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ABSTRACT |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Intratracheal instillation of lipopolysaccharide (LPS) in the rat has been used as a model of acute lung
inflammation. Among the early events in this process is a transient increase in airspace epithelial permeability which peaks 4 h after intratracheal instillation of LPS. The increased epithelial permeability
is concomitant with the influx of neutrophils into the airspaces, peaking 8 h postinstillation. We have
investigated the mechanism of this LPS-induced increase in epithelial permeability. The role of the
neutrophil in LPS-induced epithelial permeability was assessed by pretreatment with neutrophil antibody to abolish neutrophil influx, which did not affect the increase in epithelial permeability. Because
LPS instillation also induced increased tumor necrosis factor 
(TNF-) activity in bronchoalveolar lavage (BAL) fluid, and its release by cultured BAL leukocytes from treated animals, TNF- antibody was
coinstilled intratracheally with LPS in rats. TNF- antibody eliminated TNF- activity in BAL fluid, but
had no effect on LPS-induced increased epithelial permeability. Increased levels of nitric oxide (NO), measured as nitrite, were also present in BAL fluid from LPS-treated rat lungs and LPS-elicited BAL
leukocytes produced increased NO in culture. Treatment of rats with the specific NO synthase inhibitor L-NMMA significantly diminished the LPS-induced increased epithelial permeability. These data
suggest that NO is involved in LPS-induced changes in epithelial integrity. However, other mechanisms should be evoked in addition to NO to explain completely the increased epithelial permeability
produced by LPS.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Increased airspace epithelial permeability is a characteristic feature of many lung diseases, including pulmonary sarcoidosis (1), asthma (2), and adult respiratory distress syndrome (3), and also occurs in smokers (4). The mechanism of the increased airspace epithelial permeability in lung inflammation may vary according to the cause and may involve neutrophils, as been shown in the increased permeability induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine in isolated rabbit lungs (5) and in a rat model of ozone exposure (6). Cytokines, such as tumor necrosis factor (TNF), may also be involved (7, 8). We have shown that alteration in the lung oxidant/antioxidant balance, particularly depletion of the antioxidant glutathione in the airway epithelium and in the epithelial lining fluid, may be an important mechanism of cigarette smoke- induced epithelial permeability (9, 10).
Aerosolized lipopolysaccharide (LPS) as a model of acute
alveolitis increases pulmonary clearance of 99mTc-DTPA in
the rabbit (11), indicating an increase in epithelial permeability. This model also produces airspace inflammation, characterized by the influx of neutrophils (12) and elevated levels of
cytokines. A single inhalation of LPS in the guinea pig increases TNF- activity in airspace leukocyte lysate and in leukocyte cell culture supernatant (13). Ulich and colleagues (14)
found that intratracheal instillation of LPS induced TNF- mRNA expression in vivo, in whole-lung RNA preparations
as well as in both alveolar macrophages and neutrophils (15).
Nitric oxide (NO) is a highly reactive molecule that may act as a double-edged sword in lung inflammation (16), because it has been implicated in both host defense and tissue injury (17). Two major roles have been described for NO: (1) cell- cell communication mediated by the stimulation of cyclic guanosine 3',5'-monophosphate (cGMP) synthesis (18), and (2) cytotoxicity by direct or indirect interaction of the free radical NO with cellular targets (19). The activation of the L-arginine-dependent NO pathway via NO synthase is an important mechanism in both the microbiostatic capability and cytotoxicity of phagocytes. On the other hand, the accumulation of nitroso compounds in the host tissues is known to produce disruption of immunochemical homeostasis, which has been implicated in the endotoxicosis syndrome (20).
NO could also have a role in increasing airspace epithelial permeability in lung inflammation, as has been suggested in other organs. For example, inhibition of nitric oxide production leads to a reversible circulating leukocyte-independent increase in feline small intestine epithelial permeability (21).
In this study, we have investigated the role of neutrophils, TNF, and NO in the increased epithelial permeability induced by intratracheal instillation of LPS.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Intratracheal Instillation of LPS
Syngeneic Wistar-derived rats of the HAN strain (300-500 g, 12 wk of
age or more) were anesthetized using Fluothane (halothane; Zeneca,
Macclesfield, UK) inhalation in Fluotec 3 (Cyprane, Keighley, UK).
