Institutes of 1 Physiology, 2 Anesthesiology, and 3 Anatomy, and 5 Department of Surgery, University of Zurich, 8057 Zurich; and 4 Paul Scherrer Institute, 5232 Villigen, Switzerland
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ABSTRACT |
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Molecular
mechanisms of the inflammatory reaction in hypoxia-induced lung injury
are not well defined. Therefore, effects of alveolar hypoxia were
studied in rat lungs, exposing rats to 10% oxygen over periods of 1, 2, 4, 6, and 8 h. An increase in the number of macrophages in
bronchoalveolar lavage fluid of hypoxic animals was shown between 1 and
8 h. Extravasation of albumin was enhanced after 1 h and
remained increased throughout the study period. NF-B-binding
activity as well as mRNA for TNF-
, macrophage inflammatory protein
(MIP)-1
, and monocyte chemoattractant protein (MCP)-1 were increased
within the first 2 h of exposure to hypoxia. Hypoxia-inducible
factor (HIF)-1
and intercellular adhesion molecule (ICAM)-1 mRNA
were upregulated between 1 and 6 h. Elimination of
alveolar macrophages by intratracheal application of
liposome-encapsulated clodronate led to a decreased expression of
NF-
B binding activity, HIF-1
, TNF-
, ICAM-1, and MIP-1
. In
summary, alveolar hypoxia induced macrophage recruitment, an increase
in albumin leakage, and enhanced expression of inflammatory mediators,
which were mainly macrophage dependent. Alveolar macrophages appear to
have a prominent role in the inflammatory response in hypoxia-induced lung injury and the related upregulation of inflammatory mediators.
hypoxia; inflammatory mediators; lung injury
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INTRODUCTION |
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ACUTE ALVEOLAR HYPOXIA is a condition that occurs in various clinical situations. Causes are hypoventilation induced, for example, by brain injury, intoxication, or thoracic cage injury. Atelectasis results in a ventilation/perfusion mismatch and alveolar hypoxia as well. Pathophysiologically, acute exposure to hypoxia results in vasoconstriction of pulmonary arteries and a redistribution of blood flow from the basal to the apical portion of the lung (13). It is not well documented whether, at the same time, acute hypoxia also induces changes at the level of pulmonary inflammatory mediators. Most of the in vivo studies performed to date have concentrated on ischemia-reperfusion injury in organs (15, 18). However, the direct effects of hypoxia without reperfusion on tissue have not been well explored. In addition, studies performed in lungs have focused on lesions after exposure to hypoxia over several days (24, 32). The acute hypoxia-induced lung injury has not been described in detail. One potential reason might be that the lungs have not been regarded as a main target organ of hypoxia. On the basis of evidence from in vitro data, we assumed that short-term hypoxia might lead to lung injury (3).
Lung inflammatory response is regulated by the coordinated function of
cytokines, chemokines, and adhesion molecules. Cell adhesion molecules
such as the group of selectins, integrins, and the immunoglobulin gene
superfamily have been shown to play a key role in the inflammatory
response, mediating different steps of leukocyte migration through the
endothelium (11). Cytokines are best known for their
leukocyte chemoattractant activity, whereas chemokines orchestrate the
complex cellular interactions and mediate different steps of migration
of leukocytes through the endothelium (11). It has been
shown that macrophage inflammatory protein-1 (MIP-1
) plays a key
role in the IgG immune complex-induced lung injury, whereas monocyte
chemoattractant protein-1 (MCP-1) dominates the regulation of IgA
immune complex-induced lung injury (5, 14). An important
signal transduction element is the transcriptional factor nuclear
factor-
B (NF-
B). Upon stimulation by viruses, cytokines,
lipopolysaccharides (LPS), and also hypoxia, NF-
B translocates to
the nucleus. There it binds to specific promoters and induces gene
transcription of various cytokines, chemokines, and adhesion molecules
(1). Another transcriptional factor relevant to hypoxia is
hypoxia-inducible factor-1 (HIF-1), composed of the oxygen-sensitive
HIF-1
and HIF-1
subunits. Although the HIF-1
subunit is
expressed constitutively, HIF-1
expression in the lung is regulated
by the inspired oxygen concentration (35). Several target
genes of HIF-1
have been identified, including cyclooxygenase-1,
interleukin-6, and vascular endothelial growth factor
(28).
