1 Pulmonary/Critical Care Section, Providence Veterans Affairs Medical Center, Brown University School of Medicine, Providence, Rhode Island 02908; and 2 University of Maryland at Baltimore School of Pharmacy, Baltimore, Maryland 21201
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ABSTRACT |
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Accumulation of intraluminal polymorphonuclear leukocytes (PMN) is a hallmark of inflammatory diseases of the airways. Extracellular nucleotides stimulate PMN adhesion to human main pulmonary artery endothelial cells (HPAEC) by a purinoceptor-mediated mechanism. We investigated the effects of nucleotides on adhesion of freshly isolated human PMN to cultured human tracheobronchial epithelial cells (HBEC). We found that extracellular ATP and UTP were much less effective in stimulating PMN adhesion to HBEC compared with HPAEC, whereas the bacterial chemotactic peptide N-formyl-Met-Leu-Phe stimulated PMN adhesion to both cell types to an equal degree. We investigated several mechanisms that might account for decreased nucleotide-induced PMN adhesion to HBEC. The ectonucleotidase-resistant ATP analog adenosine 5'-O-(3-thiotriphosphate) was also ineffective in stimulating PMN adhesion to HBEC, indicating that degradation of ATP by ectonucleotidase(s) was not responsible for altered PMN adhesion. HBEC responded to ATP and UTP with increased intracellular calcium, indicating that these cells are capable of purinoceptor-mediated responses. We found that ATP and UTP also did not stimulate PMN adhesion to Chinese hamster ovary (CHO) cells, which had been stably transfected with the gene for hamster Muc1, a cell-associated mucin. However, ATP and UTP did stimulate adhesion of PMN to nontransfected CHO cells. These results suggested that MUC1 mucin modulates PMN adhesion to epithelium. We found that cultured HBEC expressed more mRNA and protein for MUC1 mucin than did HPAEC. We conclude that extracellular nucleotides are less effective in stimulating PMN adhesion to epithelial cells than to endothelial cells and that overexpression of hamster Muc1 mucin inhibits nucleotide-induced PMN adhesion to CHO cells.
adenosine 5'-triphosphate; uridine 5'-triphosphate; purinoceptor; polymorphonuclear leukocyte adhesion; endothelium
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INTRODUCTION |
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ACCUMULATION OF intraluminal polymorphonuclear leukocytes (PMN) is a hallmark of inflammatory airway diseases, such as chronic bronchitis, cystic fibrosis, and asthma. Adhesion of PMN to other cells is a crucial early step in PMN-mediated tissue injury. Considerable information has been gathered regarding vascular endothelial cell-PMN adhesive interactions, but less is known about bronchial epithelial cell-PMN adhesion.
Extracellular nucleotides, such as ATP and UTP, are important proinflammatory mediators, causing PMN release of superoxide anion (27), PMN aggregation (6), and PMN degranulation (30). Potential sources of extracellular nucleotides include cell lysis, platelet degranulation, and secretion via the ABC family of transporters (5, 8). We have reported that extracellular ATP and UTP increase PMN adhesion to endothelium via a purinoceptor-mediated mechanism, which is prevented by platelet-activating factor antagonists (3, 21). In the current studies, we determined the effects of extracellular ATP and UTP on PMN adhesion to monolayers of cultured human tracheobronchial epithelial cells (HBEC). We found that extracellular ATP and UTP were much less effective in stimulating PMN adhesion to tracheobronchial epithelial cells than to main pulmonary artery endothelial cells. We examined several mechanisms by which airway epithelial cells might be protected from nucleotide-induced PMN adhesion. We found that ATP and UTP were not effective in stimulating PMN adhesion to Chinese hamster ovary (CHO) cells stably transfected with the gene for hamster Muc1, a cell-associated mucin. We compared cultured pulmonary arterial endothelial cells and tracheobronchial epithelial cells for mRNA in the transmembranous domain of MUC1 and found that epithelial cells express more MUC1 mRNA than endothelial cells. In addition, we compared epithelial and endothelial cell cultures with respect to MUC1 protein expression.
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MATERIALS AND METHODS |
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Materials.