A volume of 0.2 ml of phosphate-buffered saline (PBS) containing 1 µg of LPS (Sigma, Poole, UK) or 0.2 ml of PBS alone in control experiments was instilled intratracheally into the lungs. Measurements of rat lung epithelial permeability and bronchoalveolar lavage were
carried out at various time intervals as described subsequently. In
some experiments, 50 µg/0.1 ml of anti-murine TNF- antibody (R&D
Systems Europe, Abingdon, UK) was coinstilled intratracheally, together with LPS, to abolish TNF activity in the alveolar space during
LPS-induced alveolitis.
Treatment with Neutrophil Antibody or L-NMMA
Rabbit anti-neutrophil serum (0.9 ml; Accurate Chemical & Scientific, Westbury, CT) was injected intraperitoneally 8 h before the intratracheal instillation of LPS.
To assess the role of nitric oxide (NO) in LPS-induced increased epithelial permeability, the specific inhibitor of NO synthase, NG- methyl-L-arginine (L-NMMA; Sigma) in doses of 10 and 20 mg/rat was injected into the rat peritoneal cavity immediately before intratracheal instillation of LPS. Epithelial permeability was measured 4 h thereafter.
Measurement of Rat Lung Epithelial Permeability
Rat lung epithelial permeability to 125I-labeled bovine serum albumin
([125I]BSA) was measured as previously described (9, 10). In brief, BSA Sigma was iodinated with 125I according to the modified chloramine-T method (22) and free 125I was removed by chromatography on a
Sephadex G25 column (Pharmacia, Milton Keynes, UK). To measure
epithelial permeability, 0.1 ml of PBS containing 15 µCi of [125I]BSA
was instilled intratracheally into the lungs of anesthetized rats. Five
minutes after intratracheal instillation of [125I]BSA, blood samples
were withdrawn through the femoral vein at 2 min intervals for 8 min.
The radioactivity of the [125I]BSA in each blood sample was measured
in a 
counter (Cobra II auto-gamma counting system; Packard Instruments, Meriden, CT) and the results of all blood samples were
pooled. Epithelial permeability was calculated as the percentage of the
[125I]BSA counts originally instilled, which were present in the whole-blood volume of the rat as previously described (9, 10). [125I]BSA was
separated from free 125I by precipitation with trichloroacetic acid, i.e.,
50 µL of fetal bovine serum and 2 ml of 20% trichloroacetic acid were
added to each blood sample, mixed well, and then spun at 2,500 rpm
for 10 min. The supernatant (containing free 125I) was discarded and
samples were reassessed in the counter for [125I]BSA.
In some experiments, total protein in BAL fluid was measured to assess global permeability of the airspace-blood barrier. In brief, BAL fluid was mixed with Bio-Rad solution (Bio-Rad, Munich, Germany) and incubated at room temperature for 10 min. The absorbance of the fluid was then read at 595 nm on a spectrophotometer (8700 series; Unicam, Cambridge, UK). The protein concentration was determined by comparison with a standard curve using BSA.
Bronchoalveolar Lavage and Culture of Lavaged Leukocytes
The lungs were removed from the thorax of anesthetized rats at various time intervals after LPS instillation. Four milliliters of phenol red-free Dulbecco's modified Eagle's medium (DMEM; GIBCO, Paisley, UK) containing 0.2% BSA was injected into and withdrawn from the lungs. This was followed by bronchoalveolar lavage (BAL) with four aliquots of 8 ml of PBS. BAL leukocytes were collected by spinning the lavage fluid at 1,000 rpm for 10 min and resuspending the cell pellet in medium. The supernatant from the first part of the lavage fluid (4 ml of DMEM plus 0.2% BSA) was kept separately and will be referred to as BAL fluid. To determine the total protein in BAL fluid, DMEM plus 0.2% BSA, which was normally used in the lavage procedure, was replaced with DMEM only.
To measure products released by BAL leukocytes in culture, BAL leukocytes were resuspended in DMEM plus 0.2% BSA at 1 × 106 cells/mL. The supernatants were collected after 24 h of incubation at 37° C, 5% CO2 and spun at 2,500 rpm for 10 min. To assess the effect of LPS on NO production, control alveolar macrophages were treated with LPS at 1,000 ng/ml with or without the presence of L-NMMA at 500 µM. NO levels in the cell supernatant were measured as nitrite (see the next section) 24 h thereafter.