Alveolar macrophages are situated at the air-tissue interface in the alveoli and are therefore among the first cells in contact with inhaled organisms or substances. They not only act as phagocytes but also secrete biologically active products, thereby playing an important role in regulating inflammatory reactions (29). Intratracheal instillation of liposomes containing dichloromethylene diphosphonate induces alveolar macrophage depletion (33). The liposome-encapsulated diphosphonate is ingested by phagocytic cells only and results in selective depletion of alveolar macrophages. This intervention allows us to characterize in vivo function and role of alveolar macrophages (6, 17).
The following studies were undertaken to evaluate hypoxia-induced lung injury, including changes in inflammatory mediators, and to elucidate the in vivo role of alveolar macrophages in this model. We hypothesized that alveolar macrophages might be the main source of cytokines and chemokines in hypoxia-induced lung alteration.
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MATERIALS AND METHODS |
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Animal model of hypoxia. Male Sprague-Dawley rats (250-300 g) were anesthetized with Hypnorm (fentanyl-fluanisone, 0.25 ml/kg sc) and Domitor (medetomidine hydrochloridum, 0.25 ml/kg sc). Animals were placed in a hypoxic chamber with decreasing oxygen tension from 21 to 10% within 60 min. Oxygen was substituted with nitrogen by a Digamix 2M 302/a-F pump (Woesthoff, Bochum, Germany). The gas flow rate was 37 l/min in a closed Persplex chamber. CO2 tension was maintained normal. Animals remained under 10% oxygen for 1, 2, 4, 6, and 8 h, respectively. Rehydration was ensured by intraperitoneal application of saline. At predefined time points, animals were euthanized. For bronchoalveolar lavage, 10 ml of cold phosphate-buffered saline (PBS) were gently instilled into the lungs, withdrawn, and reinstilled four times and collected. All experimental procedures (determination of extravascular albumin, analysis of interstitial neutrophil accumulation, and evaluation of expression of transcriptional factors and mRNA of various genes) were performed on the same lungs (different lobes) of the respective experimental animal.
All animals were housed in individual isolator cages within the Animal Care Facilities at the University of Zurich until the day of experimentation. The experimental protocols were approved by the animal care committees at the University of Zurich.Alveolar macrophage depletion.
Clodronate-liposomes were prepared as previously described (23,
27). Briefly, liposomes composed of 880 mg soy
phosphatidylcholine, 132 mg cholesterol, and 5 mg
DL--tocopherol were prepared in a clodronate solution
(375 mg clodronate in 10 ml Ostac; Boehringer, Mannheim, Germany) by
freeze-thawing and filter extrusion. Unencapsulated clodronate was
removed with an Amicon ultrafiltration cell, followed by size exclusion
chromatography on a Sephadex G25 column. For the in vivo experiments,
liposomes were diluted in sterile saline in a total volume of 300 µl.
Each animal received a dose of 500 µg of liposome-encapsulated
clodronate. Empty liposomes were used as controls. Rats were
anesthetized, and liposomes were administered intratracheally. Hypoxia
experiments were started 72 h later.
Bronchoalveolar lavage fluid.
Bronchoalveolar lavage fluid (BALF) was centrifuged at 2,000 rpm.
Supernatant was aliquoted and frozen at 20°C. Cell pellets from
centrifuged BALF were assessed for differential cell counts using
cytospins and Diff-Quick (Dade Behring, Duedingen, Switzerland). At the
same time, cells were identified with neutrophil- and
macrophage-specific antibodies. Cells were blocked with PBS-10% fetal
bovine serum (FBS) and incubated overnight at 4°C with primary
antibody. Neutrophils were identified with a monoclonal mouse anti-rat
neutrophil antibody (20 µg/ml), and macrophages with a monoclonal
mouse anti-rat macrophage antibody (5 µg/ml) (both from PharMingen,
San Diego, CA). A secondary fluorescein isothiocyanate-(FITC)- or
Cy-3-labeled goat anti-mouse antibody was added for 45 min at 4°C,
together with 4,6-diamidino-2-phenylindole (1:500 diluted). The
FITC-labeled antibody was diluted 1:50 in PBS-1% bovine serum albumin,
the Cy-3-labeled antibody 1:500. All washing steps were performed with
PBS. Cells were counted under an epifluorescence microscope.
