1 McDonald Research Laboratory and iCAPTURE Center, University of British Columbia, Vancouver, British Columbia V6Z 1Y6; and 2 Environmental Health Directorate, Health Canada, Ottawa, Ontario K1A 0L2, Canada
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ABSTRACT |
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We examined the
hypothesis that ambient particulate matter with a diameter of <10 µm
(PM10)-induced lung inflammation is amplified by latent
adenovirus infection. Inflammatory mediator expression in response to
PM10 exposure was compared between adenovirus
E1A-transfected A549 alveolar epithelial cells and cells transfected
with control plasmid. Messenger RNA was measured by the RNase
protection assay and protein by ELISA or immunocytochemistry.
Intercellular adhesion molecule-1 and IL-8 mRNA and protein were
increased in E1A-positive cells exposed to 500 µg/ml
PM10. Monocyte chemoattractant protein-1 mRNA and protein
were unchanged in E1A-positive cells but increased in E1A-negative
cells after 100 and 500 µg/ml PM10 exposure.
Electrophoretic mobility shift assays showed increased NF-B and
decreased specificity protein 1 nuclear binding in E1A-positive cells
exposed to PM10. These results indicate that E1A modulates
cytokine and adhesion molecule expression in epithelial cells in a
manner that could amplify PM10-induced lung inflammation.
We suggest that this amplified inflammatory response may contribute to
the pathogenesis of exacerbations of chronic obstructive pulmonary
disease associated with exposure to particulate matter air pollution.
interleukin-8; monocyte chemoattractant protein-1; intercellular
adhesion molecule-1; nuclear factor-B; transcription factor
specificity protein 1; particulate matter
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INTRODUCTION |
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RESIDENTS OF COMMUNITIES exposed to high, compared with low, levels of air pollution, especially ambient particulate matter with a diameter of <10 µm (PM10), are at a greater risk of developing lung disease. Several epidemiological studies have shown that they develop faster rates of decline in lung function and have increased hospital admissions for pneumonia and more chronic obstructive pulmonary disease (COPD) even after adjusting for individual risk factors such as cigarette smoking (10, 30). Although the precise biological mechanisms responsible for this are unclear, PM10 are known to stimulate the production of reactive oxygen species and inflammatory mediators by alveolar macrophages (3, 28, 37), airway epithelial (6, 8, 13, 27), and other lung cells (14, 24).
Cigarette smoking is the main risk factor for COPD, because it causes
peripheral lung inflammation in everyone; but only 10-20% of
chronic heavy smokers develop emphysema and airway obstruction (12). Childhood respiratory infections primarily caused by
viruses (reviewed in Ref. 25) are an independent risk
factor for the development of COPD (15), and previous work
from our laboratory has implicated the E1A gene of adenovirus in the
amplification of cigarette smoke-induced lung inflammation in both
human and animal studies (11, 25, 26, 31). Investigations
into the mechanism of this response in a human tumor cell line
with type II cell characteristics (A549 cells) have shown that
transfection with the adenovirus E1A gene increased intercellular
adhesion molecule-1 (ICAM-1) and interleukin (IL)-8 expression in
response to lipopolysaccharide stimulation (21, 22). This
upregulation correlated with increased activity of the transcription
factor nuclear factor-B (NF-
B) in these E1A-positive cells
(23).
Gilmour and coworkers (14) have shown that adenovirus E1A increased IL-8 production when A549 cells are exposed to particles. The aim of the current study was to test the hypothesis that the presence of E1A in lung epithelial cells modulates the expression of several inflammatory mediators in response to exposure to ambient particulate matter. The mRNAs for 17 candidate inflammatory mediators were monitored by an RNase protection assay that simultaneously quantifies several mRNA species in a single sample of total RNA. ELISA and immunocytochemistry were used to detect the proteins specified by these mRNAs, and an electrophoretic mobility assay (EMSA) was used to identify transcription factors that were modulated by PM10 stimulation of the E1A expressing A549 cells.
