|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
ABSTRACT |
|---|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Nitric oxide (NO) end-products (nitrate and nitrite) are present in bronchoalveolar lavage (BAL) fluid of patients with inflammatory lung diseases. Reactive oxygen-nitrogen intermediates damage macromolecules by oxidation or nitration of critical residues in proteins. The goal of this study was to measure NO end-products (nitrate+ nitrite), in BAL fluid before and after the onset of acute respiratory distress syndrome (ARDS) and to determine if these products are associated with expression of inducible nitric oxide synthase enzyme (iNOS) in BAL cells and nitration of BAL proteins. We performed bronchoalveolar lavage (BAL) in patients at risk for ARDS (n = 19), or with ARDS (n = 41) on Days 1, 3, 7, 14, and 21 after onset, and measured total nitrite (after reducing nitrate to nitrite) and protein-associated nitrotyrosine concentration in each BAL fluid sample. Cytospin preparations of BAL cells were analyzed by immunocytochemistry for iNOS and nitrotyrosine. Nitrate+nitrite were detected in BAL fluid from patients at risk for ARDS, and for as long as 21 d after the onset of ARDS. Nitrotyrosine was detectable in all BAL fluid samples for as long as 14 d after the onset of ARDS (range, 38.8 to 278.5 pmol/mg of protein), but not in BAL of normal volunteers. Alveolar macrophages of patients with ARDS were positive for iNOS and nitrotyrosine, and remained positive for as long as 14 d after onset of ARDS. The BAL nitrate+nitrite did not predict the onset of ARDS, but the concentration was significantly higher on Days 3 and 7 of ARDS in patients who died. Thus, NO end products accumulate in the lungs before and after onset of ARDS; iNOS is expressed at high levels in AM during ARDS; and nitration of intracellular and extracellular proteins occurs in the lungs in ARDS. The data support the concept that NO-dependent pathways are important in the lungs of patients before and after the onset of ARDS.
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Nitric oxide (NO) is a reactive molecule produced by nitric
oxide synthase (NOS) enzymes in a variety of cells (1). Three distinct forms of NOS have been identified; endothelial NOS
(eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS)
(2). Both eNOS and nNOS are constitutive forms of NOS
(cNOS) that produce small amounts of NO for short periods
of time when appropriately stimulated. The cNOS-derived
NO is involved in maintaining physiologic functions such as
regulating vascular tone and acting as a neurotransmitter. In
contrast, iNOS produces a large amount of NO for sustained periods of time. The NO derived from iNOS is thought to be
involved in inflammatory responses and host defense against
infection (3). Lipopolysaccharide and proinflammatory cytokines such as interferon-
, interleukin-1
(IL-1), and tumor
necrosis factor-
(TNF-) have been shown to upregulate
iNOS in human and animal cells (3, 4). NO can react with biologic targets directly or indirectly via formation of reactive intermediate products. Under aerobic conditions, NO reacts with
oxygen and superoxide radicals to form nitrogen dioxide and
peroxynitrite, respectively (5). These reactive nitrogen species
are capable of causing damage to macromolecules by oxidation of redox-active complexes or nitration of aromatic amines.
Acute respiratory distress syndrome (ARDS) is a disease process characterized by diffuse inflammation in the lung parenchyma. The involvement of inflammatory mediators in ARDS has been the subject of intense investigation (6). Oxidant-mediated tissue injury is likely to be important in the pathogenesis of ARDS (9, 10). Immunohistochemical studies have provided evidence of nitrotyrosine residues on proteins in cells in bronchoalveolar lavage fluid and lung tissue of patients with acute lung injury (11, 12). Kobayashi and colleagues (13) demonstrated that NO end-products were increased in BAL fluid from patients with ARDS after sepsis. Peroxynitrite and its reactive intermediates have been shown to inhibit pulmonary surfactant protein function by nitrating tyrosine residues (14, 15). Endotoxin-activated rat alveolar macrophages produce sufficient amounts of peroxynitrite to nitrate human SP-A in vitro, and nitration is increased in the presence of physiological concentrations of carbon dioxide and bicarbonate (16). In addition, the presence of nitrated surfactant protein A (SP-A), the most abundant surfactant apoprotein, has been demonstrated in edema fluid of patients with acute lung injury (17). These lines of evidence suggest a role for NO in the pathogenesis of acute lung injury in humans by oxidizing and/or nitrating key target proteins in the lungs.
The goals of this study were to answer the following questions: (1) are NO end-products (nitrate+nitrite) increased in the bronchoalveolar lavage fluid of patients before and after the onset of ARDS, and if so, do the concentrations change during the course of ARDS; (2) are NO end-products associated with increased protein nitration in distal air spaces of patients with ARDS; (3) do the concentrations of NO end-products in BAL fluid predict the outcome of patients at risk for ARDS, or of patients with established ARDS?
| |
METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Patient Population
All patients admitted to the intensive care unit at Harborview Medical Center (Seattle, WA) between January 1994 and March 1997 were prospectively evaluated using predetermined criteria for risk factors for ARDS, or for established ARDS (18). The patients enrolled in the at-risk group were also eligible to be enrolled in the ARDS group if they later met the criteria for ARDS.
Patients at Risk for ARDS
All intubated patients with trauma or suspected sepsis were screened.
Patients with trauma were considered to be at risk for ARDS if they
met one of the following criteria: (1) two or more of the following:
multiple fractures (two or more fractures of femur, tibia, humerus, or
stable pelvic fracture); unstable pelvic fracture; pulmonary contusion;
or massive transfusion (> 15 units in 24 h); or (2) Injury Severity
Score (ISS) > 20 plus one of the criteria in (1). Patients with sepsis
were considered to be at risk for ARDS if they had: (1) two or more
of the following signs of infection: temperature greater than or equal
to 39° C or lower than 36° C; white blood cell count greater than
14,000/mm3 or less than 4,000/mm3; a positive blood culture or a known
or strongly suspected source of infection; and (2) two or more of the
following systemic changes: systemic vascular resistance less than 800 dyne/s · cm
5; unexplained hypotension (systolic blood pressure less
than 90 mm Hg for more than 1 h); ongoing metabolic acidosis with
anion gap greater than 20 mEq/L; vasopressor use to maintain systolic blood pressure greater than 90 mm Hg; or a platelet count of less than
80,000/mm3. The at-risk patients were followed until discharge from
the intensive care unit or the onset of ARDS.
Patients with Established ARDS
All intubated patients in the intensive care unit were screened and
considered to have ARDS if they met the following criteria: (1) critical hypoxemia, with PaO2/FIO2 < 150 mm Hg, or < 200 mm Hg when
receiving 
5 cm H2O positive end-expiratory pressure (PEEP); (2)
diffuse parenchymal infiltrates involving at least 50% of three or more
quadrants on chest radiograph; (3) pulmonary artery wedge pressure
(when available) < 18 mm Hg or no clinical evidence of congestive
heart failure; and (4) no other obvious explanation for these findings.
This definition of ARDS is slightly different from American-European Consensus Conference (AECC) definition of the ARDS, but all
of our patients in the ARDS group also met the AECC criteria for
ARDS (19). All the patients were followed until death or hospital discharge. Survival was defined as hospital discharge.
