measured. The iron
concentration in LPS was 0.328 ± 0.035 part/million and that in SnPP was 0.165 ± 0.031 part/million.
RT-PCR for ferritin and HO-1 mRNAs.
Ferritin and HO-1 mRNAs were measured and normalized to GAPDH mRNA
(Figs. 9 and
10). Figure 9 shows that ferritin mRNA
did not change at either 4 or 24 h after LPS instillation compared with
the control value, which is consistent with posttranscriptional
regulation of the expression of this iron storage protein. After SnPP,
there was also no change in the amount of ferritin mRNA present in the
lung.

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Fig. 9.
Ferritin mRNA expression after LPS instillation. Ferritin mRNA,
measured by RT-PCR, did not change significantly at either 4 or 24 h
after LPS instillation compared with control media. After LPS+SnPP
instillation, ferritin mRNA also did not change significantly from the
control value at 4 or 24 h. Ferritin mRNA content was normalized to
glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
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Fig. 10.
HO-1 mRNA expression after LPS instillation. By RT-PCR, HO-1 mRNA
increased by 4 h after LPS instillation compared with control media.
HO-1 mRNA content was normalized to GAPDH. At 24 h after LPS exposure,
HO-1 mRNA was elevated above control value but was less than 4-h value.
In LPS+SnPP-treated lungs, HO-1 mRNA was further increased above
control value at 4 and 24 h. * P 0.05 compared with control value.
** P 0.05 compared with
control value.
|
|
The control lungs had a ratio of HO-1 to GAPDH mRNAs of 0.75 (Fig. 10).
After LPS instillation, there was a significant increase in HO-1 mRNA
at 4 h. By 24 h, HO-1 mRNA had decreased from 4 h but still remained
significantly increased over the control value. The combination of LPS
and SnPP significantly increased the amount of HO-1 mRNA at both 4 and
24 h after LPS instillation.
 |
DISCUSSION |
The results of our experiments show that intratracheal administration
of a large dose of LPS significantly increases two antioxidant proteins, ferritin and HO-1, in the lung. Ferritin expression increased
by 4 h, continued to increase at 16 h, and returned toward baseline at
48-96 h. This time course was in contrast to the increase in HO-1
protein and enzyme activity, which began later and was sustained for
72-96 h. We also observed that inhibition of HO-1 enzyme activity by
SnPP did not prevent the increase in lung ferritin after LPS
instillation. The ferritin mRNA content of the lung was also unaffected
by LPS, consistent with the known posttranscriptional control of its
protein translation by an IRE (1). These new results establish that, in
this model of lung injury, early expression of ferritin is independent
of HO-1 activity. Also, as the injury progresses, ferritin expression
declines to baseline despite intense HO-1 protein expression and
increased enzyme activity.
Previous study of these two proteins demonstrated that HO-1 and
ferritin can be induced in pulmonary endothelium in rats by intravenous
administration of hemoglobin. This response may be adaptive in acute
lung injury, e.g., acute respiratory distress syndrome,
where endothelial injury can occur by contact with free heme (2, 3).
Intravenous LPS also stimulates HO-1 in the rat lung (19, 20) and
increases ferritin in the liver and spleen (8, 12). Administration of
LPS via the intratracheal route generates an inflammatory response in
the lung, originating on the epithelial surface. We used this stimulus
to examine the HO-1 and ferritin responses of the lung to epithelial
injury because the alveolar surface is frequently exposed to different
forms of oxidative stress. Protection of the lung epithelium from
oxidative stress has been proposed as a function of ferritin and HO-1,
analogous to the effects of the superoxide dismutases and glutathione
(7).
