Haptoglobin reduces lung injury associated with exposure to
blood
Funmei
Yang1,
David
J.
Haile2,
Franklin G.
Berger3,
Damon C.
Herbert1,
Emily
Van
Beveren1, and
Andrew J.
Ghio4
Departments of 1 Cellular and Structural Biology and
2 Medicine, University of Texas Health Science
Center, San Antonio, Texas 78229; 3 Department of
Biological Sciences, University of South Carolina, Columbia, South
Carolina 29208; and 4 National Health and
Environmental Effects Research Laboratory, Environmental Protection
Agency, Research Triangle Park, North Carolina 27711
 |
ABSTRACT |
The biological functions of the acute-
phase protein haptoglobin (Hp) may be related to its ability to bind
hemoglobin (Hb) or to modulate immune response. Hp is expressed at a
high level in lung cells, yet its protective role(s) in the lung is not
known. With the use of transgenic mice overexpressing Hp in alveolar macrophages, we demonstrated that Hp diminished Hb-induced lung injury
when the lung was exposed to whole blood. In transgenic mouse lungs, Hb
was more efficiently removed, and the induction of stress- responsive
heme oxygenase-1 gene was significantly lower when compared with
wild-type mice. At 24 h after blood treatment, the ferritin level
that serves as an index for intracellular iron content was also lower
in alveolar macrophages in transgenic mice than in wild-type mice. We
propose that an Hp-mediated Hb catabolism process exists in alveolar
macrophages. This process is likely coupled to an iron mobilization
pathway and may be an efficient mechanism to reduce oxidative damage
associated with hemolysis.
erythrocyte; lung diseases; hemorrhage; metal transporter
protein-1; heme oxygenase-1
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INTRODUCTION |
EXTRAVASATION OF
ERYTHROCYTES into the lower respiratory tract occurs in numerous
lung injuries, including chronic bronchitis, bronchiectasis, cystic
fibrosis, lung cancer, pulmonary embolism, pneumonia, tuberculosis,
traumatic injury, and diffuse alveolar hemorrhage. After
hemolysis, the catalytic iron present in hemoglobin (Hb) can induce the
formation of reactive oxygen species and lead to oxidative damage in
lung tissues. Both free Hb and erythrocytes have been shown to induce
lung injury in experimental animal models (3, 14).
Haptoglobin (Hp), an
2-acid glycoprotein, has long been
known as the major Hb-binding protein associated with Hb catabolism. It
is produced mainly by the liver and secreted into the circulation. As a
major positive acute-phase reactant, Hp increases in plasma during
inflammation, infection, trauma, tissue damage, and malignancy. In
patients with severe hemolysis, on the other hand, Hp decreases in
plasma to a nondetectable level with the formation of Hp-Hb complexes.
The Hb scavenger receptor CD163 has recently been identified to mediate
endocytosis of Hp-Hb complexes by monocytes/macrophages (17).
In our previous studies, we identified the lung as a major site
of extrahepatic synthesis of Hp. Hp gene expression can be detected in
airway epithelial cells in mice and baboons and in alveolar macrophages
and eosinophils in humans (22, 23). As in the liver,
expression of Hp gene in the lung increases severalfold upon exposure
to inflammatory stimuli and during some diseased states, suggesting
protective roles of Hp in lung tissues. To understand the functions of
Hp in the lung, we produced transgenic mice that express the human Hp
gene at high levels in several tissues, including lung. The cell
type-specific expression of human Hp gene is maintained in the
transgenic mice. Although the endogenous mouse Hp gene is expressed in
airway epithelial cells, the human Hp transgene is expressed in
alveolar macrophages in these mice (23). In this paper, we
have used these transgenic mice to investigate the protective effects
of Hp against blood-induced lung injury. We also measured the
expression of other proteins likely involved in the Hp-mediated Hb
catabolism and iron mobilization in alveolar macrophages.
 |
MATERIALS AND METHODS |
Production of transgenic mice.
Transgenic mice carrying a 9-kb human Hp genomic DNA, which contains
the entire Hp2 gene plus 1 kb of the 5'- and 1.5 kb of the 3'-flanking
regions, were produced in a background of CB6F1, as described
previously (23). The transgene was introduced in an inbred
background C57BL/6.