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MATERIALS AND METHODS |
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Particles (PM10). PM10 particles (EHC-93) were collected by the Environmental Health Directorate, Health Canada, Ottawa. A detailed analysis of the EHC-93 has been presented elsewhere (13). Particles were suspended at a concentration of 1 mg/ml in MEM containing 10% fetal bovine serum (FBS; Life Technologies, Rockville, MD), antibiotics (100 U/ml of penicillin and 100 µg/ml of streptomycin; Sigma, St. Louis, MO), and Fungizone (1 µg/ml; GIBCO-BRL, Gaithersburg, MD) and were sonicated three times for 1 min each at maximal power on a Vibra Cell VC-50 sonicator (Sonics & Materials, Danbury, CT) before being added to the cells. The endotoxin content of this suspension was measured and reported to be very low (<0.015 EU/ml) (13).
Cell culture. A549 cells (American Type Culture Collection, Manassas, VA), a human lung epithelial cell line, had been stably transfected with the plasmid pE1Aneo, a generous gift from Dr. F. L. Graham (McMaster University, Hamilton, Canada), using polycationic liposomes as previously reported (21). This plasmid carries the promoter and entire coding region of the E1A gene of adenovirus 5. A plasmid generated from pE1Aneo by deleting a 1.8-kb BamHI-SacI fragment containing the E1A DNA was used as a transfection control (21). E1A transfectants (E1A-positive cells) analyzed in this study, E4 and E11, are two independent clones of these transfected cells. The control transfectants (E1A-negative cells) tested here, C4 and C8, are two independent clones of A549 cells transfected with the control plasmid lacking the E1A gene (21). Northern and Western blotting and immunocytochemistry showed that all E1A transfectants produced relatively high levels of E1A mRNA and E1A proteins (21). These transfectants were grown in MEM supplemented with 10% heat-inactivated FBS in 5% CO2 at 37°C.
E1A-positive cells and E1A-negative cells grown to confluence in MEM plus 10% FBS in 60-mm cell culture dishes (Falcon; Becton Dickinson, Ontario, Canada) were exposed to 2 ml of MEM containing 10% FBS, antibiotics, and Fungizone as described in Particles (PM10) or 2 ml of the PM10 preparation, which gave a final concentration of 100 or 500 µg/ml PM10.RNase protection assay.
Three, six, and nine hours after addition of PM10 to a
final concentration of 100 or 500 µg/ml, total RNA was isolated from cell cultures using a single-step phenol-chloroform extraction procedure (TRIzol; Life Technologies, Grand Island, NY). Cytokine mRNA
levels were determined with an RNase protection assay (RPA) RiboQuant
multiprobe system (PharMingen, San Diego, CA) following the
instructions of the supplier. Two customized template sets were used.
One set was for human IL-12, IL-10, ICAM-1, macrophage inflammatory
protein (MIP)-1, granulocyte colony-stimulating factor (G-CSF),
IL-8, transforming growth factor (TGF)-
1, and interferon (IFN)-
.
The second was for human regulated on activation, normal T cell
expressed, and secreted (RANTES); tumor necrosis factor (TNF)-
;
granulocyte-macrophage (GM)-CSF; IL-1
; monocyte chemoattractant
protein (MCP)-1; IL-6; leukemia inhibitory factor (LIF); vascular
endothelial growth factor (VEGF); and oncostatin M (OSM). Both template
sets also included ribosomal protein (L32) and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal controls.
In brief, 10 µg of total cellular RNA were hybridized overnight
to the [
-32P]UTP-labeled riboprobes that had been
synthesized from the supplied template sets. Single-stranded RNA and
free probe remaining after hybridization were digested by a mixture of
RNase A and T1. After the proteins were removed by phenol extraction,
the protected RNA was precipitated and analyzed on a 5% denaturing
polyacrylamide gel. After electrophoresis, the gel was dried and
subjected to autoradiography. The quantity of protected RNA was
determined by densitometry and use of the NIH Image J 1.28 software
(National Institutes of Health) available at http://rbs.info.nih.gov.
The density of the band representing the mediator mRNA of interest was
normalized to that of the internal control, GAPDH, in the same lane of
the gel, and the resulting ratio was expressed as the percentage change
from the corresponding ratio for the untreated E1A-negative cells on
the same gel. For the densitometric analysis, the RPA was repeated four
times with the first template set after 6 h of treatment and with
the second template set after 3 h.