Albumin extravasation. Extravasation of albumin was determined by a direct albumin enzyme-linked immunosorbent assay (ELISA) according to an earlier protocol (20). BALF from injured lungs (~8 ml) was analyzed with the ELISA. A coating carbonate buffer (0.1 M carbonate, pH 9.5) was used to dilute samples (1:1,000), and a standard curve was created with recombinant rat albumin (RDI, Flanders, NJ). A 96-well plate was coated with 100 µl/well and incubated overnight at 4°C. All washing steps (5 times with 200 µl/well) were performed with PBS-0.05% Tween 20. To block nonspecific binding, we added 3% dry milk in PBS for 1 h at 4°C. A first polyclonal rabbit anti-rat albumin antibody (RDI) was diluted in PBS-3% dry milk to a concentration of 10 µg/ml and incubated for 1 h at 4°C (100 µl/well). A secondary horseradish peroxidase-labeled goat anti-rabbit antibody (Sigma, Buchs, Switzerland) was added to the wells for 1 h at 4°C (100 µl/well). To develop color reaction, we added o-phenylenediamine dihydrochloride (Sigma) to the wells (200 µl/well). The reaction was stopped with 3 M H2SO4, and optical density was determined at 492 nm (ELISA reader; Bioconcept, Allschwil, Switzerland). To verify the results, BALF was also run on a SDS-PAGE gel and analyzed with Coomassie staining.
Tissue myeloperoxidase content. Lungs were homogenized in a buffer containing 50 mM potassium phosphate, 0.5% hexadecyltrimethylammonium bromide, and 5 mM ethylenediaminetetraacetic acid (EDTA), sonicated, and centrifuged as described earlier (10). Supernatant (50 µl) was added to 1,450 µl of assay buffer, consisting of 100 mM potassium phosphate, o-dianisidine hydrochloride, and 30% H2O2. The reaction was assayed every 10 s at 420 nm (ELISA reader). The results are shown as the slope of change in optical density over 360 s. Control values were defined as 1, whereas results from hypoxic lungs were normalized to the value of 1.
Nuclear extracts and electrophoretic mobility shift assay.
Nuclear extracts were prepared according to Schreiber et al.
(26). Briefly, lungs were homogenized in ice-cold
buffer A [10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM ethylene glycol-bis(-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), 1 mM
dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)]. Lysis was induced with 0.25% Nonidet P-40. After
centrifugation, nuclear pellets were resuspended in ice-cold
buffer B (20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM
EGTA, 10% glycero1, mM DTT, and 1 mM PMSF) and rocked at 4°C for
1 h. The extracts were centrifuged, and supernatants were frozen
at
70°C. The protein concentration of the extracts was determined
using the Bradford method (Bio-Rad, Hercules, CA).
Protein analysis (Western blot) for intercellular adhesion molecule-1. A crude membrane fraction from the lungs was obtained following a protocol described earlier (34). Whole lung protein (20 µg) was electrophoresed in a 12.5% SDS-polyacrylamide gel. After separation, the proteins were transblotted to a nitrocellulose membrane for 1 h at 150 V (Bio-Rad). Equal loading of proteins was confirmed by Ponceau S staining. The blot was washed in PBS and blocked with PBS-5% low fat milk-0.1% Tween 20 for 1 h at room temperature, followed by an overnight incubation with monoclonal mouse anti-rat intercellular adhesion molecule (ICAM)-1 antibody (Serotec, Oxford, England) in blocking buffer. All washing steps were performed three times with PBS-0.1% Tween 20. A secondary horseradish peroxidase-labeled anti-mouse IgG (1:5,000) in blocking buffer was added for 30 min at room temperature. Signals were detected by enhanced chemiluminescence.
RNA analysis.
Total RNA from lungs was extracted as previously described
(3) and analyzed by reverse transcriptase polymerase chain
reaction (RT-PCR) using a kit (Perkin-Elmer, Branchburg, NJ). Random
hexanucleotide primers and murine leukemia virus RT were used for cDNA
synthesis. Reverse transcription was performed with 0.8 µg of RNA at
20°C for 5 min, 42°C for 30 min, and 99°C for 5 min. Specific
primers located on separate exons were designed to assess gene
expression of TNF-, ICAM-1, MIP-1
, MCP-1, and HIF-1
, (Table
1). RT-PCR products were resolved on
1.5% agarose gels and stained with ethidium bromide. Gels were then
photographed under ultraviolet light. PCR was also performed with 18S
primers to ensure equal loading.