Tracheal instillation of blood.
Blood was collected from normal healthy adult mice by intracardiac
puncture into heparinized syringes after the animals were anesthetized
with halothane. Animals were then euthanized by further exsanguination
through the abdominal aorta. To induce lung injury, transgenic and
wild-type mice (3-4 mo old) were anesthetized with Metofane
(Pitman-Moore, Mundelein, IL) and intratracheally instilled with 50 µl of blood. The dose was determined according to the previous study
in rats (14). Control animals from each group received the
same volume of normal (0.9%) saline. Previous investigation using this
method of exposure has demonstrated a uniform distribution of instilled
material in the lung. After the instillation, all mice were allowed to
recover from the anesthesia and return to animal care facilities.
Collection of lavage fluids and lavage cells from mice.
Mice were anesthetized, euthanized, and tracheally lavaged with 1.0 ml
of normal saline. The lavage procedure was repeated twice. The combined
lavage fluid was centrifuged at 600 g for 10 min at 4°C to
separate cells from supernatant. The cells were resuspended in HBSS,
and, with a cytospin, 2 × 105 cells were then
pelleted onto slides. With the use of modified Wright's stain
(Diff-Quick stain; American Scientific Products, McGaw Park,
IL), neutrophils were enumerated, and values were expressed as
the percentage of total cells recovered.
Biochemical analysis.
Lavage protein levels were determined using the Pierce Coomassie Plus
protein assay reagent (Pierce, Rockford, IL). This assay was modified
for use in the Cobas Fara II centrifugal spectrophotometer (Hoffman-LaRoche). Bovine serum albumin served as the standard. The
lactate dehydrogenase concentration in the lavage fluid was measured
using a commercially prepared kit (Sigma Diagnostics) modified for
automated measurement. Concentrations of macrophage inflammatory
protein-2 (MIP-2) in the lavage fluids were measured by the method of
ELISA using Quantikine kits purchased from R&D Systems (Minneapolis,
MN). Total Hb in bronchoalveolar lavage supernatant was quantified by
the cyanomethemoglobin method (Sigma Chemical, St. Louis, MO).
In situ hybridization and immunohistochemistry.
Surgical specimens of human lung and normal mouse lung tissues
were obtained, quick-frozen in Tissue-Tek optimum cutting temperature compound, and stored at
80°C until used for the preparation
of cryosections. Human alveolar macrophages were obtained from
volunteers (13) and pelleted onto coated glass slides.
In situ hybridization of lung sections was conducted as previously
described (23) using antisense mouse metal transport
protein-1 (MTP-1) cRNA probes. The MTP-1 cDNA template used for the
preparation of riboprobe was described in a previous publication
(1). Immunohistochemistry for CD163 was performed using
the Vectastain Elite ABC peroxidase kit and the VIP peroxidase
substrate kit (Vector Laboratories, Burlingame, CA). Anti-CD163
antibody was a gift from Dr. Søren K. Moestrup (University of Aarhus,
Aarhus, Denmark). Immunohistochemical study for heme oxygenase-1 (HO-1)
and ferritin was performed as described previously (7,
14).
Statistics.
Data are expressed as means ± SE. Differences between multiple
groups were analyzed using analysis of variance. Two-tailed tests of
significance were employed. Significance was assumed at
P < 0.05.
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RESULTS |
The human Hp transgene is expressed in several tissues,
including liver, lung, adrenal glands, uterus, ovary, and submaxillary glands. A high level of Hp2 protein, which can be distinguished from
the mouse Hp protein, is detected in the sera of transgenic mice
(23). In most of the tissues in which Hp is produced, the human Hp2 transgene and the mouse endogenous Hp gene are expressed in
the same type of cells. In the lung, however, the human Hp transgene is
expressed in alveolar macrophages as it is in the human lung, whereas
the endogenous mouse Hp gene is expressed in airway epithelial cells.
The level of Hp protein in the lavage fluid was barely detectable in
the wild-type animals because of extensive dilution of the lung fluids.
However, an average of 15 µg/ml of Hp was found in the lavage fluids
of transgenic mice.