Exclusions
The patients were excluded from the study if they met one or more of the following criteria: age younger than 18 yr; unsupportable hypoxemia (PaO2 < 80 mm Hg with FIO2 1.0); evidence of acute myocardial infarction; cardiac arrhythmias (supraventricular tachycardia > 140 beats/min or complex ventricular ectopy); uncontrolled intracranial hypertension (intracranial pressure > 20 mm Hg); endotracheal tube internal diameter < 7.0 mm; cutaneous burns; inhalation injury; known HIV infection; or preexisting lung disease (e.g., asthma/COPD receiving daily medication, sarcoidosis, or interstitial lung disease).
Informed consent was obtained from the patient or a surrogate. The protocol was approved by the Human Subjects Review Committee of the University of Washington.
Bronchoalveolar Lavage Protocol
Patients at risk for ARDS (n = 19) underwent BAL within 24 h of the onset of risk for ARDS (Day 1) and at 48 h later (Day 3) if they were still intubated and had not developed ARDS. Patients with ARDS (n = 41) underwent BAL within 24 h of onset of ARDS (Day 1) and then on Days 3, 7, 14, and 21 if they remained intubated. For comparison, BAL also was performed on healthy volunteers who did not have any underlying lung disease.
BAL was performed by passing a flexible fiberoptic bronchoscope through the endotracheal tube in intubated patients, or orally in normal volunteers (20). Patients were preoxygenated with 100% oxygen for 15 min before and during the procedures. Five separate 30-ml aliquots of 0.89% normal saline were instilled and recovered by hand-suction with the bronchoscope wedged in the right middle lobe or the lingula.
The BAL aliquots were transported immediately to the laboratory
for analysis. The fluid was filtered through sterile filters (100 µM) to
remove mucus. Total cell counts were performed in a hemacytometer.
Differential cell counts were performed on cytospin preparations
stained with Diff-Quik (American Scientific Products, McGraw Park,
IL). Additional cytospin slides were fixed in 4% paraformaldehyde
for 10 min, washed twice in PBS, then stored at 
20° C until used for
immunocytochemical analysis. The remaining BAL fluid was spun at
200 × g for 30 min, and the supernatant was removed and stored at
70° C. Total protein was measured on an aliquot of supernatant using
the bicinchoninic acid method (Pierce Chemical Co., Rockford, IL).
Measurement of NO2 and NO3
Samples of bronchoalveolar lavage fluid were incubated with nitrate
reductase to reduce nitrate (NO3) to nitrite (NO2), and the final
concentration of nitrite was measured using the Griess reagent (Cayman Chemical Nitrate/Nitrite Assay Kit; Cayman Chemical Co., Ann
Arbor, MI). Briefly, nitrate in 100 µl of BAL fluid was converted to
nitrite by incubating BAL fluid with 10 µl of aspergillus reductase enzyme (Cayman Chemical Co.) and NADPH for 2 h at room temperature. The nitrite in BAL fluid was then converted to a deep purple azo
compound by reaction with sulfanilamide and N-(1-naphthyl)ethylenediamine. Absorbance was read at 540 nm, and NO2 concentration was determined using NaNO2 standards. The lower limit of detection was 2.5 µM (2.5 µmol/L). The values reported are the values
detected in the BAL fluids, without correction for dilution by the
BAL procedure. The efficiency of conversion of nitrate to nitrite was
tested by measuring nitrite before and after the addition of nitrate reductase to three different ARDS BAL samples, and using nitrate and
nitrite standards, as previously described (16).
Measurement of Protein-bound Nitrotyrosine in BAL Fluid
Protein-nitrotyrosine concentrations in BAL were measured by ELISA
as previously described (21). This method measures protein-bound nitrotyrosine residues rather than free nitrotyrosine. A nitrated protein
solution was prepared for use as a standard by incubating 0.1% (1 mg/
ml) bovine serum albumin in 50 mM KH2PO4 at a pH of 8 for 30 min at
37° C with 0.5 mM tetranitromethane (TNM) (Aldrich Chemical Co.,
Inc., Milwaukee, WI), an efficient nitrating agent. After adjusting the
pH to 10.0 with 3 M NaOH, the amount of nitrotyrosine present in the
TNM-treated BSA solution was measured at 430 nm (
M = 4,400 M1
cm1) and expressed as nanomoles of nitrotyrosine per milligram
BSA. Subsequently, a stock solution of the TNM-treated BSA was diluted with 0.1 M Na2CO3-NaHCO3 coating buffer at a pH of 9.6. These standard samples, along with BAL aliquots, were applied to Immulon 2 ELISA plates (Dynatech, Chantilly, VA) and allowed to bind for 1 h
at 37° C. After nonspecific binding sites were blocked with 1% BSA in
PBS, the wells were incubated for 90 min at 37° C with a mouse IgG
monoclonal antinitrotyrosine primary antibody, diluted 1:200 (a kind
gift of Joseph Beckman, Ph.D., Dept. of Anesthesiology, University of
Alabama at Birmingham), and then incubated for 30 min at 37° C with
a horseradish peroxidase-conjugated goat antimouse IgG secondary
antibody (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD),
diluted 1:1,000. After washing the plates, the peroxidase reaction product was generated using hydrogen peroxide (3.7 mM) and o-phenylenediamine (2.2 mM) (Sigma Chemical Co., St. Louis, MO). The
plate was incubated for 10 min at ambient temperature, the reaction
was stopped by the addition of sulfuric acid (2.5 mM), and the optical
density in each well was measured at 490 nm. The nitrotyrosine content per milligram protein was calculated by relating the optical absorbance values of the nitrated BSA standards measured by ELISA to the
concentration of nitrotyrosine per milligram BSA as determined spectrophotometrically. Protein concentrations were determined by the
bicinchoninic acid method. The protein nitrotyrosine content of BAL
samples was expressed as picomoles of nitrotyrosine per milligram
protein. It is unlikely that endogenous peroxidases contained in the
ARDS BAL fluid contributed to the reaction signal in this assay for
three reasons: first, when immunoassays were performed on BAL fluid
using the same methodology, but a non-peroxidase-conjugated second
antibody, no reaction product was detected in any well; second, the
BAL was diluted at least 20-fold prior to use in the assay; third, large
concentrations of hydrogen peroxide were used to develop the reaction product, which favors the action of horseradish peroxidase and
minimizes the contributions of endogenous peroxidases (e.g., myeloperoxidase and eosinophil peroxidase).
Immunocytochemical Detection of iNOS and Nitrotyrosine in BAL Cells
The presence of iNOS and nitrotyrosine in BAL cells was analyzed by immunocytochemistry. Paraformaldehyde-fixed cytospin slides were immersed in cold methanol containing 0.3% H2O2 to quench free peroxidase activity. The slides were washed and incubated in 10% normal goat serum for 1 h, then incubated overnight at 4° C with either rabbit polyclonal antihuman iNOS (Transduction Laboratories, Lexington, KY) or with murine monoclonal antinitrotyrosine IgG (from Dr. Beckman) in 5% normal goat serum containing 1% bovine serum albumin. The slides were washed with PBS, then incubated with biotinylated goat antirabbit or antimouse IgG for 45 min (Dako Co., Carpinteria, CA), washed, and developed with streptavidin-peroxidase and diamino-benzidine. Slides were counterstained with hematoxylin and examined by light microscopy. On each slide, 400 cells were counted and graded as positive or negative based on the presence or absence of the brown reaction product in the cytoplasm.
