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Little is known about neutrophil (PMN) apoptosis in the acute respiratory distress syndrome (ARDS). We used morphologic criteria to count apoptotic PMN in bronchoalveolar lavage fluid (BAL) of 35 patients on Days 1, 3, 7, 14, and 21 of ARDS and 13 patients on Days 1 and 3 of risk for ARDS. We found that the proportion of apoptotic PMN in BAL was low throughout the course of ARDS. There was no significant difference between the percentage of apoptotic PMN in patients at risk and patients with established ARDS or between patients who lived (2.4%) and patients who died (1.8%). When normal human PMN were incubated in ARDS BAL, a significantly lower proportion became apoptotic (50 ± 4%), as compared with PMN incubated in lavage fluid from normal volunteers (76 ± 7%, p < 0.05). This antiapoptotic effect of ARDS BAL was blocked by immunodepleting BAL of G-CSF and GM-CSF. We conclude that the proportion of apoptotic PMN recovered from the lungs of patients with ARDS is low throughout the course of ARDS. Furthermore, BAL from patients with ARDS prolongs survival of normal human PMN in vitro, and this effect is partially mediated by G-CSF and GM-CSF. Matute-Bello G, Liles WC, Radella F, II, Steinberg KP, Ruzinski JT, Jonas M, Chi EY, Hudson LD, Martin TR. Neutrophil apoptosis in the acute respiratory distress syndrome.
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INTRODUCTION
METHODS
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DISCUSSION
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Apoptosis, the process of programmed cell death, is now believed to play a major regulatory role in many biological
processes, including the inflammatory response (1, 2). For example, neutrophils (PMN) that migrate into an area of inflammation are believed to be removed either by necrosis, with
potential release of toxic mediators into the surrounding microenvironment, or by apoptosis. As PMN undergo apoptosis,
they lose surface adhesion molecules (3) and the ability to secrete granular contents (4). Apoptotic PMN are rapidly ingested
by macrophages before losing membrane integrity (5). Thus,
apoptosis provides a way of removing PMN from an area of inflammation with minimal damage to the surrounding tissue. Inflammatory mediators such as G-CSF, GM-CSF, IFN-
, TNF-
,
IL-2, IL-6, and glucocorticoids can modulate PMN apoptosis
(1, 2, 6), which suggests that the inflammatory response itself may influence the fate of PMN in tissue (4).
Little is known about neutrophil apoptosis in the uncontrolled inflammatory response that occurs in the acute respiratory distress syndrome (ARDS). Theoretically, inhibition of PMN apoptosis could result either in increased numbers of viable PMN, which could prolong the neutrophilic alveolitis, or in increased PMN necrosis, which could contribute to acute lung injury. Alternatively, stimulation of PMN apoptosis could be important for the resolution of the acute phase of ARDS (14).
We designed the present study to investigate PMN apoptosis in patients with ARDS or at risk for ARDS. We asked the following questions: (1) what is the proportion of apoptotic PMN in bronchoalveolar lavage (BAL) during the course of ARDS and in patients at risk for ARDS; (2) is the proportion of apoptotic PMN in BAL different in patients who live than in patients who die; and (3) does BAL fluid from patients with ARDS contain factors that modify the rate at which normal PMN undergo apoptosis in vitro?
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METHODS
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Patient Population
This study was performed as part of ongoing studies in the Seattle ARDS SCOR program. All patients admitted to the intensive care units of Harborview Medical Center (Seattle, WA) between 2/14/ 94 and 4/29/96 were prospectively evaluated and enrolled if they met predetermined criteria for either being at risk for ARDS or for having established ARDS.
Patients "at risk for ARDS." Patients with trauma were considered to be at risk for ARDS if they were intubated or on mask continuous positive airway pressure (CPAP) and had either: (1) two or more of the following: multiple fractures (two or more fractures of femur, tibia, humerus, or stable pelvis); 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 suspected sepsis were considered to be at risk for ARDS if they had: (1) two or more of the following: temperature greater than or equal to 39° C or lower than 36° C; white blood cell count greater than 14,000/ dl or less than 4,000 /dl; a positive blood culture or a known or strongly suspected source of infection; and (2) two or more of the following: systemic vascular resistance less than 800; 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 81,000 /dl.
Patients with ARDS. Patients were considered to have ARDS if they had critical hypoxemia (PaO2/FIO2 < 150 mm Hg, or < 200 mm Hg on > 5 cm H2O positive end-expiratory pressure); diffuse parenchymal infiltrates involving at least 50% of three or more quadrants on chest radiograph; pulmonary artery wedge pressure less than 18 mm Hg, or no clinical evidence of congestive heart failure; and no other obvious explanation for these findings. This definition has been used at our institution since 1985 (15). It differs slightly from the American-European Consensus Conference definition of ARDS because the oxygenation criteria are more strict, but all of our patients also meet the Consensus Conference criteria (18). We have continued to use our original definition so that longitudinal comparisons can be made in our extensive database.
Exclusions. Patients were excluded from the study for one or more of the following reasons (19): age younger than 18 yr; unsupportable hypoxemia (PaO2 < 80 mm Hg with FIO2 1.0); evidence of acute myocardial ischemia; cardiac dysrhythmias (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 on daily medication, sarcoidosis, or interstitial lung disease). All patients were followed up until death or hospital discharge. Survival was defined as hospital discharge.
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.
BAL Protocol
Patients at risk for ARDS underwent fiberoptic bronchoscopy and BAL within 24 h of the onset of risk for ARDS, then again 48 h later if they had not developed ARDS. Patients with established ARDS underwent fiberoptic bronchoscopy and BAL within 24 h of the onset of ARDS, and then again on Days 3, 7, 14, and 21. Fiberoptic bronchoscopy and BAL also were performed on healthy volunteers who were free of lung disease.