TNF and NO Assays
TNF activity in BAL fluid and in the supernatant from cultured BAL
leukocytes was measured using the L929 cell bioassay (23). In brief,
L929 cells (a gift from Dr. Julian Symonds, Northern General Hospital, Edinburgh, UK) were grown in MEM plus 10% fetal bovine serum (FBS; GIBCO) in 96-well microtiter plates (Greiner Labortechnic, Dursley, UK) overnight to form cell monolayers. The cell
monolayers were then incubated with BAL fluid or leukocyte supernatant for 16 h in the presence of 1 µg/ml actinomycin D (Sigma). The
presence of TNF in the samples causes lysis of L929 cells. The cells
that survived were stained with crystal violet methanol solution and
the optical density at 540 nm was determined with an MR650 plate
reader (Dynatech Laboratories, McLean, VA). TNF activity in the
samples was determined by comparison with a TNF- standard (Genzyme Diagnostics, Kent, UK) dilution curve. The specificity of TNF
activity in the samples was confirmed by neutralization with TNF antibody.
NO generation was determined as the accumulation of nitrite, which is the end product of NO, using a spectrophotometric assay based on the Griess reaction (24). Samples were incubated with an equal volume of the Griess reagent consisting of 1% sulfanilamide, 0.1% naphthylethylene diamine dihydrochloride and 2.5% H3PO4 at room temperature for 10 min and the absorbance at 550 nm was measured on a spectrophotometer (Unicam 8700 series, Cambridge, UK). Nitrite concentrations were determined by comparison with a sodium nitrite standard curve.
Statistical Analysis
Results were expressed as mean (SEM). Differences between mean values were assessed by analysis of variance.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Intratracheal Instillation of LPS Induces Increased Epithelial Permeability and Neutrophil Influx in Rat Lungs
The peak increase in epithelial permeability occurred 4 h after intratracheal instillation of LPS, which started to recover towards control values by 8 h (Figure 1). LPS-instillation also induced an influx of inflammatory leukocytes into the airspaces. However, the peak neutrophil influx occurred at 8 h, which was later than peak increase in epithelial permeability (Table 1). Sixteen hours after LPS, while neutrophil numbers began to fall, macrophage numbers were still rising.
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We compared the effects of LPS on epithelial permeability using two different techniques, by measuring 125I-BSA which had penetrated from the airspaces to the blood and total protein in BAL fluid, which reflects exudation from blood to airspace. Four hours after intratracheal instillation of LPS, there was a similar increase in epithelial permeability to 125I-BSA (control, 0.33 ± 0.02%; LPS, 0.49 ± 0.03%; n = 15, p < 0.001) and in total protein in BAL fluid (control, 0.33 ± 0.02 mg/ml; LPS, 0.64 ± 0.02 mg/ml; n = 6, p < 0.001).
Role of Neutrophils in LPS-induced Increased Epithelial Permeability
Epithelial permeability was measured after circulating neutrophils were depleted by neutrophil antibody in LPS-instilled rats. The administration of neutrophil antibody totally depleted peripheral blood neutrophils (data not shown) and abolished the neutrophil influx induced by LPS instillation, but did not affect the increase in epithelial permeability (Figure 2).
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Role of TNF in LPS-induced Epithelial Permeability
There was no detectable TNF activity in BAL fluid in control
rats. LPS instillation produced a dramatic increase in TNF in BAL fluid, the levels peaking 1 h after instillation (Figure 3). TNF released by cultured BAL leukocytes obtained from
LPS-instilled rat lungs was also higher than that produced by
control BAL leukocytes. The greatest amount of TNF- was
produced by leukocytes obtained from rats 1 h after LPS instillation (Figure 3).
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To investigate the relationship between elevated TNF levels in the alveolar space and increased epithelial permeability in LPS-treated rats, TNF antibody was coinstilled with LPS and measurements of epithelial permeability were made 4 h thereafter. The LPS-induced increase in TNF in BAL fluid was abolished by treatment with TNF antibody (Figure 4). However, the potential for inflammatory leukocytes to release TNF in culture was not influenced by the presence of TNF antibody in the alveolar space. BAL leukocyte differential cell counts remained the same in rats treated with LPS plus TNF antibody compared with rats treated with LPS alone (Table 2). Epithelial permeability induced by LPS instillation was not affected by coinstillation of TNF antibody (Figure 4). In animals treated with a combination of neutrophil antibody and LPS, TNF activity both in BAL fluid and in leukocyte supernatant increased, compared with rats treated with LPS alone (Table 3).
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Role of NO in LPS-induced Epithelial Permeability
Nitrite levels increased in BAL fluid after LPS instillation (Figure 5). The potential of leukocytes in culture to produce NO was also enhanced by LPS instillation, as shown by elevated nitrite levels in the supernatant from cells obtained by BAL and cultured for 24 h. The greatest release of NO by leukocytes in culture was from those obtained by BAL 2 h after LPS instillation (Figure 5).