Data Analysis
The data are presented as box plots showing medians and the 10th, 25th, 75th, and 90th percentiles. Comparisons between more than two groups were made using the Kruskall-Wallis one-way analysis of variance for nonparametric data. Secondary comparisons were made using the Mann-Whitney U test. A p value of < 0.05 was accepted as significant.
| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Patient Population
Between February, 1994 and March, 1997, 60 patients were enrolled in the study (Table 1). Nineteen patients met at-risk criteria for ARDS; seven of these patients subsequently developed ARDS. Of the patients at risk, eight had a sepsis risk and 11 had a trauma risk. The patients at risk had significant gas exchange abnormalities, consistent with AECC criteria for acute lung injury (19), but none of these patients met oxygenation or radiographic criteria for ARDS. Forty-one patients met criteria for ARDS. The primary risk factors for ARDS were sepsis (n = 10), trauma (n = 15), and "other" (n = 11), including gastric aspiration, pancreatitis, drug overdose, and massive transfusion. The mortality rate was 10% in patients at risk for ARDS and 22% in patients with ARDS. The number of patients who remained in the study declined with time, reflecting the loss of patients who either recovered or died. The mortality declined with time in patients with persistent ARDS who remained mechanically ventilated (Table 1).
|
Nitrate+Nitrite in BAL Fluids
The concentration of nitrate+nitrite (NOx) was barely above
background in BAL fluid from normal subjects (range, 2.5 to
4.3 µM; median, 2.5 µM). In patients at risk for ARDS, the
NOx concentration in BAL fluid from Days 1 and 3 after onset
of the risk factor was significantly higher than in normal subjects (p 
0.002) (Figure 1). In patients with ARDS, the NOx
concentration was significantly higher than normal, and it remained elevated throughout the course of ARDS (p 0.005)
(Figure 1). In patients with ARDS, the majority of the products detected were in the form of nitrate (> 90% nitrate, and < 10% nitrite). In comparing patients at risk for ARDS and patients with established ARDS, there was no statistically significant difference in NOx concentration, either at the onset of
ARDS or at any subsequent time. However, the patients studied on Day 21 after onset of ARDS had the lowest concentrations of NOx in BAL fluid.
|
NOx Concentration and Clinical Risk Factors for ARDS
The NOx concentration in the BAL fluid of patients on Day 1 of ARDS is shown in Figure 4, with patients grouped by the major clinical risk factor associated with ARDS. The patients with sepsis had a significantly higher concentration of NOx than did patients with trauma (p = 0.001) or other causes of ARDS (p = 0.04).
|
NOx Concentration and Outcome of Patients at Risk for ARDS
Seven of the at-risk patients subsequently developed ARDS (37%). There was no statistically significant difference in BAL NOx concentration in the at-risk patients who did or did not develop ARDS (Figure 2).
|
NOx Concentration and Outcome of Patients with Established ARDS
On Day 1 of ARDS, the NOx concentrations were similar in
patients who lived or died (Figure 3). On Day 3 of ARDS
there was a trend toward higher NOx in patients who died, and
on Day 7 of ARDS, the BAL NOx concentrations were significantly higher in the patients who died (p = 0.034) (Figure 3).
These conclusions are limited by the small number of patients
who died (n = 10 on Day 3; n = 3 on Day 7). In order to determine whether these trends would be consistent in a larger
number of patients, we measured NOx using the same methods in BAL fluids that had been stored frozen (80° C) from
an earlier group of patients with established ARDS. We studied these patients in order to investigate inflammatory mechanisms in the lungs of patients with persistent ARDS, and the
details of this series have appeared in prior reports (7, 22).
These patients were enrolled using the same clinical criteria as
the patients in the current series; however, the BAL program was designed to study patients on Days 3, 7, 14, or 21 of established ARDS, and some of the patients did not have serial
BAL samples. It was not possible to combine the data from
the two series at all times, as there were no at-risk patients in
this group, and none of the patients were studied on Day 1 of
ARDS. Therefore the combined analyses were limited to patients studied in the two series on Days 3 and 7 of established
ARDS.
|
The combined data from the prior and the current series
are shown in Table 2. The mean NOx values at Days 3 and 7 were similar in the prior and the current series of patients,
even though the BAL fluids from the prior series had been
stored frozen for as long as 5 yr. When the two series were
combined, the NOx concentrations in the BAL were significantly higher on Days 3 and 7 in the patients who subsequently died, confirming the trends that we found in the current series for Days 3 and 7. Furthermore, in septic patients who died, the BAL NOx concentrations were significantly
higher on Days 3 and 7 of ARDS than in survivors (p 0.035 for lived versus died on both days). This was not true in the
patients with trauma. In patients with other risks for ARDS,
there was a trend for those who died to have higher BAL NOx
concentrations, but this did not reach statistical significance on
either Day 3 (p = 0.066) or Day 7 (p = 0.065).
|
Nitrotyrosine in BAL Fluids
Protein-bound nitrotyrosine was detected in BAL fluid from patients at risk and with established ARDS for as long as 14 d and was significantly higher than normal at all times (p < 0.05) (Figure 5). The nitrotyrosine concentration did not differ in patients at risk and with established ARDS. The median nitrotyrosine concentrations were the same on Days 1 and 14 of ARDS, and did not decline with time. These data provide only an estimate of the nitrotyrosine content, as BAL nitrated proteins may have different affinities for the nitrotyrosine antibody, as compared with the nitrated albumin standard that was used.
|
Immunocytochemical Staining for iNOS and Nitrotyrosine in BAL Cells
Neither iNOS nor nitrotyrosine residues were detectable in normal human AM. In contrast, iNOS and nitrotyrosine were detectable in AM from patients at risk and from patients with established ARDS (Figures 6-8). The number of AM labeling for iNOS was low at the onset of risk for ARDS, but it increased at Day 3 of risk and was maximal at the onset of ARDS, when more than 75% of the AM were iNOS positive. The iNOS labeling was still detectable on Day 7 of ARDS, but it declined to almost background by Day 14. Similarly, the intracellular nitrotyrosine labeling in AM was low at the onset of risk for ARDS, but increased dramatically by Day 3 of risk for ARDS (p = 0.02). At the onset of ARDS, more than 70% of the AM labeled for nitrotyrosine, and this declined over time in patients with persistent ARDS.
|
|
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
The major goals of this study were, first, to determine whether stable metabolites of NO, measured as nitrate+nitrite (NOx), accumulate in the BAL fluid before and/or after the onset of ARDS; second, to determine whether NOx accumulation is associated with evidence of protein nitration and iNOS expression in BAL cells; third, whether NOx accumulation differs in patients who live or die. The data provide the first evidence that NO-related species accumulate in patients at risk before the onset of ARDS and that NO production occurs in a sustained manner during the course of ARDS. Furthermore, the immunocytochemistry data provide strong evidence that iNOS is expressed in human alveolar macrophages before as well as after the onset of ARDS, and that iNOS expression persists in the lungs of patients with sustained ARDS. The NOx accumulation and iNOS expression are associated with protein nitration in the BAL fluid. In addition, nitrotyrosine residues were detected in alveolar macrophages during the course of ARDS, suggesting that intracellular protein nitration occurs in AM when iNOS is induced. The NOx concentrations were significantly higher on Days 3 and 7 of ARDS in patients who later died, particularly in the subgroup with sepsis. Taken together, these data support an important role for NO-dependent reactions in the lungs before the onset and during the course of ARDS.