The temporal pattern of response of the lung to intratracheal LPS
instillation provides important information about the sequence of
events leading to ferritin and HO-1 expression. Initially, upregulation
of ferritin occurs in the bronchial epithelium, and by 4 and 16 h,
labeling of inflammatory cells for ferritin, especially macrophages, is intense. At 16 and 24 h, ferritin staining is no
longer apparent in bronchial epithelium but is prominent in the
alveolar regions of the lung. The early intense staining for ferritin
in the bronchial epithelium is likely due to the high initial exposure
of this region to intratracheal endotoxin. The localization of ferritin
apparently shifts over time after the injury from the airway and
macrophages to the alveolar wall region. This shift could reflect
cycling of the ferritin from the epithelial to the endothelial
compartment for transport of the storage protein from the lung to the
liver. The alternative possibility that the progressive increase in
HO-1 stimulates ferritin synthesis in the alveolar region by releasing
iron is ruled out by the SnPP experiments.
The precise mechanism by which LPS causes the initial increase in
ferritin content in the lung is not completely defined; however, our
finding of a rapid ferritin increase after LPS instillation with no
change in mRNA suggests activation of the IRE and posttranscriptional protein synthesis. LPS could effect a disequilibrium in
metal handling, producing an increase in available iron that would then activate the IRE. Indeed, LPS and other cytokines such as
interleukin-1
, tumor necrosis factor-
, and interferon-
have
been shown to alter iron handling (10) and to increase the ferritin
content of other tissues, including the liver and spleen (8, 12).
Although LPS can bind measurable amounts of iron (6), the amount of iron we measured was too small to cause a dramatic increase in ferritin
content. It is more likely that ferritin induction in the lung after
LPS instillation is a direct response of the IRE to the release of
cytokines or other inflammatory mediators.
The induction of HO-1 by LPS in the lung follows that of the ferritin.
We show that HO-1 protein induction follows an increase in HO-1
steady-state mRNA, as has been shown for hemoglobin by other
investigators (4, 20). By Western analysis and immunohistochemistry, HO-1 protein expression increases at 24 h, and the signals continue to
intensify at 48 and 72 h. The stimulus for this continued HO-1 expression is not known, but it could be the result of an ongoing requirement to catabolize heme derived from erythrocytes that appear in
the lung after injury by LPS. Another possibility is that activated
polymorphonuclear leukocytes, which are capable of oxidizing hemoglobin
to methemoglobin, provide a potent stimulus for HO-1 production and
activity (3). Either of these mechanisms may be operative in the acute
lung injury after LPS exposure, as significant inflammatory cell
infiltrate and hemorrhage occur after LPS instillation. Another notable
finding is the increase in HO-1 activity 4 h after LPS instillation,
before the increase in protein. The HO-1 antibody is sensitive to <10
ng of protein, which argues against a lack of sensitivity of our
immunodetection methods. This suggests a direct effect of LPS on the
enzyme activity that has not previously been considered.
It has been demonstrated in vitro that heme degradation by HO-1
releases iron that, in turn, induces ferritin (9, 24). This sequence of
events has been proposed as one of the cytoprotective functions of HO-1
(7, 9, 24). This mechanism, however, does not seem to be a major factor
in the lung after LPS exposure because of the different time course of
induction and localization of these two proteins. At 72 h after LPS
instillation, when HO-1 expression was greatest, ferritin content had
returned almost to baseline. Our experiments also clearly show that the
early increase in ferritin in the lung is not dependent on HO-1 enzyme activity because it occurred even when HO-1 was totally inhibited for
up to 24 h by SnPP. Quantitative measurements of ferritin expression
after LPS+SnPP instillation also demonstrate that ferritin content in
the lung is higher than after LPS alone. We also ruled out iron
contamination of SnPP as the cause of ferritin induction. Although the
mechanisms for this difference remain to be explored, they include
positive feedback on ferritin by inhibition of HO, e.g., by substrate
(e.g., heme), a direct effect of SnPP on ferritin (e.g., through the
porphyrin center), or release of ferritin expression from product
inhibition (e.g., carbon monoxide).