To determine whether an elevated level of Hp in the lung can
attenuate lung injury associated with exposure to the blood, mice were
intratracheally instilled with a small volume of whole blood (50 µl)
derived from normal healthy mice. Twenty-four hours after treatment,
lung tissues and lavage fluids were collected and analyzed. The
transgenic mice consistently displayed a higher degree of tolerance to
blood-induced lung injury compared with wild-type animals. As shown in
Fig. 1, the commonly used indexes for
lung tissue injury, i.e., levels of total protein and lactate dehydrogenase in the lavage fluids, were elevated in both transgenic and wild-type mice after the animals were exposed to blood. However, the change was significantly less in the transgenic mice than in the
wild-type mice.

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Fig. 1.
Comparison of the degree of lung injuries between
wild-type and haptoglobin (Hp) transgenic mice after intratracheal
instillation of blood. Two indexes, lactate dehydrogenase (LDH)
activity (A) and total protein (B) in the lavage
fluids were measured. Results from both measurements indicated that Hp
transgenic mice are more tolerant than wild-type mice to lung injuries
caused by exposure to blood. * Significantly higher in blood-treated
than in saline-treated mice in both genotypes. ** Significantly
lower in transgenic mice than in wild-type mice in the blood-treated
groups. P < 0.05, n = 6.
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Additionally, a lower degree of lung inflammation was found in
transgenic mice compared with wild-type mice after blood treatment. The
level of the major inflammatory cytokine MIP-2 was lower in the
transgenic mouse lungs than in the wild-type mouse lungs (Fig. 2). We also studied the profiles of
lavage cells to determine whether there is a difference in the degree
of inflammatory cell infiltration between wild-type and transgenic
mice. After blood treatment, the percent of neutrophils in wild-type
mice is twice as high as in transgenic mice (Table
1). The total cell number was also higher
in wild-type than in transgenic mice. There was no significant
difference in the numbers of total cells or neutrophils between
wild-type and transgenic mice in response to saline treatment.

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Fig. 2.
Levels of macrophage inflammatory protein-2 (MIP-2) in
the lung after exposure to blood. MIP-2 was significantly higher in the
wild-type than in the transgenic mice 24 h after the mice were
intratracheally instilled with whole blood. * Significantly higher
in blood-treated than in saline-treated mice in both genotypes.
** Significantly lower in transgenic mice than in wild-type mice in
the blood-treated groups. P < 0.05, n = 6.
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Table 1.
Comparison of neutrophil infiltration in wild-type and Hp- transgenic
mouse lungs after intratracheal instillation of whole blood
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As predicted, the amount of Hb retained in the lung after blood
treatment was much lower in transgenic mice than in wild-type mice
(Fig. 3). To identify the lung cells that
are involved in the clearance of Hb in the lung, we studied the
expression of the Hb scavenging protein CD163 in the lung. This
protein, a monocyte/macrophage cell surface receptor, was recently
shown to bind Hp-Hb complexes. An immunohistochemical experiment was
performed on human lung sections, freshly isolated human alveolar
macrophages, and baboon lung sections using an antibody against human
CD163 that cross-reacts with baboon, but not mouse, antigen. A strong
immunostain for CD163 was localized in alveolar macrophages but not in
any other lung cell (Fig. 4). These
results indicated that alveolar macrophages are the major cells
responsible for Hb catabolism in the lung via CD163-mediated
endocytosis.

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Fig. 3.
Comparison of the lavage hemoglobin (Hb) in wild-type and
Hp transgenic mice after tracheal instillation of blood. Lavage fluids
were collected 24 h after treatment. * Significantly higher in
blood-treated than in saline-treated mice in both genotypes.
** Significantly lower in transgenic mice than in wild-type mice in
the blood-treated groups. P < 0.05, n = 6.
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Fig. 4.
Immunohistochemistry for CD163 in alveolar macrophages.
Human lung sections (A and B), freshly isolated
human alveolar macrophages (C and D), and baboon
lung sections (E and F) were reacted with
anti-human CD163 antibody (A, C, and
E) or control rabbit IgG (B, D, and
F). Specific immunostaining can be seen in alveolar
macrophages (arrows) but not other cells in the lung. Original
magnification was ~×400.