Nitric oxide combines with superoxide anion to form the
highly reactive intermediate, peroxynitrite (ONOO), a strong
nitrating species (25, 26), and evidence for peroxynitrite formation has been detected in the lungs of patients with ARDS (12). Other pathways also exist for the production of reactive oxygen-nitrogen intermediates capable of nitrating, oxidizing and chlorinating protein in the alveolar space. For example,
iNOS and myeloperoxidase (MPO) colocalize in neutrophils
(27), and NO and nitrite (NO2) are produced together with
HOCl at inflammatory foci (1), suggesting that NO2 and
HOCl may interact. Eiserich and colleagues (28) found that NO2 reacts with HOCl to form a reactive intermediate, possibly nitryl chloride (Cl-NO2), that is capable of nitrating, chlorinating, and oxidizing tyrosine residues in proteins (28). Also,
van der Vliet and colleagues (29) demonstrated the nitration
of phenolic compounds, including tyrosine, by the products of
NO2 + H2O2, catalyzed by heme peroxidases such as MPO
(29). The physiological relevance of these nitration reactions
is supported by studies using neutrophils as the source of
MPO and H2O2 (30), and by the observation that free extracellular MPO and H2O2 are present in the BAL and expired
breath of patients with ARDS (31, 32).
Tyrosine residues in proteins are particularly sensitive to nitration, and we detected substantial amounts of protein- nitrotyrosine in BAL fluid and in alveolar macrophages before and after the onset of ARDS. This extends prior studies in which protein-nitrotyrosine residues were detected by immunocytochemistry in the BAL cells of five patients with ARDS and lung tissue from patients with acute lung injury (11, 12). The formation of nitrotyrosine residues modifies protein function. SP-A can be nitrated by NO-dependent reactions in vitro, and the nitrated form of SP-A is less effective in aggregating lipids and binding mannose (14, 21). Nitration of SP-A in edema fluid from patients with ARDS has recently been reported (17). NO metabolites and nitrotyrosine have been identified in the lungs of patients with other inflammatory disorders, including asthma, idiopathic pulmonary fibrosis, cystic fibrosis, and obliterative bronchiolitis after lung transplantation (reviewed in Reference 5).
There are several sources of NO in the lungs, but the contribution of human alveolar macrophages to NO production
has been open to question. Constitutive or inducible forms of
NOS have been detected in airway epithelial cells, Type II
pneumocytes, macrophages, neutrophils, mast cells, vascular
endothelial cells, and smooth muscle cells (5). In rodents, alveolar macrophages produce large quantities of NO when stimulated (4, 33, 34), and NO production is increased significantly
in the presence of physiological concentrations of carbon dioxide
and bicarbonate (16). In contrast, normal human alveolar
macrophages produce little or no nitric oxide in vitro, even
when stimulated with interferon gamma (IFN-) and other inflammatory cytokines (35). However, iNOS was readily detectable in alveolar macrophages recovered from the lungs of
patients with tuberculosis, suggesting a role for NO in host defense against intracellular pathogens in humans (36). In a prior
study, iNOS was detected in alveolar macrophages of patients
with ARDS after sepsis, but not in AM from patients without
lung injury (13). Thus, while normal human alveolar macrophages are poor sources of NO, the present in vitro study and
prior evidence suggest that at inflammatory foci, iNOS and NO
production are induced in macrophages by as yet undetermined mechanisms.
We found that human alveolar macrophages from the lungs
of patients at risk expressed iNOS, and that iNOS expression
was increased in macrophages at the onset of ARDS. The
iNOS was detectable in alveolar macrophages for as long as
14 d after the onset of ARDS. Thus, the inflammatory milieu
in the lungs induced iNOS in the alveolar macrophages, but
the factor(s) responsible remain uncertain. We have identified
a number of acute response cytokines in these fluids, but in
contrast to an earlier report, IFN- was not detectable by immunoassay (7, 37). It is likely that the iNOS induced in the alveolar macrophages was biologically active, because NOx species were readily detectable in BAL fluids and immunocytochemical studies indicated that virtually all of the AM recovered on Day 1 of ARDS contained large amounts of nitrotyrosine. We did not measure the activity of the alveolar
macrophages recovered from these patients, but evidence
from prior studies has shown that alveolar macrophages are
active in ARDS. For example, the AM recovered from patients with ARDS produce cytokines spontaneously and when stimulated with bacterial endotoxin (13, 38, 39). These observations suggest that the large amounts of intracellular nitrotyrosine that we detected do not necessarily inhibit macrophage
function in the lungs, although further studies of macrophage
function are needed to confirm this point.
We found high concentrations of the NO metabolites, nitrate and nitrite, in the BAL fluid not only in patients with sustained ARDS, but also in the patients at risk for ARDS. Thus, the oxidant stress detected at the onset of ARDS in prior studies actually begins when patients are at risk, before the clinically defined syndrome is recognized (9). In addition, the evidence of oxidant stress, as reflected by NOx species and nitrotyrosine formation, persists for as long as ARDS is clinically evident. The concentrations of nitrate and nitrite reported here were measured in undiluted BAL fluid, which probably dilutes alveolar epithelial fluid as much as 100-fold, so the actual concentrations in the lungs are likely to be much higher. Zhu and colleagues (17) have reported nitrate+nitrite concentrations in excess of 100 µM in the undiluted edema fluid of patients with ARDS.
The NOx concentration in the BAL of patients at risk did
not predict the onset of ARDS, and it did not predict the ultimate outcome on Day 1 of ARDS. However, on Days 3 and 7 of ARDS, there were trends for the NOx concentrations to be
higher in BAL in patients who died. These trends became significant when we combined the data from this series with similar measurements in stored BAL fluids from an earlier series
of patients with persistent ARDS (Table 2). In addition, the
BAL NOx was significantly higher in patients with sepsis who
died, but this effect was not seen in patients with trauma. We
combined the data from these two series on Days 3 and 7 of
ARDS because the patients had been identified by the same
study team using the same defining criteria for ARDS. In addition, the BAL NOx concentrations were very similar in the two series, even though the BAL fluids had been stored frozen for variable periods of time. In both series, the BAL NOx concentration was higher on Days 3 and 7 of ARDS in the patients who later died, and the differences became statistically
significant when the number of analyzable patients was increased by combining the two series. In the prior series of patients, the BAL concentrations of IL-1, MCP-1, TFG-, and
PCPIII on Day 7 after the onset of ARDS also were significantly higher in patients who died (7, 23, 24). Taken together,
these pieces of evidence suggest that patients with persistent
oxidant stress and intense inflammation in the lungs have the
worst prognosis.
The mortality rate in this series was 10% in the patients at risk and 22% in patients at the onset of ARDS. The mortality rate actually declined with time on the ventilator in the patients with persistent ARDS (Table 1). This is a lower mortality rate than in other studies of ARDS, and a lower mortality rate than in our earlier series of patients with ARDS (Table 2). In part, this reflects the declining mortality rate in all patients with ARDS at our institution (40). It also reflects the fact that the sickest patients were excluded from the bronchoalveolar lavage procedure, usually for safety concerns, so that the mortality in this study is lower than the mortality in the entire ARDS population at our institution (40). The number of patients with sustained ARDS declined with time because some improved, and some died. This is a limitation of all studies using serial bronchoalveolar lavage. Nevertheless, we believe that this patient population is representative of the patients with ARDS, and that the findings in patients with persistent ARDS are an accurate description of the events in the lungs of patients who remain mechanically ventilated.