ATP, UTP, ,
-methylene ATP, and
N-formyl-Met-Leu-Phe chemotactic
peptide (fMLP) were obtained from Sigma Chemical (St. Louis, MO).
Adenosine
5'-O-(3-thiotriphosphate)
(ATP
S) and murine anti-von Willebrand factor antibody were supplied
by Boehringer Mannheim (Indianapolis, IN). Fura 2-AM (acetoxymethyl
ester) was obtained from Molecular Probes (Eugene, OR). Antibody
to MUC1 (C-20) was obtained from Santa Cruz Biotechnology (Santa
Cruz, CA).
Culture of human pulmonary artery endothelial cells. Human main pulmonary artery endothelial cells (HPAEC) were obtained from Clonetics (San Diego, CA) and grown in endothelial cell growth medium (Clonetics) containing 2% fetal bovine serum (FBS). Primary cultures reached confluence in 7-9 days and were subcultured every 7-10 days using trypsin-EDTA. Cultures were refed 24 h after subculturing and routinely every 48 h.
Endothelial cells were identified by typical phase-contrast "cobblestone" morphology, by immunofluorescence to factor VIII antigen, and by uptake of acetylated low-density lipoprotein labeled with fluorescent 1,1'-dioctadecyl-1,3,3,3',3'-tetramethylindocarbocyanine perchlorate (Biomedical Technologies, Stoughton, MA).Culture of HBEC. HBEC were obtained from Clonetics and grown in epithelial cell growth medium containing 0.1 µg/ml of retinoic acid [bronchial epithelial growth medium (BEGM)]. Primary cultures reached confluence in 7-9 days and were subcultured every 7-10 days using trypsin-EDTA. Cultures were refed with BEGM 24 h after subculturing and routinely every 48 h.
Isolation of human neutrophils. Heparinized human blood was obtained by venipuncture from male and female healthy volunteers from whom informed consent had been obtained under a protocol approved by the Brown University and Providence Veterans Affairs Medical Center Institutional Review Boards. All human blood was handled in accord with "universal precautions" to prevent accidental infection of technical personnel with bloodborne viruses. PMN were isolated using a Hypaque-Ficoll (Pharmacia, Piscataway, NJ) centrifugation technique, as previously described (3).
51Cr labeling of neutrophils and adherence to endothelium. Neutrophil adherence was assessed with 51Cr-labeled cells in a manner similar to that described by Parker et al. (21). PMN (2 × 107/ml) suspended in sterile PBS-10 mM glucose were labeled with Na251CrO4 (50 µCi) for 60 min at 37°C. Cells were then washed two times with sterile PBS-0.1% BSA and resuspended in MEM containing 0.1% BSA (MEM-BSA) at 2 × 106 cells/ml.
Confluent monolayers of HPAEC, HBEC, or CHO cells in 24-well plates were washed two times with MEM-BSA, followed by the addition to each well of 0.25 ml of MEM-BSA with or without nucleotides or fMLP. The 51Cr-labeled PMN (5 × 105/0.25 ml) were added to each monolayer, and the plates were then incubated at 37°C in 95% air-5% CO2. After 15 min, the nonadherent cells were removed with gentle agitation, the monolayers were then washed one time with 0.5 ml MEM, and the washes were combined with the monolayer supernatant for subsequent 51Cr counting. The endothelial or epithelial cells and adherent PMN were lysed with 0.5 ml of 1 N NaOH, and the lysate plus one PBS wash were combined for 51Cr counting. Percent adherence was calculated as the ratio of counts per minute (cpm) adherent to cpm adherent plus cpm nonadherent × 100.Intracellular calcium concentration.
HBEC were cultured on glass coverslips previously coated with Cell-Tak
(Collaborative Research, Bedford, MA). The fura 2 technique was used to
assess changes in intracellular calcium caused by 100 µM
,
-methylene ATP, ATP, or UTP. The slides were incubated with 3 µM fura 2 in HEPES-buffered saline, pH 7.35, for 60 min at room
temperature, rinsed with HEPES-buffered saline, and then positioned in
a cuvette in a dual-beam fluorescence spectrophotometer in
HEPES-buffered saline at 37°C. Baseline fluorescence at excitation 340 and 380 nm and emission 505 nm were measured, ligands were added to
the cuvette, and changes in fluorescence were monitored over 5 min.
