Abstract
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.
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-γ, interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) 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 (6-8). 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?
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.
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.
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 /Fi O2 < 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.
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 Fi O2 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.
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).
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).
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 (ɛ M = 4,400 M−1 cm−1) 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).
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.
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.
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).
| At Risk | ARDS | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Day 1 | Day 3 | Day 1 | Day 3 | Day 7 | Day 14 | Day 21 | ||||||||
| n | 19 | 14 | 36 | 41 | 30 | 16 | 11 | |||||||
| Age, yr | 48.0 | 48.2 | 42.5 | 45.1 | 46.3 | 44.6 | 42.6 | |||||||
| Sex, % male | 58 | 64 | 67 | 59 | 63 | 50 | 46 | |||||||
| Primary risk | ||||||||||||||
| Sepsis, n (%) | 8 (42.1) | 4 (28.6) | 10 (27.8) | 15 (36.6) | 10 (33.3) | 4 (25.0) | 3 (27.3) | |||||||
| Trauma, n (%) | 11 (57.9) | 10 (71.4) | 15 (41.7) | 15 (36.6) | 14 (46.7) | 8 (50.0) | 5 (45.5) | |||||||
| Other, n (%) | 0 (0) | 0 | 11 (30.6) | 11 (26.8) | 6 (20.0) | 4 (25.0) | 3 (27.3) | |||||||
| Apache II Score | 21.8 ± 1.4 | 14.6 ± 2.1 | 21.1 ± 1.1 | 21.0 ± 1.1 | 18.8 ± 1.4 | 18.9 ± 1.3 | 15.5 ± 1.8 | |||||||
| Po 2/Fi O2 ratio | 209.6 ± 23.8‡ | 243.8 ± 30.0 | 152.5 ± 8.3‡ | 162.7 ± 8.8 | 203.1 ± 14.0 | 197.2 ± 13.1 | 207.7 ± 18.7 | |||||||
| Mortality, n (%) | 2 (10) | 1 (7) | 8 (22) | 8 (20) | 3 (10) | 1 (7) | 0 | |||||||
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.
Fig. 1. Nitrate and nitrite concentration (NOx) in BAL from normal volunteers (NL), patients at risk for ARDS (RISK) and patients with established ARDS (ARDS) studied at sequential times. The horizontal axis shows the patient group and the day on which the BAL was performed. The numbers in parentheses above the horizontal axis are the number of subjects in each group. The data are presented as box plots showing the 10th, 25th, 75th, and 90th percentiles and the median. *p ⩽ 0.005 versus normal subjects.
[More] [Minimize]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).
Fig. 4. Nitrate and nitrite concentration (NOx) in BAL of patients with established ARDS on Day 1 after the onset of ARDS, grouped by the risk factor for ARDS. The horizontal axis shows the major risk groups for patients with ARDS. The numbers in parentheses above the horizontal axis are the number of subjects in each group. *p = 0.001 for sepsis versus trauma groups; **p = 0.04 for sepsis versus other groups.
[More] [Minimize]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).
Fig. 2. Nitrate and nitrite concentration (NOx) in BAL from patients at risk for ARDS on Day 1 after the onset of the risk factor, separated by those who developed ARDS (closed circles) or those who did not develop ARDS (open circles). Each point represents one patient. The bars are median values. The numbers in parentheses above the horizontal axis are the number of patients in each group. There is no statistically significant difference between the groups.
[More] [Minimize]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, 22-24). 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.
Fig. 3. Nitrate and nitrite concentration (NOx) in BAL of patients with established ARDS who lived or died. The numbers in parentheses above the horizontal axis are the number of patients in each group. *p = 0.034 for lived versus died on Day 7 after onset of ARDS. Only one patient alive on Day 14 died. None of the patients alive on Day 21 died.
[More] [Minimize]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).
| ARDS, Day 3 | ARDS, Day 7 | |||||||
|---|---|---|---|---|---|---|---|---|
| Lived | Died | Lived | Died | |||||
| 1996 series (n) (7) | 28 | 22 (44%) | 20 | 26 (56.5%) | ||||
| BAL NOX | 5.75 ± 0.63 | 8.14 ± 1.17 | 6.86 ± 0.89 | 8.31 ± 1.01 | ||||
| p Value | 0.08 | 0.28 | ||||||
| Current series (n) | 33 | 8 (19.5%) | 27 | 3 (10%) | ||||
| BAL NOX | 6.55 ± 0.58 | 9.92 ± 2.41 | 5.16 ± 0.46 | 21.3 ± 13.9 | ||||
| p Value | 0.24 | 0.034 | ||||||
| Combined series (n) | 61 | 30 (33%) | 47 | 29 (38.2%) | ||||
| BAL NOX | 6.18 ± 0.43 | 8.61 ± 1.06 | 5.89 ± 0.47 | 9.66 ± 1.67 | ||||
| p Value | 0.039 | 0.037 | ||||||
| Sepsis (n) | 15 | 11 | 11 | 12 | ||||
| BAL NOX | 6.18 ± 0.88 | 10.91 ± 1.88 | 5.76 ± 0.86 | 12.01 ± 3.54 | ||||
| p Value | 0.035 | 0.029 | ||||||
| Trauma (n) | 24 | 8 | 24 | 8 | ||||
| BAL NOX | 6.76 ± 0.75 | 5.26 ± 0.99 | 6.04 ± 0.75 | 5.30 ± 0.62 | ||||
| p Value | NS | NS | ||||||
| Other (n) | 22 | 11 | 12 | 9 | ||||
| BAL NOX | 5.42 ± 0.61 | 8.77 ± 1.86 | 5.87 ± 0.84 | 10.41 ± 2.29 | ||||
| p Value | 0.066 | 0.065 | ||||||
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.
Fig. 5. Nitrotyrosine concentration in BAL fluid from patients at risk and with established ARDS. The numbers in parentheses above the horizontal axis are the number of subjects in each group. *p < 0.05 for normals versus “at risk” and Days 1, 7, and 14 of ARDS. The bars represent the median values.
[More] [Minimize]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.
Fig. 6. Measurement of iNOS and nitrotyrosine positive alveolar macrophages in cytospin preparations of ARDS BAL cells. (A) iNOS positive alveolar macrophages (% of total AM). (B) Nitrotyrosine positive AM (% of total AM). *p = 0.02 for Risk Day 1 versus Risk Day 3. **p = 0.034 for Risk Day 1 versus ARDS Day 1.
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Fig. 7. Analysis of iNOS in BAL cells by immunocytochemistry. (A) Normal alveolar macrophages showing no labeling for iNOS. (B) Day 1 of Risk. (C ) Day 1 of ARDS. (D) Day 7 of ARDS. Control preparations using cells from a patient on Day 1 of ARDS labeled with the secondary antibody only showed no immunolabeling (similar to the normal alveolar macrophages shown in (A).
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Fig. 8. Analysis of nitrotyrosine residues in BAL cells by immunocytochemistry. (A) normal alveolar macrophages showing no labeling for nitrotyrosine. (B), Day 1 of Risk. (C ), Day 1 of ARDS. (D), Day 7 of ARDS. Control preparations of cells from patients on Day 1 of ARDS incubated with an excess of nitrotyrosine showed minimal labeling (similar to (A).
[More] [Minimize]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-γ) 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-γ 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-1β, MCP-1, TFG-α, 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.
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.
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