In summary, we have found that products of NO accumulate in the lungs of patients before and after the onset of ARDS. This is associated with iNOS expression in alveolar macrophages, and nitrotyrosine formation in soluble proteins and BAL leukocytes. In patients with persistent ARDS, high concentrations of NO products appear to be associated with a higher mortality, but this needs to be confirmed in a larger series of patients. Oxidant stress mediated by NO appears to be an important factor in the pathogenesis of lung injury in patients at risk as well as in patients with established ARDS.
| |
Footnotes |
|---|
Correspondence and requests for reprints should be addressed to Thomas R. Martin, M.D., Pulmonary Research Labs, 151L, Seattle VA Medical Center, 1660 S. Columbian Way, Seattle, WA 98108. E-mail: trmartin{at}u.washington.edu
(Received in original form April 20, 2000 and in revised form June 16, 2000).
Acknowledgments:
Supported in part by grants HL-30542, GM-37696, AI-29103, HL-51173, and
HL-31197 from the National Institutes of Health and by the Medical Research
Service of the U.S. Department of Veterans Affairs.
| |
References |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Moncada S,
Higgs A.
The L-arginine-nitric oxide pathway.
N Engl J Med
1993;
329:
2002-2012
2. Nathan C, Xie QW. Nitric oxide synthases: roles, tolls, and controls. Cells 1994; 78: 915-918 [Medline].
3. Nussler AK, Billiar TR. Inflammation, immunoregulation, and inducible nitric oxide synthase. J Leukoc Biol 1993; 54: 171-178 [Abstract].
4.
Liu HW,
Anand A,
Bloch K,
Christiani D,
Krandin R.
Expression of inducible nitric oxid synthase by macrophages in rat lung.
Am J Respir
Crit Care Med
1997;
156:
223-228
5.
van der Vliet A,
Eiserich JP,
Shigenaga MK,
Cross CE.
Reactive nitrogen species and tyrosine nitration in the respiratory tract: epiphenomena or a pathobiologic mechanism of disease?
Am J Respir Crit Care
Med
1999;
160:
1-9
6.
Meduri GU,
Kohler G,
Headley S,
Tolley E,
Stentz F,
Postlethwaite A.
Inflammatory cytokines in the BAL of patients with ARDS.
Chest
1995;
108:
1303-1314
7. Goodman RB, Strieter RM, Steinberg KP, Milberg JA, Martin DP, Maunder RJ, Kunkel SL, Walz A, Hudson LD, Martin TR. Inflammatory cytokines in patients with persistence of the acute respiratory distress syndrome. Am J Respir Crit Care Med 1996; 154: 602-611 [Abstract].
8. Chollet-Martin S, Jourdain B, Gibert C, Elbim C, Chastre J, Gougerot-Pocidalo MA. Interactions between neutrophils and cytokines in blood and alveolar spaces during ARDS. Am J Respir Crit Care Med 1996; 154: 594-601 [Abstract].
9. Cochrane CG, Spragg R, Revak SD. Pathogenesis of the adult respiratory distress syndrome: evidence of oxidant activity in bronchoalveolar lavage fluid. J Clin Invest 1983; 71: 754-761 .
10. Lamb NJ, Gutteridge JM, Baker C, Evans TW, Quinlan GJ. Oxidative damage to proteins of bronchoalveolar lavage fluid in patients with acute respiratory distress syndrome: evidence for neutrophil-mediated hydroxylation, nitration, and chlorination. Crit Care Med 1999; 27: 1738-1744 [Medline].
11. Haddad IY, Pataki G, Hu P, Galliani C, Beckman JS, Matalon S. Quantitation of nitrotyrosine levels in lung sections of patients and animals with acute lung injury. J Clin Invest 1994; 94: 2407-2413 .
12. Kooy NW, Royall JA, Ye YZ, Kelly DR, Beckman JS. Evidence for in vivo peroxynitrite production in human acute lung injury. Am J Respir Crit Care Med 1995; 151: 1250-1254 [Abstract].
13.
Kobayashi A,
Hashimoto S,
Kooguchi K,
Kitamura Y,
Onodera H,
Urata Y,
Ashihara T.
Expression of inducible nitric oxide synthase
and inflammatory cytokines in alveolar macrophages of ARDS following sepsis.
Chest
1998;
113:
1632-1639
14.
Haddad IY,
Zhu S,
Ischiropoulos H,
Matalon S.
Nitration of surfactant
protein A results in decreased ability to aggregate lipids.
Am J Physiol
1996;
270:
L281-L288
15.
Haddad IY,
Crow JP,
Hu P,
Ye Y,
Beckman J,
Matalon S.
Concurrent
generation of nitric oxide and superoxide damages surfactant protein
A.
Am J Physiol
1994;
267:
L242-L249
16.
Zhu S,
Basiouny KF,
Crow JP,
Matalon S.
Carbon dioxide enhances nitration of surfactant protein A by activated alveolar macrophages.
Am J Physiol
2000;
278:
L1025-L1031
17.
Zhu S,
Ware LB,
Geiser T,
Matthay MA,
Matalon S.
Increased levels of nitrate and surfactant protein A nitration in the pulmonary edema fluid of
patients with acute lung injury.
Am J Respir Crit Care Med
2001;
163:
166-172
18.
Matute-Bello G,
Liles WC,
Radella FR II,
Steinberg KP,
Ruzinski JT,
Jonas M,
Chi EY,
Hudson LD,
Martin TR.
Neutrophil apoptosis in
the acute respiratory distress syndrome.
Am J Respir Crit Care Med
1997;
156:
1969-1977
19. Bernard GR, Artigas A, Brigham KL, Carlet J, Falke K, Hudson LD, Lamy M, LeGall JR, Morris A, Spragg R. The American-European Consensus Conference on ARDS: definitions, mechanisms, relevant outcomes and clinical trial coordination. Am J Respir Crit Care Med 1994; 149: 818-824 [Abstract].
20. Steinberg KP, Mitchell DR, Maunder RJ, Milberg JA, Whitcomb ME, Hudson LD. Safety of bronchoalveolar lavage in patients with adult respiratory distress syndrome. Am Rev Respir Dis 1993; 148: 556-561 [Medline].
21. Zhu S, Haddad IY, Matalon S. Nitration of surfactant protein A (SP-A) tyrosine residues results in decreased mannose binding ability. Arch Biochem Biophys 1996; 333: 282-290 [Medline].
22. Steinberg KP, Milberg JA, Martin TR, Maunder RJ, Cockrill BA, Hudson LD. Evolution of bronchoalveolar cell populations in the adult respiratory distress syndrome. Am J Respir Crit Care Med 1994; 150: 113-122 [Abstract].
23.
Clark JG,
Milberg JA,
Steinberg KP,
Hudson LD.
Type III procollagen
peptide in the adult respiratory distress syndrome: association of increased peptide levels in bronchoalveolar lavage fluid with increased
risk for death.
Ann Intern Med
1995;
122:
17-23
24.
Madtes DK,
Klima LD,
Rubenfeld G,
Milberg JA,
Steinberg KP,
Martin TR,
Hudson LD,
Clark JG.