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In vitro assay with alveolar macrophages.
Alveolar macrophages were collected as described previously
(3). Cells (1 × 107 cells/plate) were
cultured for 1 h in DMEM with glutamax (Life Technologies, Basel,
Switzerland) supplemented with 10% FBS. After becoming adherent, they
were exposed to 5% oxygen for 5 h, while control cells stayed at
21%. A cell incubator (Bioblock, Ittigen, Switzerland) adjustable to
different oxygen concentrations by insufflation of nitrogen was used as
a hypoxic cell chamber (3). Supernatant was collected and
analyzed with a TNF- and MCP-1 enzyme-linked ELISA purchased from PharMingen.
Statistical analysis. All experiments were performed at least five times. Albumin ELISA contained four to six replicates with five different animals. Each data point in the graphs represents mean ± SE. Analysis of variance (ANOVA) with post-ANOVA comparison was performed to assess statistical significance of differences.
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RESULTS |
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Depletion of macrophages. Before experiments with clodronate-liposomes were started, different depletion conditions were evaluated. Depletion time was varied between 24 and 72 h, with the most effective depletion observed after 72 h. Several concentrations of clodronate were used (100, 500, 1,000, and 1,500 µg). Optimal depletion was seen at a dose of 500 µg, whereas with increased concentrations of clodronate, enhanced neutrophil accumulation was observed. This phenomenon of neutrophil recruitment induced by clodronate is a known observation (6). Depletion was verified with macrophage staining in BALF and tissue (data not shown).
BALF cells.
As seen in Fig. 1A, the cell
number increased after 1 h (170% increase, P < 0.05), being upregulated over a period of 8 h (300% at 2 h,
240% at 4 h, 350% at 6 h, and 150% at 8 h;
P < 0.05 for all values compared with control). This
increase appeared to be almost exclusively due to macrophages as
determined by fluorescence staining. Neutrophils were seen after 4 and
6 h, but the contribution to total cell count was only 13% at
4 h and 8% at 6 h (P < 0.05). After
macrophage depletion, total cell number did not change quantitatively under hypoxia, but the total ratio of cell types changed (Fig. 1B). The increase of the number of neutrophils compared with
nondepleted animals after 2 or 4 h of hypoxia was due to
clodronate, as verified in normoxic control liposome and clodronate
animals (6).
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Hypoxia-induced neutrophil activation.
Tissue myeloperoxidase (MPO) increased by 230% at 1 h
(P < 0.01) and by 35-80% between 2 and 8 h
(P < 0.05) (Fig. 2).
Intervention with intratracheally applied clodronate-liposomes did
not have an effect on MPO values at 2 and 4 h of hypoxia (data not
shown).
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Extravasation.
Exposure to 10% oxygen caused a 100% increase in albumin
extravasation as early as 1 and 2 h (P < 0.05),
as assessed by the transpulmonary flux of albumin (Fig.
3A). After 4 h of
hypoxia, albumin concentration even increased by 200%
(P < 0.005). A similar result was seen in the
Coomassie staining of a SDS-PAGE gel performed with BALF (Fig.
3B). To evaluate the influence of alveolar macrophages on
vascular permeability, we measured albumin in BALF of normoxic and
hypoxic control liposome and clodronate animals. However, there was no
difference in vascular permeability between hypoxic animals with or
without macrophages (data not shown).
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Determination of NF-B.
NF-
B as an essential activator of many inflammatory mediators
was determined by EMSA. As seen in Fig.
4A, a peak in NF-
B DNA-binding activity was observed at 1 and 2 h. NF-
B in
LPS-injured lungs was assessed as positive control (4).
Increased NF-
B binding was completely inhibited by preincubation of
nuclear extracts with an excess of unlabeled consensus oligonucleotide
competitor. Clodronate-liposome pretreated animals showed less whole
lung NF-
B binding activity compared with control liposome animals after 2 h exposure to hypoxia (Fig. 4B).
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Determination of mRNA for HIF-1.
RT-PCR of whole lung HIF-1
showed a baseline expression of HIF-1
in control lungs. This is also known from mouse brain and skeletal
muscle with HIF-1
expression in normoxic animals (31). HIF-1
expression was increased after 1 h of hypoxia compared with control animals and remained upregulated (Fig.