Immunocytochemistry. To demonstrate cell surface ICAM-1 expression, we grew E1A-positive cells and E1A-negative cells on coverslips in six-well plates and incubated them with 500 µg/ml of PM10 for 24 h. Cells were fixed with 1% paraformaldehyde for 10 min, and immunocytochemistry was performed by the alkaline phosphatase antialkaline phosphatase method using mouse anti-human CD54 monoclonal antibody (Immunotech, Marseille, France) to identify ICAM-1.
ELISA measurements.
Cells were seeded at 0.8 × 106 cells/60-mm
plate. Three days later we added either 2 ml of medium alone
(control) or medium with PM10 suspension to give a final
concentration of 100 or 500 µg/ml. After being cultured for 24 h, supernatant was removed, centrifuged, filtered through a syringe
filter with pore size of 0.22 µm (Corning, Cambridge, MA), and stored
at 70°C. IL-8 and MCP-1 levels were measured by the Cytokine Core
Laboratory (Baltimore, MD) using an ELISA based on a
biotin-streptavidin-peroxidase detection system as previously described
(13). These values were corrected for cell numbers at the
time of collection, because each cell type had a different morphology
and growth rate. Each condition was tested six times, the measurements
for the two cytokines in each of the six supernatants were done in
triplicate, and the values reported are the mean of the six experiments.
EMSA.
E1A-positive and E1A-negative cells grown to confluence in 60-mm dishes
were exposed to culture medium or to medium with 500 µg/ml
PM10 for 1 and 2 h. After being washed twice with PBS,
the nuclear proteins were extracted as previously described
(23) except for the use of 1:500 diluted proteinase
inhibitor cocktail (Sigma, St. Louis, MO) in the place of
phenylmethylsulfonyl fluoride and leupeptin. EMSA of specific nuclear
proteins was carried out as previously described (23).
Briefly, 10 µg of the nuclear protein extract were incubated at 4°C
for 30 min with four volumes of binding buffer (10 mM
Tris · HCl, 50 mM NaCl, 1 mM EDTA, 1 mM
dithiothreitol, and 4% glycerol) and 1.5 µg of poly(dI-dC) (Boehringer Mannheim, Mannheim, Germany) followed by the addition of
[-32P]ATP end-labeled double-strand consensus
oligonucleotides for NF-
B: 5'-AGT TGA GGG GAC TTT CCC AGG-3', AP-1:
5'-CGC TTG ATG AGT CAG CCG GAA-3', or specificity protein 1 (Sp1):
5'-ATT CGA TCG GGG CGG GGC GAG-3' (Promega, Madison, WI). Further
incubation for 20 min allowed the formation of protein-DNA complexes.
For competition assays, excess unlabeled double-stranded
oligonucleotides (3.5 pmol) of sequence identical with or unrelated to
that of the labeled probe were added to the nuclear extract before
addition of the labeled probe. The protein-DNA complexes were separated on a nondenaturing 6% polyacrylamide gel. Gels were dried under vacuum
and autoradiographed.
Statistical analysis. Data are expressed as the means ± SE. The minimum number of replicates for all measurement was at least four. Differences between multiple groups were compared by one-way analysis of variance (ANOVA). The post hoc test for multiple comparison was the Fisher's protected least significant difference test. Because of the small sample size of the RPA (n = 4), the densitometric results from this assay were also analyzed by the nonparametric Kruskal-Wallis one-way ANOVA. Significance was assumed at P < 0.05.
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RESULTS |
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Effect of PM10 on expression of cytokine and ICAM-1
mRNAs.