Elevated transforming growth factor-
levels in bronchoalveolar lavage fluid in patients with acute respiratory distress syndrome.
Am J Respir Crit Care Med
1998;
158:
424-430
25.
Pryor WA,
Squadrito GL.
The chemistry of peroxynitrite: a product
from the reaction of nitric oxide with superoxide.
Am J Physiol
1995;
268:
L699-L722
26.
Beckman JS,
Koppenol WH.
Nitric oxide, superoxide, and peroxynitrate: the good, the bad, and ugly.
Am J Physiol
1996;
271:
C1424-C1437
27.
Evans TJ,
Buttery LD,
Carpenter A,
Springall DR,
Polak JM,
Cohen J.
Cytokine-treated human nuetrophils contain inducible nitric oxide
synthase that produces nitration of ingested bacteria.
Proc Natl Acad
Sci USA
1996;
93:
9553-9558
28.
Eisherich JP,
Cross CE,
Jones AD,
Halliwell B,
van der Vliet A.
Formation of nitrating and chlorinating species by reaction of nitrite with hypochlorous acid: a novel mechanisms for nitric oxide-mediated protein modification.
J Biol Chem
1996;
271:
19199-19208
29.
van der Vliet A,
Eiserich JP,
Halliwell B,
Cross CE.
Formation of reactive nitrogen species during peroxidase-catalyzed oxidation of nitrite:
a potential additional mechanism of nitric oxide-dependent toxicity.
J
Biol Chem
1997;
272:
7617-7625
30. Eisherich JP, Hristova M, Cross CE, Jones AD, Freeman BA, Halliwell B, van der Vliet A. Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils. Nature 1998; 391: 393-397 [Medline].
31. Weiland JE, Davis WB, Holter JF, Mohammed JR, Dorinsky PM, Gadek JE. Lung neutrophils in the adult respiratory distress syndrome: clinical and pathophysiologic significance. Am Rev Respir Dis 133:218-225.
32. Baldwin SR, Simon RH, Gram CM, Ketai CH, Boxer LA, Devall LJ. Oxidant activity in expired breath from patients with adult respiratory distress syndrome. Lancet 1986; 1: 11-14 [Medline].
33. Skerrett SJ, Martin TR. Roles for tumor necrosis factor alpha and nitric oxide in resistance of rat alvellar macrophages to Legionella pneumophila. Infect Immun 1996; 64: 3236-3243 [Abstract].
34.
Polsinelli T,
Meltzer MS,
Fortier AH.
Nitric oxide-independent killing of
Francisella tularensis by IFN--stimulated murine alveolar macrophages.
J Immunol
1994;
153:
1238-1245
[Abstract].
35. Albina JE. On the expression of nitric oxide synthase by human macrophages: why no NO. J Leukoc Biol 1995; 58: 643-649 [Abstract].
36.
Nicolson S,
da Gloria Bonecini-Almeida M,
Lapa e Silva JR,
Nathan C,
Xie Q,
Mumvord R,
Weidner JR,
Calaycay J,
Geng J,
Boechat N, et al
.
Inducible nitric oxide synthase in pulmonary alveolar macrophages
from patients with tuberculosis.
J Exp Med
1996;
183:
2293-2302
37. Suter PM, Suter S, Girardin E, Roux-Lombard P, Grau GE, Dayer JM. High bronchoalveolar levels of tumor necrosis factor and its inhibitors, interleukin-1, interferon, and elastase, in patients with adult respiratory distress syndrome after trauma, shock, or sepsis. Am Rev Respir Dis 1992; 145: 1016-1022 [Medline].
38. Jacobs RF, Tabor DR, Burks AW, Campbell GD. Elevated interleukin-1 release by human alveolar macrophages during the adult respiratory distress syndrome. Am Rev Respir Dis 1989; 140: 1686-1692 [Medline].
39. Donnelly SC, Haslett C, Reid PT, Grant IS, Wallace WAH, Metz CN, Bruce LJ, Bucala R. Regulatory role for macrophage migration inhibitory factor in the acute respiratory distress syndrome. Nat Med 1997; 3: 320-323 [Medline].
40.
Milberg JA,
Davis DR,
Steinberg KP,
Hudson LD.
Improved survival of
patients with acute respiratory distress syndrome (ARDS): 1983-1993.
JAMA
1995;
273:
306-309
This article has been cited by other articles:
 |
|
 |
|
  A. Lazrak, I. Nita, D. Subramaniyam, S. Wei, W. Song, H.-L. Ji, S. Janciauskiene, and S. Matalon {alpha}1-Antitrypsin Inhibits Epithelial Na+ Transport In Vitro and In Vivo Am. J. Respir. Cell Mol. Biol., September 1, 2009; 41(3): 261 - 270. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. L. Zemans, S. P. Colgan, and G. P. Downey Transepithelial Migration of Neutrophils: Mechanisms and Implications for Acute Lung Injury Am. J. Respir. Cell Mol. Biol., May 1, 2009; 40(5): 519 - 535. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Matute-Bello, C. W. Frevert, and T. R. Martin Animal models of acute lung injury Am J Physiol Lung Cell Mol Physiol, September 1, 2008; 295(3): L379 - L399. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Chen, C. A. Bosworth, T. Pico, J. F. Collawn, K. Varga, Z. Gao, J. P. Clancy, J. A. Fortenberry, J. R. Lancaster Jr., and S. Matalon DETANO and Nitrated Lipids Increase Chloride Secretion across Lung Airway Cells Am. J. Respir. Cell Mol. Biol., August 1, 2008; 39(2): 150 - 162. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. L. Lagan, D. D. Melley, T. W. Evans, and G. J. Quinlan Pathogenesis of the systemic inflammatory syndrome and acute lung injury: role of iron mobilization and decompartmentalization Am J Physiol Lung Cell Mol Physiol, February 1, 2008; 294(2): L161 - L174. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Song and S. Matalon Modulation of alveolar fluid clearance by reactive oxygen-nitrogen intermediates Am J Physiol Lung Cell Mol Physiol, October 1, 2007; 293(4): L855 - L858. [Full Text] [PDF]
|
|
|
|
 |
|
 S. Heemskerk, R. Masereeuw, H. van der Hoeven, and P. Pickkers Renal Effects of Nitric Oxide during Sepsis: Another Two-Edged Sword? Am. J. Respir. Crit. Care Med., August 15, 2007; 176(4): 419a - 420. [Full Text] [PDF]
|
|
|
|
|
|
B. M. Rotoli, V. Dall'Asta, A. Barilli, R. D'Ippolito, A. Tipa, D. Olivieri, G. C. Gazzola, and O. Bussolati Alveolar Macrophages from Normal Subjects Lack the NOS-Related System y+ for Arginine Transport Am. J. Respir. Cell Mol. Biol., July 1, 2007; 37(1): 105 - 112. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Geny, H. Khun, C. Fitting, L. Zarantonelli, C. Mazuet, N. Cayet, M. Szatanik, M.-C. Prevost, J.-M. Cavaillon, M. Huerre, et al. Clostridium sordellii Lethal Toxin Kills Mice by Inducing a Major Increase in Lung Vascular Permeability Am. J. Pathol., March 1, 2007; 170(3): 1003 - 1017. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. T. Forrester and J. S. Stamler A Classification Scheme for Redox-Based Modifications of Proteins Am. J. Respir. Cell Mol. Biol., February 1, 2007; 36(2): 135 - 137. [Full Text] [PDF]
|
|
|
|
|
|