Background fluorescence of an unstained slide was also determined and
subsequently subtracted from the experimental data. The same slit width
was used for all experiments. The ratio of fluorescence at the two
excitation wavelengths was calculated, and the peak ratio was compared
with baseline as a measure of changes in intracellular calcium caused
by the various ligands.
Transfection of CHO cells. The hamster tracheal surface epithelial Muc1 mucin gene was isolated as described by Park et al. (20), and stable transfectants and vector controls of CHO cells were prepared (11).
Culture of CHO cells. CHO cells were fed a 1:1 mixture of F-12 medium and Dulbecco's modified Eagle medium (F-12-DMEM, GIBCO BRL, Gaithersburg, MD) with 5% FBS every 48-72 h. Cells were subcultured with trypsin-EDTA every 10-14 days. CHO-Muc1 cells were handled in an identical manner except that F-12-DMEM with 5% FBS was supplemented with geneticin at 300 µg/ml to prevent emergence of a nontransfected strain.
Generation of human MUC1 cDNA probe. Cultures of HBEC were harvested in PBS, and total RNA was isolated. Reverse transcription-polymerase chain reaction was used to isolate and amplify cDNA of the sequence nucleotides 3020-3595 of MUC1, as sequenced by Hollingsworth et al. (10). This cDNA corresponds to a 192-amino acid peptide, which spans the membranous portion of MUC1 including a small fragment of the extracellular domain. The upstream (sense) primer was 5'-TGCTCTAGAGCACAACCCCAGCCAGCAAGAGCACT-3' and the downstream (antisense) primer was 5'-CCGCTCGAGCGGCCAGCGCAACCAGAACACAGAC-3', synthesized by GIBCO BRL and included Xba I and Xho I sites, respectively. The resulting 645-bp cDNA fragment was subcloned into pBluescript (Stratagene, LaJolla, CA), with the use of unique Xba I and Xho I sites. The sequence of the MUC1 cDNA fragment was confirmed using an ABI 377 automatic DNA sequencer.
Northern blot analysis. Total RNA was isolated from HBEC and HPAEC incubated with various ligands for 15 min. RNA (20 µg) was resolved by 1% agarose-formaldehyde gel electrophoresis. The RNA was transferred by vacuum blot to Genescreen Plus membranes (NEN Life Sciences, Boston, MA), and the blot was ultraviolet cross-linked and hybridized with random-primed MUC1 cDNA probes at 69°C for 1 h in QuickHyb solution (Stratagene). Blots were washed under high stringency. Autoradiographs were analyzed by densitometry. Blots were normalized for equal loading using glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Western immunoblot analysis. Confluent cultures of HPAEC and HBEC were washed with PBS and scraped, and then cells were lysed with (in mM) 50 HEPES buffer, pH 7.0, 150 NaCl, 5 EGTA, 5 EDTA, 20 NaF, 20 sodium pyrophosphate, 1 Na3VO4, and 1 phenylmethylsulfonyl fluoride, and 1% Triton X-100 (RIPA buffer). Protein (200 µg total) was immunoprecipitated with 0.8 µg of goat anti-human polyclonal antibody directed against the carboxy terminus (constant sequence) of MUC1 in 500 µl of RIPA buffer for 1 h at 4°C with shaking. Protein G agarose (50 µl, Pierce) was added in a 50% slurry in RIPA and incubated overnight at 4°C with shaking. Immunoprecipitates were washed two times with RIPA buffer (500 µl) and one time with high-salt (350 mM NaCl) RIPA buffer. Samples were resuspended in Laemmli buffer and subjected to electrophoresis using a 7.5% polyacrylamide gel, with equal loading of protein into all lanes. The proteins were electrophoretically transferred to nitrocellulose and incubated for 30 min in blocking buffer (PBS-0.05% Tween-3% BSA). The blots were then probed with MUC1 antibody (1:1,000 dilution) in PBS-0.05% Tween-3% BSA for 1 h at room temperature. The blots were rinsed two times with PBS-0.05% Tween and incubated for 1 h at room temperature with 1:3,000 donkey anti-goat secondary antibody conjugated to horseradish peroxidase (Jackson Immunoresearch Laboratory, West Grove, PA). The proteins of interest were visualized by the enhanced chemiluminescence method as described by the manufacturer (Amersham). Blots were analyzed by scanning densitometry.