Representative autoradiographs of cytokine mRNA expression by
E1A-negative cells and -positive cells after incubation in medium alone
(control) or with 100 or 500 µg/ml of PM10 suspension are shown in Fig. 1. In the absence of
stimulation, ICAM-1 and IL-8 mRNA levels were greater in E1A-positive
cells than in -negative cells (Fig. 1A). After 6 h of
incubation, E1A-positive cells increased expression of both mRNAs in a
PM10-dose-dependent manner. In E1A-negative cells, IL-8
mRNA levels increased with dose of PM10, whereas ICAM-1 mRNA was difficult to assess because of very low levels of expression. These IL-8 and ICAM-1 mRNA levels were all lower than those of the
E1A-positive cells. TGF-1 mRNA was constitutively expressed with no
differences between control and PM10 stimulation in both cell types (Fig. 1A). Basal levels of MCP-1 mRNA were
greater in E1A-negative cells than in E1A-positive cells (Fig.
1B). In E1A-negative cells this mRNA expression was
increased by PM10 stimulation in a dose-dependent manner
after 3 h of incubation, while E1A-positive cells did not show
this effect. LIF and VEGF mRNAs were constitutively expressed by both
cell types with a small increase in LIF and little or no difference in
VEGF expression with PM10 stimulation (Fig.
1B). Similar results as those reported above for all the
mRNAs were also found after 3 or 6 h and after 9 h of
incubation (data not shown). In both E1A-negative and E1A-positive cells, PM10 at either concentration did not induce mRNA
expression of IL-12, IL-10, MIP-1
, G-CSF, IFN-
, RANTES, TNF-
,
GM-CSF, IL-1
, IL-6, and OSM after 3, 6, and 9 h of incubation
(Fig. 1, A and B, and data not shown).
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Effect of PM10 on expression of surface ICAM-1.
Figure 3 shows E1A-negative and
E1A-positive cells immunocytochemically stained with CD54 to identify
ICAM-1 expression. In the absence of stimulation E1A-negative and
E1A-positive cells rarely expressed ICAM-1 on the cell surface (Fig. 3,
A and C). After stimulation with 500 µg/ml
PM10 for 24 h, E1A-positive cells stained positively
for ICAM-1 (Fig. 3D), while E1A-negative cells were
unaffected (Fig. 3B).
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Effect of PM10 on release of cytokines.
The numbers of E1A-negative cells and E1A-positive cells at the time
the supernatants were collected for ELISA were 3.0 ± 0.2 ×106 cells/well (n = 6) and 4.9 ± 0.2 ×106 cells/well (n = 6), respectively.
The cytokine concentrations measured by ELISA were corrected for these
cell numbers. Figure 4 shows IL-8 and
MCP-1 protein levels in supernatants of E1A-negative and E1A-positive
cells incubated for 24 h with medium alone (control) or with 100 or 500 µg/ml PM10 suspension. Basal IL-8 protein levels were similar in both cell types. IL-8 production in response to 500 µg/ml PM10 by E1A-positive cells was significantly higher than that of the corresponding unstimulated or 100 µg/ml
PM10 stimulated cells and of the E1A-negative cells with or
without stimulation (Fig. 4A). In contrast, basal MCP-1
production was significantly higher in E1A-negative cells than positive
cells. MCP-1 production by E1A-negative cells increased significantly after 500 µg/ml PM10 stimulation, and this value was
higher than those of the E1A-positive cells with or without stimulation
(Fig. 4B).
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Effect of PM10 on NF-B, activator protein-1, and Sp1
binding activity.
Representative autoradiographs of EMSA to detect nuclear NF-
B,
activator protein (AP)-1, and Sp1 binding activity in E1A-negative and
-positive cells after 2 h of incubation in medium alone or 500 µg/ml PM10 suspension are shown in Fig.
5. The basal level of NF-
B binding
activity in E1A-positive cells was higher than in E1A-negative cells.
After PM10 exposure, the binding activity of NF-
B was
weakly increased in the nucleus of E1A-negative cells but, in contrast,
very strongly increased in E1A-positive cells (Fig. 5A).