D. E. McClintock, L. B. Ware, M. D. Eisner, N. Wickersham, B. T. Thompson, M. A. Matthay, and the National Heart, Lung, and Blood Institute ARDS Higher Urine Nitric Oxide Is Associated with Improved Outcomes in Patients with Acute Lung Injury Am. J. Respir. Crit. Care Med., February 1, 2007; 175(3): 256 - 262. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-W. Jin, L. Zhang, Q.-Q. Lian, D. Liu, P. Wu, S.-L. Yao, and D.-Y. Ye Posttreatment with Aspirin-Triggered Lipoxin A4 Analog Attenuates Lipopolysaccharide-Induced Acute Lung Injury in Mice: The Role of Heme Oxygenase-1 Anesth. Analg., February 1, 2007; 104(2): 369 - 377. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Sheffield, S. Mabry, D. W. Thibeault, and W. E. Truog Pulmonary Nitric Oxide Synthases and Nitrotyrosine: Findings During Lung Development and in Chronic Lung Disease of Prematurity Pediatrics, September 1, 2006; 118(3): 1056 - 1064. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. S. Farley, L. F. Wang, H. M. Razavi, C. Law, M. Rohan, D. G. McCormack, and S. Mehta Effects of macrophage inducible nitric oxide synthase in murine septic lung injury Am J Physiol Lung Cell Mol Physiol, June 1, 2006; 290(6): L1164 - L1172. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Neumann, N. Gertzberg, E. Vaughan, J. Weisbrot, R. Woodburn, W. Lambert, and A. Johnson Peroxynitrite mediates TNF-{alpha}-induced endothelial barrier dysfunction and nitration of actin Am J Physiol Lung Cell Mol Physiol, April 1, 2006; 290(4): L674 - L684. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 ATS Workshop Proceedings: Exhaled Nitric Oxide and Nitric Oxide Oxidative Metabolism in Exhaled Breath Condensate. Proceedings of the ATS, January 1, 2006; 3(2): 131 - 145. [Full Text] [PDF]
|
|
|
|
|
|
X. Peng, R.-E. E. Abdulnour, S. Sammani, S.-F. Ma, E. J. Han, E. J. Hasan, R. Tuder, J. G. N. Garcia, and P. M. Hassoun Inducible Nitric Oxide Synthase Contributes to Ventilator-induced Lung Injury Am. J. Respir. Crit. Care Med., August 15, 2005; 172(4): 470 - 479. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-C. T. Huang, Z. Li, L. E. Brighton, J. L. Carson, S. Becker, and J. M. Soukup 3-Nitrotyrosine attenuates respiratory syncytial virus infection in human bronchial epithelial cell line Am J Physiol Lung Cell Mol Physiol, May 1, 2005; 288(5): L988 - L996. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. D. Lang, M. Figueroa, K. D. Sanders, M. Aslan, Y. Liu, P. Chumley, and B. A. Freeman Hypercapnia via Reduced Rate and Tidal Volume Contributes to Lipopolysaccharide-induced Lung Injury Am. J. Respir. Crit. Care Med., January 15, 2005; 171(2): 147 - 157. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. D. Swain, T. W. Wright, P. M. Degel, F. Gigliotti, and A. G. Harmsen Neither Neutrophils nor Reactive Oxygen Species Contribute to Tissue Damage during Pneumocystis Pneumonia in Mice Infect. Immun., October 1, 2004; 72(10): 5722 - 5732. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Sparkman and V. Boggaram Nitric oxide increases IL-8 gene transcription and mRNA stability to enhance IL-8 gene expression in lung epithelial cells Am J Physiol Lung Cell Mol Physiol, October 1, 2004; 287(4): L764 - L773. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Whitsett, C. J. Bachurski, K. C. Barnes, P. A. Bunn Jr., L. M. Case, D. N. Cook, D. Crooks, M. W. Duncan, L. Dwyer-Nield, R. C. Elston, et al. Functional Genomics of Lung Disease Am. J. Respir. Cell Mol. Biol., August 1, 2004; 31(2/S1): S1 - S81. [Full Text] [PDF]
|
|
|
|
|
|
A. J. Gow, C. R. Farkouh, D. A. Munson, M. A. Posencheg, and H. Ischiropoulos Biological significance of nitric oxide-mediated protein modifications Am J Physiol Lung Cell Mol Physiol, August 1, 2004; 287(2): L262 - L268. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. M. Razavi, L. F. Wang, S. Weicker, M. Rohan, C. Law, D. G. McCormack, and S. Mehta Pulmonary Neutrophil Infiltration in Murine Sepsis: Role of Inducible Nitric Oxide Synthase Am. J. Respir. Crit. Care Med., August 1, 2004; 170(3): 227 - 233. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Hashimoto, T. Kawabe, K. Imaizumi, T. Hara, M. Okamoto, K. Kojima, K. Shimokata, and Y. Hasegawa CD40 Plays a Crucial Role in Lipopolysaccharide-Induced Acute Lung Injury Am. J. Respir. Cell Mol. Biol., June 1, 2004; 30(6): 808 - 815. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Chen, C. M. Fuller, T. R. Kleyman, and S. Matalon Mutations in the extracellular loop of {alpha}-rENaC alter sensitivity to amiloride and reactive species Am J Physiol Renal Physiol, June 1, 2004; 286(6): F1202 - F1208. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. C. Bailey, K. A. Da Silva, J. F. Lewis, K. Rodriguez-Capote, F. Possmayer, and R. A. W. Veldhuizen Physiological and inflammatory response to instillation of an oxidized surfactant in a rat model of surfactant deficiency J Appl Physiol, May 1, 2004; 96(5): 1674 - 1680. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. M. Wright, L. A. Ginger, N. Kosila, N. D. Elkins, B. Essary, J. L. McManaman, and J. E. Repine Mononuclear Phagocyte Xanthine Oxidoreductase Contributes to Cytokine-Induced Acute Lung Injury Am. J. Respir. Cell Mol. Biol., April 1, 2004; 30(4): 479 - 490. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. V. Usatyuk and V. Natarajan Role of Mitogen-activated Protein Kinases in 4-Hydroxy-2-nonenal-induced Actin Remodeling and Barrier Function in Endothelial Cells J. Biol. Chem., March 19, 2004; 279(12): 11789 - 11797. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Gao, B. X. Zeng, L. J. Zhou, and S. Y. Yuan Protective effects of early treatment with propofol on endotoxin-induced acute lung injury in rats Br. J. Anaesth., February 1, 2004; 92(2): 277 - 279. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Han, M. P. Fink, T. Uchiyama, R. Yang, and R. L. Delude Increased iNOS activity is essential for pulmonary epithelial tight junction dysfunction in endotoxemic mice Am J Physiol Lung Cell Mol Physiol, February 1, 2004; 286(2): L259 - L267. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. G. Laffey, D. Honan, N. Hopkins, J.-M. Hyvelin, J. F. Boylan, and P. McLoughlin Hypercapnic Acidosis Attenuates Endotoxin-induced Acute Lung Injury Am. J. Respir. Crit. Care Med., January 1, 2004; 169(1): 46 - 56. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Matalon, K. M. Hardiman, L. Jain, D. C. Eaton, M. Kotlikoff, J. P. Eu, J. Sun, G. Meissner, and J. S. Stamler Regulation of ion channel structure and function by reactive oxygen-nitrogen species Am J Physiol Lung Cell Mol Physiol, December 1, 2003; 285(6): L1184 - L1189. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Salinas, L. Sparkman, K. Berhane, and V. Boggaram Nitric oxide inhibits surfactant protein B gene expression in lung epithelial cells Am J Physiol Lung Cell Mol Physiol, November 1, 2003; 285(5): L1153 - L1165. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-W. Chow, M. T. Herrera Abreu, T. Suzuki, and G. P. Downey Oxidative Stress and Acute Lung Injury Am. J. Respir. Cell Mol. Biol., October 1, 2003; 29(4): 427 - 431. [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Lowson Nitric Oxide Signaling and Clinical Alternatives to Nitric Oxide Seminars in Cardiothoracic and Vascular Anesthesia, September 1, 2003; 7(3): 239 - 252. [Abstract] [PDF]