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The leukocytes from rat by BAL 4 h after LPS instillation produced a significantly greater amount of NO in culture (11.59 ± 1.25 µM/million cells) than did control BAL leukocytes (4.61 ± 0.83 µM/million cells, n = 3; p < 0.05) and this in vivo LPS-stimulated enhancement of NO production by BAL leukocytes was inhibited by the presence of L-NMMA, the specific inhibitor of NO synthase in culture (3.19 ± 0.17 µM/ million cells, p < 0.001).
Studies in vitro also showed that both spontaneous and LPS-induced NO production by control alveolar macrophages was dramatically inhibited by L-NMMA (Table 4).
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To assess the role of NO in LPS-induced epithelial permeability, L-NMMA was given intraperitoneally before LPS instillation in rats. Four hours after administration to rats of 10 mg of L-NMMA and LPS, there was a significant reduction in
epithelial permeability compared with LPS treatment alone,
either measured as epithelial permeability to [125I]BSA (p < 0.001) or as total protein in BAL fluid (p < 0.001) (Figure 6),
although the cellular influx in BAL remained the same (Table
5). To investigate the dose response of L-NMMA, we doubled the dose of L-NMMA to 20 mg/rat and found that although
there was a further reduction in epithelial permeability as
measured by total protein in BAL (
20.6% change compared
with L-NMMA in a dose of 10 mg/rat), the protein level was
still significantly higher than that of the vehicle control
(126.1% change compared with PBS control).
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As in our previous studies (9), there was good agreement
between the results of epithelial permeability assessed by total 125I ([125I]BSA plus free 125I) or bound 125I ([125I]BSA only): total 125I: control
0.98 (0.03%), LPS only1.74 (0.07%), LPS
plus L-NMMA1.297 (0.066%), mean (SEM), n = 15; bound
125I: control0.338 (0.012%), LPS only0.594 (0.023%), LPS
plus L-NMMA0.440 (0.029%), mean (SEM), n = 15.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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This study shows that intratracheal instillation of LPS causes an acute and transient alveolitis, characterized by recruitment of inflammatory leukocytes, which are predominantly neutrophils, and a transient increase in airspace epithelial permeability. These data agree with the findings of Brown and coworkers (11), who found in the rabbit that aerosolized LPS increased the pulmonary clearance of 99mTc-DTPA. Xing and coworkers (15) also studied the cellular response to intratracheal LPS and showed that 1 h after instillation in the rat, the bronchoalveolar lavage cell population was 98% alveolar macrophages, whereas by 6 h 88% of BAL cells were neutrophils. Thomas and colleagues (12) also found that intratracheal instillation of LPS induced a neutrophil influx into the airspace that peaked between 6 to 12 h, and a monocyte influx that peaked 24 h after instillation. Although the time courses of the neutrophil influx in these studies are different compared with our observations, the patterns of neutrophil and macrophage recruitment in response to LPS instillation are similar to these previous studies.
The role of neutrophils in increasing epithelial permeability varies depending on the stimulus. Bhalla and coworkers (6) found that depletion of neutrophils in the rat prevented the increase in tracheal permeability induced by ozone exposure. Hyde and colleagues (25) found that there was a strong relationship between epithelial necrosis, which resulted in increased epithelial permeability and the emigration and retention of neutrophils in the lung in an ozone exposure study in rhesus monkeys. O'Byrne and colleagues (26) suggested that ozone-induced hyperresponsiveness may depend on the mobilization of neutrophils into the airways. The present study shows that depletion of neutrophils with neutrophil antibody did not affect LPS-induced increased epithelial permeability, suggesting that neutrophils were not involved in this event.
LPS has been shown to stimulate increased production of
TNF by BAL leukocytes and other residents lungs cells, such
as type II epithelial cells in vivo and in vitro. Intratracheal instillation of LPS markedly increases TNF- in lung tissue (12,
15). Xing and colleagues (15) found that increased levels of
TNF in the lungs were present at 30 min and peaked within 6 h
of LPS instillation. At an early time point (1 h), macrophages
were the major source of TNF-, and at later time points (6 and 12 h) neutrophils were the predominant cell type that released TNF-. In the present study, LPS-induced TNF production by mixed leukocytes obtained by BAL peaked 1 h after LPS instillation, at a time when macrophages were the
predominant cells in BAL. Depletion of neutrophils in LPS-treated rats increased TNF activity in BAL fluid and TNF production by BAL leukocytes in vitro. Therefore at this time
point alveolar macrophages are the major source of TNF production, confirming the work of Xing and coworkers (15).