Despite inactivating HO, SnPP caused significant increases in lung HO-1
protein and mRNA above control values. To explain this observation, it
is tempting to invoke a negative feedback mechanism where enzyme
inhibition (with loss of product) increased enzyme expression. The time
course of induction of HO-1 mRNA by SnPP, however, was so similar to
that of LPS that it suggests that a direct stimulus by the
protoporphyrin is superimposed on that of LPS. SnPP contains a heme
moiety that could stimulate production of the enzyme.
In conclusion, we have demonstrated a distinct difference in the time
course and localization of ferritin in relation to HO-1 in the lung
during acute alveolar inflammation after intratracheal LPS
instillation. We have shown that under these conditions, changes in the
expression of ferritin protein does not depend on HO-1. Further studies
will be required to understand the cellular mechanisms leading to
induction of these proteins after LPS instillation and the protective
roles of ferritin and HO-1 in inflammatory lung injury.
 |
FOOTNOTES |
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: M. S. Carraway, Division of Pulmonary
and Critical Care Medicine, Duke Univ. Medical Center, Box 3315, Durham, NC 27710.
Received 9 January 1998; accepted in final form 14 May 1998.
 |
REFERENCES |
1.
Aziz, N.,
and
H. N. Munro.
Iron regulates ferritin mRNA translation through a segment of its 5' untranslated region.
Proc. Natl. Acad. Sci. USA
84:
8478-8482,
1987[Abstract].
2.
Balla, G.,
H. S. Jacob,
J. Balla,
M. Rosenberg,
K. A. Nath,
F. Apple,
J. W. Eaton,
and
G. M. Vercellotti.
Ferritin: a cytoprotective antioxidant strategy of endothelium.
J. Biol. Chem.
267:
18148-18153,
1992[Abstract/Free Full Text].
3.
Balla, J.,
H. S. Jacob,
G. Balla,
K. A. Nath,
J. W. Eaton,
and
G. M. Vercellotti.
Endothelial cell heme uptake from heme proteins: induction of sensitization and desensitization to oxidant damage.
Proc. Natl. Acad. Sci. USA
90:
9285-9289,
1993[Abstract].
4.
Balla, J.,
K. 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].
5.
Camhi, S. L.,
J. Alam,
L. Otterbein,
S. L. Sylvester,
and
A. M. K. Choi.
Induction of heme oxygenase-1 gene expression by lipopolysaccharide is mediated by AP-1 activation.
Am. J. Respir. Cell Mol. Biol.
13:
387-398,
1995[Abstract].
6.
Coughlin, R. T.,
S. Tonsager,
and
E. J. McGroarty.
Quantitation of metal cations bound to membranes and extracted lipopolysaccharide of Escherichia coli.
Biochemistry
22:
2002-2007,
1983[Medline].
7.
Choi, A. M. K.,
and
J. Alam.
Heme oxygenase-1: function, regulation, and implication of a novel stress-inducible protein in oxidant-induced lung injury.
Am. J. Respir. Cell Mol. Biol.
15:
9-19,
1996[Abstract].
8.
El-Shobaki, F.,
and
W. Rummel.
Mucosal iron binding proteins and the inhibition of iron absorption by endotoxin.
Blut
50:
95-101,
1985[Medline].
9.
Eisenstein, R. S.,
D. Garcia-Mayol,
W. Pettingell,
and
H. N. Munro.
Regulation of ferritin and heme oxygenase synthesis in rat fibroblasts by different forms of iron.
Proc. Natl. Acad. Sci. USA
88:
688-692,
1991[Abstract].
10.
Fahmy, M.,
and
S. P. Young.
Modulation of iron metabolism in monocyte cell line U937 by inflammatory cytokines: changes in transferrin uptake, iron handling, and ferritin mRNA.
Biochem. J.
926:
175-181,
1993.
11.
Frevert, C. W.,
A. E. Warner,
and
L. Kobzik.
Defective pulmonary recruitment of neutrophils in a rat model of endotoxemia.
Am. J. Respir. Cell Mol. Biol.