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Because the safe sequestration of iron is crucial for lung
defense, we studied the molecular mechanisms involved in iron recycling after hemolysis in the lung. After endocytosis, iron can be released from Hb catalyzed by heme oxygenase. The inducible form of heme oxygenase, HO-1, is known to be activated by its substrate heme and numerous stress stimuli (reviewed in Ref. 16). To
investigate the response of the HO-1 gene in lung cells in vivo to
blood exposure, we performed immunohistochemical analysis of HO-1 on
lung specimens collected 4, 8, 16, and 24 h after intratracheal
instillation of blood. HO-1 expression was increased in alveolar
macrophages, endothelial cells, and alveolar epithelial cells, but not
in airway epithelial cells, in both wild-type and transgenic mice at
4 h after blood exposure (Table 2).
HO-1 levels peaked at 4-8 h after exposure and declined
after that. At all times, the level of HO-1 was much higher in
wild-type than in transgenic mice. This was particularly noticeable in
alveolar macrophages (Table 2). By 24 h after blood treatment, the
HO-1 expression in transgenic mouse lung had decreased to the same
level as in the untreated wild-type or transgenic mouse lung. However,
it remained high in the alveolar macrophages in wild-type mice (Table 2
and Fig. 5). There was also a more
obvious increase in the number of alveolar macrophages in wild-type
(Fig. 5B) than in transgenic mouse lungs after blood
exposure. This is in agreement with our observations for the profiles
of lavage cells in these lungs (see Table 1).

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Fig. 5.
Activation of heme oxygenase-1 (HO-1) gene expression in mouse lung
after blood exposure. Immunohistochemical study for HO-1 protein was
performed on lung paraffin sections derived from wild-type mice
(A and B) and transgenic mice (C and
D) exposed to saline (A and C) or
blood (B and D) for 24 h. HO-1 protein level
remained elevated in alveolar macrophages (arrows) in wild-type mice
(B) but not in transgenic mice (D). Original
magnification was ~×400.
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After Hb is catabolized in alveolar macrophages, free iron
released from heme can be sequestered in ferritin or mobilized to
extracellular space. Previous studies have shown that macrophages are
capable of releasing iron in the form of ferritin and transferrin (4, 9, 20), especially during iron overload (20,
21). To investigate the molecular mechanism underlining this
process in alveolar macrophages, we studied the expression of an iron export protein, MTP-1 (also known as ferroportin 1, Ireg1),
that has recently been shown to be responsible for iron export in
zebrafish yolk sac, Xenopus oocytes (11), and
cultured cells (1). We found that MTP-1 is expressed in
alveolar macrophages and airway epithelial cells in human lungs
(unpublished observations). By using the technique of in situ
hybridization, we demonstrated that a high level of MTP-1 mRNA was
present mainly in the alveolar macrophage in the mouse lung (Fig.
6). This result suggests MTP-1 plays a
role in iron metabolism in alveolar macrophages after Hb
catabolism.

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Fig. 6.
Metal transport protein-1 (MTP-1) gene expression in
alveolar macrophages of mouse lung. MTP-1 mRNA was detected by the in
situ hybridization technique. A bright field picture (A) and
a dark field picture (B) are shown. Hybridization signals
(silver grains) can be seen specifically in alveolar macrophages
(arrows). Original magnification was ~×400 for A and
×100 for B.
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To investigate the efficiency of iron mobilization in alveolar
macrophages, we conducted immunohistochemical analysis of ferritin, which reflects the level of intracellular iron, in wild-type and transgenic mouse lungs 24 h after blood exposure. The results are
summarized in Table 3. In the
endothelium, airway epithelium, and alveolar epithelium, the ferritin
level was not significantly different between transgenic and wild-type
mice whether the animals were treated with saline or blood. In alveolar
macrophages, the ferritin level was the same between transgenic and
wild-type mice treated with saline. There was a marked increase in both
transgenic and wild-type mice after blood exposure. However, the level
of ferritin in alveolar macrophages was much greater in wild-type mice
than in transgenic mice (Fig. 7),
suggesting a higher efficiency of Hb catabolism and iron mobilization
in transgenic mice than in wild-type mice.

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Fig. 7.