TNF- has a wide range of biological pathological properties
that include changing the integrity of epithelium or endothelium. Increased vascular permeability induced by TNF can be
either dependent (27) or independent of neutrophils (28). In
in vitro studies, TNF increases the permeability of endothelial
and epithelial cell monolayers (7, 29). In a previous study we
also showed that TNF- is responsible for producing increased epithelial permeability in a bacterial-induced acute alveolitis (30). In the present study, administration of TNF antibody abolished the LPS-induced increase in TNF in BAL fluid. However, this was not accompanied by a reduction of
the increase in epithelial permeability. Thus, these data do not
support a role for TNF in increasing epithelial permeability in
LPS-induced alveolitis. Although TNF does not appear to
contribute to the alteration in epithelial permeability in this
study, it may play a role in recruiting neutrophils to the airspaces (31). However, abolition of TNF had no effect on neutrophil recruitment in this model of acute alveolitis.
In this study, we found an increase in the levels of NO in BAL fluid after intratracheal instillation of LPS. Furthermore, BAL leukocytes from rats 4 h after treatment with LPS produced more NO than control BAL leukocytes. The NO production by leukocytes both from control and LPS-treated animals could be inhibited by coincubation with NO synthase inhibitor, L-NMMA, in culture, suggesting that the increased NO production occurred by activation of NO synthase utilizing L-arginine. NO is a highly reactive molecule that has both protective and injurious properties (17, 20). Kubes (21) has shown that local intraarterial infusion of the NO synthase inhibitor, L-NAME, increased epithelial permeability in the feline small intestine and that this effect was completely reversed by infusion with either sodium nitroprusside (an exogenous source of NO) or L-arginine. In a rat model, Ischiropoulos and coworkers (32) found that injection of L-NAME immediately after exposure to wood and polyvinylchloride smoke abolished the increase in concentrations of circulating nitrate levels and lung myeloperoxidase activity, reflecting a reduction in neutrophil activation and also decreased airspace epithelial permeability. Our data suggest that NO in the bronchoalveolar space during LPS-induced alveolitis may be, at least partially, responsible for the increased epithelial permeability, but did not influence neutrophil influx. Activated alveolar macrophages may not be the only source of NO. We have shown that rat type II epithelial cells produce NO in culture in response to LPS stimulation, which is also inhibitable by another NO synthase inhibitor, L-NAME (33), although the magnitude of NO production by type II epithelial cells is much smaller than that by rat alveolar macrophages. Because there was incomplete abolition of LPS-induced epithelial permeability by administration of L-NMMA with both doses that were used, other factors may be implicated in increasing epithelial permeability. In previous studies we have shown that during exposure to cigarette smoke, there is increased epithelial permeability resulting from a decrease in intra- and extracellular glutathione in epithelial lining fluid (9, 10). In this study, we also found a 60% decrease in total glutathione in BAL fluid after LPS intratracheal instillation (data not shown). Thus it is possible that this fall in the reduced form of glutathione may have contributed to the increase in epithelial permeability.
There is a complex interaction between cytokines and NO.
A mixture of TNF-, IL-1
, and interferon increased inducible NO synthase mRNA expression and NO production in
the human type II epithelial cell line, A549, and in primary
cultures of human bronchial epithelial cells (17, 34). Jorens
and colleagues (35) found that rat alveolar macrophages incubated with LPS, recombinant interferon , but not with
recombinant TNF-, induced NO production. On the other
hand, NO may itself influence cytokine production. LPS-
induced TNF- production by human neutrophils was increased by exposure to sodium nitroprusside, an exogenous
source of NO (36). NO-releasing agents (sodium nitroprusside, 3-morpholino-sydnonimine) also enhanced IL-1-induced
TNF synthesis in human mononuclear cells (37). The interaction between TNF and NO in this model requires further investigation.
In summary, intratracheal instillation of LPS in the rat produces a neutrophil alveolitis, transiently increases airspace epithelial permeability, and increases TNF activity and NO levels in BAL fluid and in supernatant of BAL leukocytes in
culture. However, neither neutrophils nor TNF- appears to
have a role in the development of LPS-induced increased epithelial permeability, whereas NO is partly responsible for this
LPS-induced epithelial permeability.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Professor W. MacNee, Respiratory Medicine Unit, Department of Medicine, Royal Infirmary, Edinburgh, EH3 9YW Scotland, UK. E-mail: w.macnee{at}ed.ac.uk
(Received in original form May 28, 1996 and in revised form October 13, 1997).
Acknowledgments: Supported by the Medical Research Council and the Norman Salvesen Emphysema Research Trust.
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References |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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