5A). Interestingly, macrophage
depletion led to a downregulation (Fig. 5B).
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Upregulation of rat mRNA for TNF-, ICAM-1, MIP-1
, and MCP-1
in whole lung.
To confirm equal loading, we chose 18S as an internal standard. mRNA
for TNF-
increased after 1 and 2 h, leveling off within 4 h. ICAM-1 mRNA exhibited a significant increase after 1 h of hypoxia, which persisted up to 8 h after injury. MIP-1
mRNA and mRNA for MCP-1 both peaked between 1 and 2 h (Fig.
6A). Macrophage depletion lead
to a clear downregulation of mRNA for TNF-
, ICAM-1, and MIP-1
compared with control liposome animals (Fig. 6B), whereas mRNA for MCP-1 was not changed (data not shown).
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TNF- and MCP-1 production of alveolar macrophages under hypoxic
conditions.
To analyze the in vitro expression pattern of TNF-
and MCP-1 of
alveolar macrophages, we exposed cells to hypoxia for 5 h. TNF-
and MCP-1 proteins were measured in the supernatant. As seen in Fig.
7A TNF-
was increased from
54 up to 89 pg/ml (68% increase, P < 0.01), whereas
MCP-1 production during hypoxia was enhanced by 50% (P < 0.01) (Fig. 7B).
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DISCUSSION |
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These studies demonstrate for the first time that acute exposure
of rats to moderate hypoxia results in a mild lung injury. The injury
was characterized by the accumulation of macrophages, a modest
neutrophil influx, and an increased accumulation of extravascular albumin. On the level of inflammatory mediators, DNA-binding activity of NF-B and expression of mRNA for HIF-1
, TNF-
, ICAM-1,
MIP-1
, and MCP-1 were increased.
Compared with other acute inflammatory processes in the lung such as LPS-induced or immune complex-induced lung injury, only a slight increase of mRNA and protein for ICAM-1 under hypoxia was observed (4, 19, 25). Whole lung mRNA for ICAM-1 was increased by 300% in the LPS lung injury and by 250% in the IgG immune complex-induced lung injury. In the hypoxia-induced lesion, however, only a 77% increase of mRNA for ICAM-1 was measured. Our previous in vitro studies support these data: alveolar epithelial cells (AEC) under LPS stimulation showed a 600% increase of mRNA for ICAM-1, whereas hypoxia led to a 100% upregulation (3). One reason for the observed differences could be the severity of the injury, which is high in the IgG- and LPS-induced inflammation models with capillary leakage and a recruitment of neutrophils. The hypoxia-induced injury, however, results in a mild lesion. Another explanation could be a different functional role of ICAM-1 in the hypoxia-induced lung injury. O'Brien et al. (21) recently showed that ICAM-1 on AEC played an important role in host defense against Klebsiella pneumoniae. A protective function of the upregulated adhesion molecule ICAM-1 could also play a role in the hypoxic lesion. Furthermore, it could be hypothesized that the inflammatory changes caused by hypoxia represent a priming condition and gain in severity in conjunction with an ensuing superimposed injury. It could be shown in a cell model with alveolar macrophages that LPS stimulation under hypoxia increased injury compared with LPS stimulation alone (16). Similar results were seen in a sepsis model of lung injury. Under sublethal conditions, there was no evidence of an inflammatory response in lungs of animals with cecal ligation/puncture-induced sepsis alone. However, after a direct intrapulmonary insult, enhanced lung injury in septic animals was observed (7).
The fact that vascular leakage is increased by chronic hypoxia is not new. Stelzner et al. (30) showed in vivo increased pulmonary extravasation of albumin in rat lungs after 48 h of exposure to hypobaric hypoxia. However, short-term exposure to hypoxia (1, 3, 6, and 13 h) did not cause significant increases in extravasation. These findings are not in accordance with our results. Differences between the experimental systems may partly explain this, since we were exclusively investigating under decreased oxygen concentrations. Stelzner et al., however, applied hypobaric hypoxia. Although we determined extravascular albumin, our results, however, do not explain the origin of the albumin (exudate, transudate). Theoretically, the interstitial accumulation of albumin might be due to an enhanced inflammation-induced vascular permeability, but also to hypoxic vasoconstriction with transudation. This question was previously analyzed with the help of a special experimental setup (30). Stelzner et al. measured hemodynamic changes in the pulmonary vascular system as well as protein leakage in hypoxic rat lungs. Although mean pulmonary pressures in hypoxic animals were increased compared with pressure in normoxic animals, no significant differences in protein leakage index were seen in the two different groups, assuming that hypoxic vasoconstriction is not responsible for increased extravasation of albumin.