Basal AP-1 binding activity in the two cell types was similar, and this
binding was weakly increased after PM10 stimulation of both
cells (Fig. 5B). In contrast to NF-
B, E1A-negative cells showed a higher basal level of Sp1 binding activity than E1A-positive cells. After PM10 stimulation, binding of this
transcription factor was increased in E1A-negative cells as well as in
E1A-positive cells (Fig. 5C). Specific binding was abolished
by competition with excess unlabeled oligonucleotide specific for each
transcription factor, whereas the nonspecific competitor was
ineffective (Fig. 5, A-C, two lanes at
right). Similar results were obtained when these experiments
were repeated twice for NF-
B and AP-1 binding and three times for
Sp1 after 3 h of PM10 stimulation and once for each of
these transcription factors after 1 h of stimulation (data not
presented).
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DISCUSSION |
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In the present study we showed that exposure of lung epithelial
cells to ambient particulate matter stimulates these cells to produce
and release a variety of proinflammatory mediators and that this
response is further modified by adenovirus E1A. Our data confirmed an
earlier report by Gilmour and coworkers (14), who found
that E1A increased IL-8 expression by these cells. We also showed that
E1A upregulated ICAM-1 expression and that the increased ICAM-1 and
IL-8 expression was associated with increased activation of the
transcription factor NF-B. In contrast, the increased MCP-1
expression in response to particulate matter was attenuated by E1A, and
this attenuation was associated with decreased activation of the
transcription factor Sp1. We suspect that E1A modulation of the
inflammatory response of epithelial cells to ambient particles
compromises particle processing and increases the particle burden in
the lung.
ICAM-1 and IL-8 are major inflammatory mediators important for the site-specific recruitment of neutrophils and have been implicated in neutrophil-mediated injury (32). They coordinate neutrophil migration by promoting firm adhesion of these cells to the endothelium and epithelium and by providing a chemotactic gradient, respectively. The recruitment and activation of neutrophils to the lung are thought to play an important role in the pathogenesis of COPD by creating an imbalance between neutrophil proteases and the antiproteases that results in proteolytic lung tissue damage (2). ICAM-1 has been reported to be upregulated in the epithelium of patients with chronic bronchitis (reviewed in Ref. 21), and this adhesion molecule is overexpressed as emphysematous lung destruction progresses (31). Studies have shown that particulate matter, such as diesel exhaust particle and silica, stimulates airway epithelial cells to increase ICAM-1 expression (8, 17). Excessive amounts of IL-8 are also present in the airways of patients with COPD (7, 20), and IL-8 production by airway epithelial cells is increased by PM10 exposure (6, 8, 13). This suggests that ICAM-1 and IL-8 might be crucial in regulating the local inflammatory response induced by PM10 exposure, and our results suggest that latent adenoviral infection might amplify the lung inflammation by increasing the expression of these mediators that promote excess neutrophil recruitment.
The expression of ICAM-1 and IL-8 is regulated by redox-sensitive
transcription factors such as NF-B and AP-1 (reviewed in Ref.
23), which are activated in lung cells in response to
particles (18). The results presented here show that the
levels of binding of nuclear NF-
B are enhanced in E1A-positive cells
following PM10 stimulation. This result is consistent with
previous reports using different stimuli, including particles
(14, 23), suggesting that NF-
B activation is
responsible for the upregulation of ICAM-1 and IL-8 genes in
E1A-positive cells. Our results also show that AP-1 binding activity
was relatively high in both E1A-positive as well as negative cells
before stimulation with a small increase in both cell types after
PM10 stimulation. In contrast Gilmour and colleagues
(14) showed a significant elevation of AP-1 binding activity in response to PM10 stimulation in E1A-positive
cells compared with negative cells. This discrepancy could be due to differences in dose (500 vs. 100 µg/ml PM10) or treatment
time (2 vs. 18 h) between our studies and Gilmour's,
respectively. Another variable that could influence the biological
response and thus explain this discrepancy is the composition of the
particles (37). Gilmour and colleagues (14)
used ambient particles with unspecified physical and biochemical
properties, whereas the current study used well-characterized ambient
particles collected over a major North American city (3, 28,
37).