Immunohistochemical analysis of ferritin in mouse lungs
after blood exposure. Immunostaining of ferritin was performed on
paraffin sections derived from mouse lungs exposed to blood for 24 h. There is diffuse uptake of the antibody for ferritin by all cell
types in the lower respiratory tract. However, large, intra-alveolar
macrophages (arrows) that stain strongly for the antibody are evident
in the wild-type animals (A) but not in the transgenics
(B). Original magnification was ~×400.
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DISCUSSION |
In numerous acute and chronic lung injuries, frequently, red blood
cells can be present in the lower respiratory tract. Extravasated erythrocytes can be removed by phagocytosis or destroyed by proteases, resulting in the release of Hb and reactive iron. These byproducts potentially participate in oxidant generation and contribute to injury
(15, 24). Ghio et al. (14) has shown that
intratracheal instillation of whole blood in the rat can induce a
neutrophilic lung injury with a marked increase in both TNF-
and
MIP-2. This injury is also associated with a disruption of iron
equilibrium, which was made evident by quantifying iron and staining
for Hb and ferritin. Nevertheless, the clearance processes for both
erythrocytes and Hb in the lung are considered to be very efficient,
and after a few days, little free Hb remains after exposure to whole
blood (14).
As a major acute-phase protein, Hp has been shown to have several
biological functions, including a role in Hb catabolism (10). In previous studies, we have found that the airway
epithelium is a major site of Hp gene expression in baboons and mice.
In humans, the Hp gene is not expressed in normal healthy lung but is
greatly activated in alveolar macrophages in diseased lung tissues. In
mouse lungs, alveolar macrophages can also be induced to synthesize Hp
during inflammation (unpublished observations). The roles of Hp in lung
health and lung defense have not been elucidated. Results from this
study indicate that a major pathway for the removal of Hb in the lung
involves the formation of Hp-Hb complexes. Using a transgenic mouse
system, we have shown that overexpression of Hp in the lung promotes Hb
removal after intratracheal instillation of whole blood. This was
accompanied by a lower degree of neutrophilic lung injury and
inflammation in transgenic compared with wild-type mice. After blood
exposure, the increase of both TNF-
(data not shown) and MIP-2 was
significantly less in transgenic than in wild-type mice. We have
conducted a similar study using wild-type mice and Hp knockout mice
generated by Lim et al. (18). In this experiment, the
wild-type mice were much more tolerant to blood-induced lung injury
than the Hp knockout mice (unpublished observations), providing
further support for the protective effects of Hp during lung injury.
The fate of the Hp-Hb complexes in the lung is not clear. In the
circulation, the Hp-Hb complexes are delivered to the parenchymal cells
of the liver where Hb is broken down to bilirubin. A mechanism, which
could be responsible for the transport of high-molecular-weight Hp-Hb
complexes from respiratory tract to circulation, is yet to be
established. In this study, we have shown that CD163, which was
recently identified as an Hp-Hb scavenging receptor (17), is present in alveolar macrophages but not other lung cells. CD163 has
a high affinity for Hp-Hb complexes but does not bind to Hp or Hb.
CD163-mediated endocytosis of Hp-Hb complexes by alveolar macrophages
could be the major pathway responsible for the removal of Hb in the
lung. Increased production of Hp by alveolar macrophages and airway
epithelial cells at the site of inflammation could contribute
significantly to the clearance of Hb and thus protect the lower
respiratory tract against Hb-mediated oxidative damage. In humans,
there are two different genetic alleles for Hp, the Hp1 and Hp2.
Interestingly, complexes of Hb and multimeric Hp (Hp 2-2 phenotype)
exhibit a 10-fold higher functional affinity for CD163 than do
complexes of Hb and dimeric Hp (Hp 1-1 phenotype) (17). It
is not known whether individuals with the Hp1-1 type are more
susceptible to Hb-induced lung injury and inflammation.
In alveolar macrophages, Hb catabolism involves the key enzyme
heme oxygenase. Expression of the inducible form of this protein, HO-1,
is known to be elevated in the lung during injury, inflammation, hyperoxia, and other stressful conditions. In this study, we have shown
that HO-1 was induced at the early time points in alveolar macrophages
in both transgenic and wild-type mouse lungs but diminished at later
time points in transgenic mice. Because HO-1 gene expression is induced
by its substrate heme in Hb, these results support our hypothesis that
Hb is removed and catabolized more efficiently in transgenic mice than
in wild-type mice. It is likely that the elevated level of Hp in the
transgenic mouse lung, especially in the vicinity of alveolar
macrophages, promotes a speedy clearance of Hb from the alveolar space.