Statistics. Data are expressed as means ± SE. Groups were analyzed for significant differences with analysis of variance and Fisher's least significant difference multiple comparison test (Statview). Differences were considered significant at P < 0.05.
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RESULTS |
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As previously reported (3, 21), both ATP and UTP increased adhesion of
human PMN to HPAEC (Fig. 1). However, the
same concentration (100 µM) of these nucleotides had considerably
less effect on adhesion of human PMN to HBEC (Fig. 1). In contrast, the
bacterial chemotactic peptide fMLP was equally effective in stimulating
PMN adhesion to both HPAEC and HBEC (Fig. 1). Thus the freshly isolated
human PMN used in these studies were capable of stimulation of
adhesion. Dose responses of ATP and UTP effects on PMN adhesion to HBEC
revealed little effect at concentrations less than 100 µM (Fig.
2), whereas our previous studies had shown increased adhesion to HPAEC at concentrations as low as 1 µM (21). Thus the nucleotides ATP and UTP were less effective in stimulating PMN
adhesion to tracheobronchial epithelial cells than to pulmonary artery
endothelial cells.
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One possible explanation for this difference might be more effective
ectonucleotidase activities on HBEC, which could hydrolyze ATP and UTP,
thereby preventing a purinoceptor-mediated adhesive interaction. To
test for this possibility, we compared the proadhesive effectiveness of
ATPS, a nonmetabolizable ATP analog. We found that ATP
S
stimulated PMN adhesion to HPAEC but not to HBEC (Fig. 3). Thus it is unlikely that enhanced
ectonucleotidase activity is the cause of the decreased
nucleotide-induced adhesion of PMN to HBEC.
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Another possible explanation for our results was that the
tracheobronchial cells that we utilized did not bear purinoceptors and
thus were resistant to purinoceptor-mediated adhesions stimulated by
ATP and UTP. To test for this possibility, we determined the effects of
ATP, UTP, and the P2X analog ,
-methylene ATP on intracellular calcium concentrations, as assessed by fura 2. As is shown in Fig.
4, both ATP and UTP, but not
,
-methylene ATP, increased intracellular calcium in HBEC, an
effect consistent with purinoceptor-mediated responses (5). Thus lack
of purinoceptors on HBEC did not explain the differences between HPAEC
and HBEC.
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Another possible difference between HPAEC and HBEC is the presence of
cell-associated mucin(s) on the apical surface of HBEC. Cell-associated
mucin, such as MUC1, might serve to protect the surface of HBEC from
PMN adhesion. To test for this possibility, we assessed the effect of
ATP and UTP on PMN adhesion to CHO cells, which had been stably
transfected with the gene for hamster tracheal Muc1 or vector controls.
We found that both ATP and UTP effectively stimulated PMN adhesion to
vector control CHO cells but not to CHO cells which had been
transfected with the gene for hamster Muc1 (Fig.
5). However, fMLP was effective in
stimulating PMN adhesion to both control and transfected CHO cells
(Fig. 5).
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In other experiments, we compared HPAEC and HBEC for expression of MUC1
mRNA. We found that both cell types express mRNA for MUC1, assessed
using a cDNA probe for the membranous portion of the molecule (Fig.
6A).
When normalized for loading of mRNA into lanes using GAPDH, HBEC
expressed more mRNA for MUC1 than HPAEC (ratio of densitometric
measurement, MUC1/GAPDH: HPAEC 0.220, HBEC 0.334).
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We demonstrated that bronchial epithelial cells express more MUC1 protein by Western blot analysis of MUC1 mucin immunoprecipitated using antibody to the constant carboxy terminus of MUC1 (Fig. 6B). These results were confirmed by densitometry (HPAEC 0.297, HBEC 0.528).