MCP-1 is a potent monocyte/macrophage chemoattractant that also attracts T lymphocytes and basophils to inflammatory sites (1). Several studies have shown that the conducting airways and lung parenchyma of patients with severe COPD are infiltrated with macrophages and T lymphocytes, as well as neutrophils and B cells (31). Macrophages appear to play a critical role in the pathogenesis of COPD by releasing multiple proteinases, especially matrix metalloproteinases, which by breaking down connective tissue in the lung parenchyma produce emphysema (2, 33). By drawing macrophages into the lung, MCP-1 is implicated in disease progression in COPD, and studies on human lung tissue (7) and in a mouse model (16) support this notion. Boitelle and colleagues (4) showed that increased concentrations of MCP-1 are found in bronchoalveolar lavage fluid from patients with coal workers' pneumoconiosis and that macrophages, fibroblasts, and type II cells are involved in the enhanced production of MCP-1. This suggests that MCP-1 production in the lung following particle exposure results in the recruitment of monocytes from the circulating blood into the peripheral airways. These monocytes become activated macrophages that generate multiple proteinases and proinflammatory cytokines involved in the development of lesions associated with COPD (33).
Interestingly, our results show that adenovirus E1A represses MCP-1 expression induced by PM10 stimulation. This observation seems to contradict the hypothesis that E1A contributes to the pathogenesis of COPD, because an increase in the numbers of macrophages is one of the features of this disease (9, 29, 31). However, localized expression of E1A in widely separated epithelial cells could increase the particle burden in these regions of the lung. Inhaled particles deposited deep in the lung are cleared primarily as intracellular particles within macrophages via the tracheo-bronchial route with minor clearance by transport to the regional lymph nodes (5). Therefore, localized repression of macrophage mobilization could interfere with particle clearance in areas where the adenoviral E1A decreased MCP-1 expression. The overstimulation of the alveolar epithelium by this increased particle burden could result in excess production of IL-8 and increase neutrophil traffic. Furthermore, MCP-1 has been shown to protect mice from lethal endotoxemia possibly by decreasing the production of proinflammatory cytokines such as IL-12 and TNF and by promoting anti-inflammatory cytokine IL-10 production (39). Repression of MCP-1 by E1A could amplify the lung inflammation induced by PM10 exposure by removing these protective mechanisms. Therefore, the apparent discrepancy between the well-documented macrophage accumulation in COPD and the results of our studies showing E1A repression of MCP-1 expression in lung epithelial cells could be reconciled by the widespread distribution of E1A-positive cells and might help explain why the disease is localized to certain areas of the lung.
In our studies, adenovirus E1A repression of MCP-1 expression in response to PM10 stimulation was preceded by reduced Sp1 binding activity. Sp1 is one of the first sequence-specific eukaryotic transcription factors to be purified and cloned (19), and Sp1 binding sites or GC boxes are found in numerous genes including that of MCP-1. Ueda and colleagues (36) reported that removal of the GC box resulted in complete loss of basal MCP-1 transcription, whereas overexpression of Sp1 increased its transcription, suggesting that Sp1 controls MCP-1 transcription via the GC box. Other studies have shown that the expression of the murine JE gene that is highly homologous to the human MCP-1 was severely reduced in cells expressing adenovirus E1A (35), that E1A binds the transcriptional coactivator p300, and that this coactivator collaborates with Sp1 in activating gene expression (38). Suzuki and colleagues (34) showed that E1A can induce expression of G10BP, which competes with Sp1 for binding to the G-rich DNA sequences and can therefore inhibit binding of Sp1 to its DNA response element. Collectively, these data suggest that the downregulation of MCP-1 induced by adenovirus in the studies reported here may result from competition for coactivator by E1A and the consequent repression of Sp1 activity.
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ACKNOWLEDGEMENTS |
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This work was supported by Medical Research Council Canada Grants 4219 and 7246, the British Columbia Lung Association, and National Heart, Lung, and Blood Institute Grant HL-66569-03. S. F. van Eeden is the recipient of the American Lung Association Career Investigators Award.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. Hayashi, McDonald Research Laboratory, Univ. of British Columbia, St. Paul's Hospital, 1081 Burrard St., Vancouver, BC, Canada, V6Z 1Y6 (E-mail: shayashi{at}mrl.ubc.ca).
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.00197.2002
Received 19 June 2002; accepted in final form 10 October 2002.
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