An interesting finding in our studies was the transient increase of
mainly macrophages in BALF under hypoxia. Compared with other models
such as LPS- or IgG-induced lung injury, neutrophil recruitment was
minimal. To evaluate whether these alveolar macrophages were the main
source of mRNA of inflammatory mediators in the case of hypoxia-induced
lung injury, we performed macrophage depletion. Theoretically, other
potential sources such as interstitial macrophages or nonmacrophage
cell types might be responsible for the production of mRNA of
inflammatory mediators. Macrophage depletion, however, showed that mRNA
of the mediators were produced in alveolar macrophages or at least
induced by them. Hypoxia-induced NF-B activation in whole lung
tissue seems to be macrophage dependent as well. This implies that
products of activated alveolar macrophages are required to stimulate
nuclear translocation of NF-
B, which has been shown in the IgG model
(17). The same hypothesis can also be applied to enhanced
whole lung HIF-1
, which is also attenuated by macrophage depletion.
Again, activation of HIF-1
might be triggered by alveolar
macrophages or even produced by macrophages. The fact that mRNA of
inflammatory mediators is not completely abolished suggests the
presence of other sources for inflammatory mediators such as
interstitial macrophages, AEC, or endothelial cells. It is well known
that AEC are able to endocytose liposomes (12). In view of
the less than perfectly complete macrophage depletion, a small residual
fraction of alveolar macrophages could be another source of
inflammatory mediators. Uneven distribution of the intratracheal
liposomes cannot be fully excluded in this context. An interesting
finding was the observation that macrophage depletion did not affect
MCP-1 expression compared with other inflammatory mediators.
Macrophages thus do not appear to be a main source of MCP-1. Epithelial
cells might be a more important production site for MCP-1 as has been
previously shown in the kidney in tubular epithelial cells
(2).
A very important aspect of the data in this model is not only the inflammatory component, but also physiological mechanisms. Alveolar hypoxia might increase ventilation and therefore cause a mechanical distension of lung tissue. Few publications examine the impact of hyperpnea on inflammatory mechanisms. Most of these studies, however, were performed with dry air, and experiments evaluated the effect of repetitive hyperpnea (8, 9, 22). Their results do not correlate with our experiments. A potential impact of hyperpnea in our model cannot be fully excluded.
MPO data provided valuable information. It could be shown that hypoxia induced a slight interstitial accumulation of neutrophils at an early time point of inflammation. Compared with other models of lung inflammation such as LPS-induced lung injury, however, the magnitude of neutrophil infiltration was small. These results support our interpretation that neutrophils are not the main effector cells in this model.
To further demonstrate the importance of alveolar macrophages in
the hypoxia-induced inflammation, we studied the in vitro expression
pattern of TNF- of alveolar macrophages. Interestingly, the increase
of TNF-
production was not impressive compared with the in vivo
data. Therefore, we assume that not only the direct input of alveolar
macrophages is central in the hypoxic inflammatory reaction but also
the indirect influence of these cells on other cell types in the lung.
Similar results were also seen in IgG lung injury model
(17).
In conclusion, this study demonstrates that acute hypoxia results in inflammatory changes in the lung representing a mild lung injury, whereby alveolar macrophages are the main effector cells during this inflammatory response. The precise interaction of effector cells with target cells in this model of acute hypoxia will be further investigated.
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ACKNOWLEDGEMENTS |
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The authors thank Christian Gasser for technical assistance.
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FOOTNOTES |
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This study was supported by Swiss National Science Foundation Grant 31-55702.98 and the Gebert Rüf Stiftung, Switzerland.
Address for reprint requests and other correspondence: B. Beck-Schimmer, Inst. of Anesthesiology, Inst. of Physiology, Univ. Zurich-Irchel, Winterthurerstr. 190, CH-8057 Zurich, Switzerland (E-mail: bbeck{at}physiol.unizh.ch).
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 11, 2002;10.1152/ajplung.00158.2002
Received 6 June 2002; accepted in final form 9 October 2002.
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