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DISCUSSION |
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In these studies, we demonstrate that the extracellular nucleotides ATP and UTP are much less effective in stimulating adhesion of human PMN to cultured monolayers of HBEC than to monolayers of HPAEC. However, the bacterial chemotactic peptide fMLP stimulates PMN adhesion to both cell types.
In our previous studies, we found that adenosine was much less
effective than nucleotides in stimulating PMN adhesion to endothelial cells and that the pattern of nucleotide enhancement of PMN adhesion indicated that the effect was mediated via a P2Y2
purinoceptor (3). Therefore, one possible explanation for decreased
nucleotide-induced adhesion to epithelial cells is increased
ectonucleotidase activity, which could effectively lower the
concentration of extracellular nucleotide ligand. Our current results
indicate that the blunted adhesion to epithelial cells is not due to
enhanced ectonucleotidase activity because the nonmetabolizable
nucleotide ATPS was also ineffective in stimulating PMN adhesion to
epithelial monolayers. The epithelial cells responded to ATP and UTP,
but not to
,
-methylene ATP, with increased intracellular calcium
concentrations, as would be expected for epithelium bearing
P2Y2 purinoceptors (5, 13, 18).
We reasoned that one possible explanation for the decreased PMN adhesion to epithelium might be more abundant mucin associated with cells or secreted into the medium. Secretion of mucins by differentiated epithelial cells is stimulated by ATP and UTP via a P2Y2 purinoceptor-mediated mechanism (1, 13, 15, 18). Secretion occurs rapidly, with maximal effect reported within 30 min (15, 18). Among the physiological effects of mucins are decreased cell-matrix adhesion (29), decreased homophilic cell-cell adhesion (16, 28), and decreased adhesion of leukocytes to activated endothelium (31). Mucins are large, complex glycoproteins consisting of a protein core that is highly glycosylated via O-glycosidic bonds between N-acetylgalactosamine of oligosaccharides and serine and threonine in the protein core (24). The molecules are highly anionic due to sulfation of oligosaccharides and terminal sialation (26). Nine mucin genes have been identified, MUC1-MUC8, to date. Respiratory tract epithelia have been reported to express MUC1, MUC2, MUC 4, MUC5AC, MUC5B, and MUC8 (12, 24). Analysis of amino acid sequences indicates that mucins may exist in secreted or cell membrane-associated forms. MUC1 mucin, also called episialin, is a cell-associated mucin with cytoplasmic, transmembrane, and extracellular domains (7, 24). It is expressed by a variety of epithelial cells, including hamster (20) and human (10) tracheobronchial epithelial cells. The highly glycosylated extracellular domain of MUC1 mucin is estimated to extend as far as 200-500 nm from the cell surface (9). Cell-associated mucins, such as MUC1, might impair leukocyte adhesion via several possible mechanisms including steric hindrance, charge repulsion due to the anionic nature of the sulfated and sialated extracellular domains, and interaction of sugars with lectinlike adhesion molecules, thereby blocking adhesion receptors.
We tested the effects of cell-associated mucin on PMN adhesion by assessing PMN adhesion to CHO cells, which had been transfected with the gene for hamster Muc1 and which overexpressed Muc1 mucin (11). We found that extracellular ATP and UTP were ineffective in stimulating PMN adhesion to CHO cells, which had been transfected with the gene for hamster Muc1, whereas both nucleotides stimulated PMN adhesion to vector control CHO cells. There were no differences in PMN adhesion stimulated by the bacterial chemotactic peptide fMLP. These results indicate that overexpression of hamster Muc1 mucin inhibits PMN adhesion but that this effect is unique to nucleotide-induced PMN adhesion. This is similar to our results comparing endothelial and epithelial cells in that only nucleotide-induced adhesion was decreased to epithelial cells. The mechanism of inhibition of adhesion by Muc1 mucin is not due to simple steric hindrance, inasmuch as we would expect similar inhibition of fMLP-induced PMN adhesion. It is unlikely that nucleotides changed synthesis of mucin, inasmuch as only a 15-min incubation was required for PMN adhesion. It is also unlikely that nucleotides stimulated secretion of Muc1 mucin, inasmuch as Meerzaman et al. (17) have reported that the majority of mucins secreted by hamster tracheal surface epithelial cells in response to ATP are not Muc1. Another possible explanation for the differences between nucleotide- and fMLP-induced PMN adhesion is removal of cell-associated mucin in the presence of fMLP. Degranulation of PMN in the presence of fMLP might release proteolytic enzymes capable of removal of the extracellular domain of cell-associated mucin (14). ATP also causes degranulation of PMN, although it is less effective than fMLP (30). Finally, it is possible that extracellular ATP and UTP change cell-surface display of Muc1 mucin such that PMN adhesion is impaired.