11:
716-723,
1994[Abstract].
12.
Kumagai, M, A,
and
S. Okada.
Mobilization of iron and iron-related proteins in rat spleen after intravenous injection of lipopolysaccharide (LPS).
Pathol. Res. Pract.
188:
931-941,
1992[Medline].
13.
Klausner, R. D.,
T. A. Rouault,
and
J. B. Harford.
Regulating the fate of mRNA: the control of cellular iron metabolism.
Cell
72:
19-28,
1993[Medline].
14.
Lee, P. J.,
J. Alam,
G. W. Weigand,
and
A. M. K. Choi.
Overexpression of heme oxygenase-1 in human pulmonary epithelial cells results in cell growth arrest and increased resistance to hyperoxia.
Proc. Natl. Acad. Sci. USA
93:
10393-10398,
1996[Abstract/Free Full Text].
15.
Lowry, O. H.,
N. J. Rosebrough,
A. L. Farr,
and
R. J. Randall.
Protein measurement with the Folin phenol reagent.
J. Biol. Chem.
193:
265-275,
1951[Free Full Text].
16.
Maines, M. D.
Heme oxygenase: function, multiplicity, regulatory mechanisms, and clinical applications.
FASEB J.
2:
2557-2568,
1988[Abstract/Free Full Text].
17.
McCoubrey, W. K., Jr.,
T. J. Huang,
and
M. D. Maines.
Isolation and characterization of a cDNA from the rat brain that encodes hemoprotein heme oxygenase-3.
Eur. J. Biochem.
247:
725-732,
1997[Abstract].
18.
Nathens, A. B.,
R. Bitar,
C. Davreux,
M. Bujard,
J. C. Marshall,
A. P. B. Dackiw,
R. W. G. Watson,
and
O. D. Rotstein.
Pyrrolidine dithiocarbamate attenuates endotoxin-induced acute lung injury.
Am. J. Respir. Cell Mol. Biol.
17:
608-616,
1997[Abstract/Free Full Text].
19.
Otterbein, L.,
B. Y. Chin,
S. L. Otterbein,
V. C. Lowe,
H. E. Fessler,
and
A. M. K. Choi.
Mechanism of hemoglobin-induced protection against endotoxemia in rats: a ferritin-independent pathway.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L268-L275,
1997[Abstract/Free Full Text].
20.
Otterbein, L.,
S. L. Sylvester,
and
A. M. K. Choi.
Hemoglobin provides protection against lethal endotoxemia in rats: the role of heme oxygense-1.
Am. J. Respir. Cell Mol. Biol.
13:
595-601,
1995[Abstract].
21.
Taylor, J.,
M. S. Carraway,
and
C. A. Piantadosi.
Lung specific induction of heme oxygenase-1 and hyperoxic lung injury.
Am. J. Physiol.
274 (Lung Cell. Mol. Physiol. 18):
L582-L590,
1998[Abstract/Free Full Text].
22.
Tenhunen, R.,
H. S. Marver,
and
R. Schmid.
The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase.
Proc. Natl. Acad. Sci. USA
61:
748-755,
1968[Medline].
23.
Tenhunen, R.,
H. S. Marver,
and
R. Schmid.
Microsomal heme oxygenase.
J. Biol. Chem.
244:
6388-6394,
1969[Abstract/Free Full Text].
24.
Vile, G. F.,
S. Basu-Modak,
C. Waltner,
and
R. M. Tyrrell.
Heme oxygenase-1 mediates an adaptive response to oxidative stress in human skin fibroblasts.
Proc. Natl. Acad. Sci. USA
91:
2607-2610,
1994[Abstract].
25.
Vogt, B. A.,
A. J. Croatt,
G. M. Vercellotti,
and
K. A. Nath.
Acquired resistance to acute oxidative stress: possible role of heme oxygenase and ferritin.
Lab. Invest.
72:
474-483,
1995[Medline].
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