|
|
|
|
 |
|
 C. Gessner, S. Hammerschmidt, H. Kuhn, T. Lange, L. Engelmann, J. Schauer, and H. Wirtz Exhaled Breath Condensate Nitrite and Its Relation to Tidal Volume in Acute Lung Injury Chest, September 1, 2003; 124(3): 1046 - 1052. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Christofidou-Solomidou, A. Scherpereel, R. Wiewrodt, K. Ng, T. Sweitzer, E. Arguiri, V. Shuvaev, C. C. Solomides, S. M. Albelda, and V. R. Muzykantov PECAM-directed delivery of catalase to endothelium protects against pulmonary vascular oxidative stress Am J Physiol Lung Cell Mol Physiol, August 1, 2003; 285(2): L283 - L292. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Agorreta, M. Garayoa, L. M. Montuenga, and J. J. Zulueta Effects of Acute Hypoxia and Lipopolysaccharide on Nitric Oxide Synthase-2 Expression in Acute Lung Injury Am. J. Respir. Crit. Care Med., August 1, 2003; 168(3): 287 - 296. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Nabeyrat, G. E. Jones, P. S. Fenwick, P. J. Barnes, and L. E. Donnelly Mitogen-activated protein kinases mediate peroxynitrite-induced cell death in human bronchial epithelial cells Am J Physiol Lung Cell Mol Physiol, June 1, 2003; 284(6): L1112 - L1120. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Frank, J.-F. Pittet, H. Lee, M. Godzich, and M. A. Matthay High tidal volume ventilation induces NOS2 and impairs cAMP- dependent air space fluid clearance Am J Physiol Lung Cell Mol Physiol, May 1, 2003; 284(5): L791 - L798. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Yamamoto, T. Koizumi, T. Kaneki, K. Fujimoto, K. Kubo, and T. Honda Direct hemoperfusion with polymyxin B-immobilized fiber improves shock and hypoxemia during endotoxemia in anesthetized sheep Innate Immunity, December 1, 2002; 8(6): 419 - 426. [Abstract] [PDF]
|
|
|
|
|
|
J. D. Lang, P. J. McArdle, P. J. O'Reilly, and S. Matalon Oxidant-Antioxidant Balance in Acute Lung Injury Chest, December 1, 2002; 122 (2009): 314S - 320S. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Koval Sharing signals: connecting lung epithelial cells with gap junction channels Am J Physiol Lung Cell Mol Physiol, November 1, 2002; 283(5): L875 - L893. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Bebok, K. Varga, J. K. Hicks, C. J. Venglarik, T. Kovacs, L. Chen, K. M. Hardiman, J. F. Collawn, E. J. Sorscher, and S. Matalon Reactive Oxygen Nitrogen Species Decrease Cystic Fibrosis Transmembrane Conductance Regulator Expression and cAMP-mediated Cl- Secretion in Airway Epithelia J. Biol. Chem., November 1, 2002; 277(45): 43041 - 43049. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Tobin The Official Copy of AJRCCM Is Posted but Not Printed Am. J. Respir. Crit. Care Med., October 1, 2002; 166(7): 905 - 906. [Full Text]
|
|
|
|
 |
|
 I. C. Davis, A. J. Zajac, K. B. Nolte, J. Botten, B. Hjelle, and S. Matalon Elevated Generation of Reactive Oxygen/Nitrogen Species in Hantavirus Cardiopulmonary Syndrome J. Virol., July 17, 2002; 76(16): 8347 - 8359. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T.C. Bailey, C. Cavanagh, S. Mehta, J.F. Lewis, and R.A.W. Veldhuizen Sepsis and hyperoxia effects on the pulmonary surfactant system in wild-type and iNOS knockout mice Eur. Respir. J., July 1, 2002; 20(1): 177 - 182. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Matthay, H. G. Folkesson, and C. Clerici Lung Epithelial Fluid Transport and the Resolution of Pulmonary Edema Physiol Rev, July 1, 2002; 82(3): 569 - 600. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. F. Wang, M. Patel, H. M. Razavi, S. Weicker, M. G. Joseph, D. G. McCormack, and S. Mehta Role of Inducible Nitric Oxide Synthase in Pulmonary Microvascular Protein Leak in Murine Sepsis Am. J. Respir. Crit. Care Med., June 15, 2002; 165(12): 1634 - 1639. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. TOBIN Critical Care Medicine in AJRCCM 2001 Am. J. Respir. Crit. Care Med., March 1, 2002; 165(5): 565 - 583. [Full Text] [PDF]
|
|
|
|
|
|
M. Christofidou-Solomidou, S. Kennel, A. Scherpereel, R. Wiewrodt, C. C. Solomides, G. G. Pietra, J.-C. Murciano, S. A. Shah, H. Ischiropoulos, S. M. Albelda, et al. Vascular Immunotargeting of Glucose Oxidase to the Endothelial Antigens Induces Distinct Forms of Oxidant Acute Lung Injury : Targeting to Thrombomodulin, But Not to PECAM-1, Causes Pulmonary Thrombosis and Neutrophil Transmigration Am. J. Pathol., March 1, 2002; 160(3): 1155 - 1169. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Hickman-Davis, J. R. Lindsey, and S. Matalon Cyclophosphamide Decreases Nitrotyrosine Formation and Inhibits Nitric Oxide Production by Alveolar Macrophages in Mycoplasmosis Infect. Immun., October 1, 2001; 69(10): 6401 - 6410. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Coffey, S. M. Phare, and M. Peters-Golden Peroxynitrite-Induced Nitrotyrosination of Proteins Is Blocked by Direct 5-Lipoxygenase Inhibitor Zileuton J. Pharmacol. Exp. Ther., October 1, 2001; 299(1): 198 - 203. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Baldus, L. Castro, J. P. Eiserich, and B. A. Freeman Is {middle dot}NO News Bad News in Acute Respiratory Distress Syndrome? Am. J. Respir. Crit. Care Med., February 1, 2001; 163(2): 308 - 310. [Full Text]
|
|
|
|
|
|
J. M. Hickman-Davis, P. O'Reilly, I. C. Davis, J. Peti-Peterdi, G. Davis, K. R. Young, R. B. Devlin, and S. Matalon Killing of Klebsiella pneumoniae by human alveolar macrophages Am J Physiol Lung Cell Mol Physiol, May 1, 2002; 282(5): L944 - L956. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Proc. Am. Thorac. Soc. | Am. J. Respir. Cell Mol. Biol. |