Thus it appeared that the presence of cell-associated MUC1 mucin might explain the differences in nucleotide-stimulated PMN adhesion between epithelial and endothelial cells. In support of this, cultured epithelial cells expressed more MUC1 mRNA and protein than cultured pulmonary artery endothelial cells. We were surprised to find MUC1 mRNA and protein expression by pulmonary vascular endothelial cells. However, MUC1 immunohistochemical staining has been reported on high endothelial cells of postsinusoidal venules of lymph nodes, on the luminal surface of mesothelium, and on nonepithelial malignant cells (9). Thus it appears that cells other than epithelium are capable of expression of MUC1 mucin.
Airway inflammation is an important factor in the pathogenesis of
chronic bronchitis. Chronic bronchitis with increased PMN in early
aliquots of bronchial alveolar lavage fluid ("bronchial" sample)
had more sputum production and lower airflow rates than those with
lesser numbers of PMN (23). In airways, PMN migrate across the
basolateral epithelial surface from blood vessels into the airway
lumen. Our studies address PMN adhesion to the apical surfaces of HBEC.
However, PMN adhesion to the luminal (apical) surface of bronchial
epithelium might exacerbate epithelial injury. Also, inhibition of PMN
adhesion to the apical surface of tracheobronchial epithelium would
prevent emigration of PMN back into the airway wall. Several factors
have been reported to enhance PMN adhesion to tracheobronchial
epithelial cells, including phorbol myristate acetate (22), cytokines
(interferon-, tumor necrosis factor-
) (2), serum (25), and
substance P (4). On the other hand, our results suggest that MUC1 mucin
might have a protective effect toward PMN adhesion to epithelium.
Potential sources of extracellular nucleotides in airways include release from neurons, secretion from epithelium via membrane transporters, and lysis of necrotic cells (8). The latter may be especially important because cytoplasmic levels of ATP are in the millimolar range (8).
Inhaled UTP is a potential mucokinetic agent (19). If extracellular nucleotides enhance secretion or display of mucin(s) protective against PMN adhesion, then inhaled nucleotides might possess anti-inflammatory effects also.
In summary, we report that extracellular ATP and UTP are much less effective in stimulating PMN adhesion to tracheobronchial epithelium than to pulmonary artery endothelium. We also show that overexpression of hamster Muc1 mucin is protective against PMN adhesion in response to extracellular ATP and UTP. We show that epithelial cells express more MUC1 mRNA and protein than endothelial cells. We speculate that the reason for decreased nucleotide-induced PMN adhesion to epithelium is enhanced cell surface display of MUC1 mucin. We further speculate that inhaled nucleotides might be protective against PMN adhesion to the apical surface of airway epithelium.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the Cystic Fibrosis Foundation, the Dean of Brown University School of Medicine, and Veterans Affairs Merit Review and National Heart, Lung, and Blood Institute Grant HL-47125.
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FOOTNOTES |
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Some of the reported studies were presented at the annual meeting of the American Thoracic Society in May 1997 and were published in abstract form (Am. J. Respir. Crit. Care Med. 155: A361, 1997).
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 and other correspondence: S. Rounds, Pulmonary/Critical Care Section, Providence VA Medical Center, 830 Chalkstone Ave., Providence, RI 02908 (E-mail: sharon_rounds{at}brown.edu) .
Received 14 September 1998; accepted in final form 24 June 1999.
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