Shortly after blood exposure, this protective effect allowed the
transgenic mice to activate only the needed level of HO-1, which was
much lower than that required in the wild-type mice. The structure of
the heme molecule and the mechanism involved in the induction of HO-1
by heme are not clear. Alternative explanations for a lower degree of
HO-1 induction in transgenic mice could include the
compartmentalization of Hp-Hb complexes in alveolar macrophages in
these mice. Disregarding the mechanism involved, the level of HO-1
induction appeared to serve as an excellent index for Hb-associated
oxidative stress. The anti-inflammatory effect of Hp in transgenic
mouse lungs was also evident by a lower level of inflammatory
cytokines, such as MIP-2 and TNF-
.
In alveolar macrophages, iron released from Hb may either enhance
the production of ferritin and intracellular iron storage or be
excreted as a metal bound to ferritin and/or transferrin. Iron efflux
in alveolar macrophages has been shown to increase during iron overload
(21) and may reflect a cellular protective mechanism
against cell death as a result of oxidative stress (12). The molecular mechanism(s) involved in intracellular iron trafficking and iron transport across the cell membrane is not completely understood. Our finding that the iron exporter MTP-1 is expressed at a
high level in alveolar macrophages suggests that MTP-1 may be another
key protein that participates in an iron detoxification (recycling)
process in alveolar macrophages. Our proposed model for this process is
illustrated in Fig. 8. In this model, Hb
released during hemolysis is first captured by Hp synthesized locally
at a high level by alveolar macrophages (in human) or airway epithelial cells (in mice and baboon). The Hp-Hb complexes are taken up by alveolar macrophages through CD163-mediated endocytosis. After Hb is
degraded, iron can be sequestered in the cells or released in less
reactive forms as transferrin and/or ferritin. Some of these
iron-containing proteins may be captured within mucus and may
subsequently be expelled from the lung. A high concentration of
iron-containing protein has been found in sputum from patients with
lung diseases (2). Interestingly, all the key proteins participating in this Hb catabolism and iron detoxification process are
elevated during inflammation (5, 6, 8, 19, unpublished observations), further suggesting their importance in lung defense.

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Fig. 8.
Proposed model for hemoglobin
catabolism and iron mobilization in the human lung. Hb-Hp complexes are
taken up by alveolar macrophages through a scavenger receptor (CD163).
After endocytosis, iron is released from Hb by heme oxygenase. Through
the iron transporter MTP-1, iron can be exported to the extracellular
space in inactive protein complexes. This process is initiated by Hp
secreted by alveolar macrophages and results in the safe sequestration
of bioactive iron and the reduction of oxidative stress associated with
hemolysis in the lung.
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Using transgenic mice overexpressing Hp, we have shown that an
increased level of Hp can promote Hb clearance and attenuate blood-induced lung injury and inflammation. Our results suggest that Hb
catabolism in alveolar macrophages is linked to iron mobilization to
ensure the safe sequestration of iron derived from Hb. Alveolar macrophages appear to produce all the key proteins and enzymes needed
in this catabolic pathway. Hp and other proteins involved in iron
mobilization may play important roles in lung defense.
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ACKNOWLEDGEMENTS |
The authors thank Dr. Søren K. Moestrup (University of Aarhus,
Aarhus, Denmark) for helpful discussion and for sharing the anti-CD163
antibody with us. We also thank the San Antonio Cancer Institute
Pathology Core Laboratory for help with the preparation of tissue
sections and Renee Judkins of Laboratory Animal Resources (University
of Texas Health Science Center) for supplying mouse blood from the
pathogen-free facility. Karen Barbour (University of South Carolina)
provided technical assistance.
 |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grants DK-53079 and DK-3386 and a Morrison Trust
research grant.
Address for reprint requests and other correspondence: F. Yang, Dept. of Cellular and Structural Biology, Univ. of Texas Health Science Center at San Antonio, San Antonio, TX 78229.
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. Section 1734 solely to indicate this fact.
First published October 18, 2002;10.1152/ajplung.00115.2002
Received 16 April 2002; accepted in final form 11 October 2002.
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