Bronchoscopy was performed using a fiberoptic bronchoscope inserted through the endotracheal tube in intubated patients while breathing 100% oxygen, or transorally in normal volunteers, as described (20). The bronchoscope was wedged in the right middle lobe or the lingula, and five separate 30 ml aliquots of 0.9% NaCl at 21° C were instilled and recovered by gentle hand suction. The BAL recovery averaged 75 ml (50% return) with a range of 23 to 110 ml.
The BAL aliquots were transported immediately to the laboratory
for processing. The fluid was pooled and filtered through gauze moistened with 0.9% NaCl to remove mucus. Total cell counts were performed with a hemacytometer. Cell differential counts were performed
on cytospin preparations stained with modified Wright-Giemsa (Diff-Quik; American Scientific Products, McGaw Park, IL). The slides
were stored in the dark at 21° C. The remainder of the lavage fluid was
spun at 200 × g for 30 min, and the supernatant was removed aseptically, and aliquots were stored in polypropylene tubes at 
70° C. The
total protein content of the BAL fluid was measured on an aliquot of
the supernatant using the bicinchoninic acid method (Pierce Chemical
Co., Rockford, IL).
Measurement of Apoptotic PMN
Normal PMN were recovered from venous blood of normal volunteers, and the PMN were separated using density gradient centrifugation (Polymorphoprep; Nycomed Pharma AS, Oslo, Norway), followed by hypotonic lysis of erythrocytes. The resulting preparation contained > 98% leukocytes of which > 95% were PMN. PMN were incubated in RPMI-1640 + L-glutamine, 292 µg/ml + penicillin, 100 U/ml and streptomycin, 100 µg/ml.
Morphologic criteria for apoptosis. In order to determine the proportion of apoptotic cells in normal peripheral blood PMN or in PMN recovered from the BAL fluids, the cytospin slides were evaluated independently by two investigators. On each slide, at least 200 PMN were graded for apoptosis using predetermined morphologic criteria. The PMN were considered apoptotic if they showed dense condensation of chromatin in the form of either a single nucleus or nuclear fragments not connected by strands (6, 21). In addition, PMN that were inside of macrophages were considered apoptotic. The results are expressed as the percentage of PMN on each slide that met the criteria for apoptosis.
Flow cytometry protocol. To measure cell membrane changes associated with apoptosis, normal peripheral blood PMN and PMN from three patients on day 1 of ARDS were labeled with annexin V-FITC (Apoptest-FITC kit; Nexins Research BV, Hoeven, The Netherlands), which labels membrane phosphatidylserine residues, and analyzed by flow cytometry (22, 23). PMN were washed twice with ice cold PBS and resuspended at a concentration of 1 × 106 cells/ml in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4). Then 445 µl were transferred to a 5 ml culture tube and 5 µl of annexin V-FITC and 50 µl of propidium iodide (100 µg/ml) were added. Cells were incubated for 10 min at room temperature in the dark and analyzed within 1 h by flow cytometry (Becton-Dickinson FACScan; San Jose, CA) with gating set on forward scatter and side scatter to identify PMN and exclude cell debris.
DNA electrophoresis. To identify the characteristic DNA fragmentation typical of apoptosis, normal PMN were washed once with PBS,
gently resuspended in 0.5 ml of lysis buffer (50 mM tris, 10 mM
EDTA, 1% sodium dodecyl sulfate and 250 µg/ml proteinase K) and
incubated for 16 h at 37° C/5% CO2. The lysate was extracted twice
with phenol/chloroform/isoamyl alcohol (25:24:1 v/v/v) and precipitated with 1 volume of ethanol and 0.1 volume of 3.5 M sodium acetate at 70° C overnight. The DNA was washed once with 70% ethanol, lyophilized and then resuspended in 30 µL TBE buffer (89 mM
Tris, 89 mM boric acid, 2 mM EDTA, pH 8.0) containing 250 µg/ml
RNAase and incubated at 65° C for 5 min. Then 0.1 volume of loading
buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF, 30% glycerol in water) was added to each sample, and electrophoresis was performed in a 1.2% agarose gel at 50 volts for 3 h. After staining with
ethidium bromide, the DNA was visualized with UV light and photographed.
Electron microscopy. To determine the prevalence of macrophages with ingested apoptotic PMN, we performed electron microscopy on the BAL cell pellets from three patients with ARDS. BAL samples containing at least 2 × 10 6 cells were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 2 h, washed in the same buffer, and post-fixed in 2% potassium-ferrocyanide and 2% osmium tetroxide in distilled water for 4 h at room temperature. The cells were rinsed with distilled water, then block stained with 0.5% uranyl acetate for 20 min and again rinsed in distilled water. The samples were embedded in 2% agar in 0.1 M sodium cacodylate buffer. After hardening the agar on ice, a pellet containing the lavage cells was cut from the block, dehydrated in a graded series of ethanol solutions and embedded in Medcast (Ted Pella Inc., Redding, CA) into four to six BEEM capsules. Thin sections were cut from two randomly selected blocks with a diamond knife using an LKB Nova ultramicrotome and collected on parlodion coated 200 mesh copper grids (Electron Microscopy Sciences, Fort Washington, PA). The sections were stained with uranyl acetate and lead citrate and examined with a JEOL TEM 1200 EX at a magnification of ×3,000 or higher. Only cells with a recognizable nucleus were included in the analysis. For each BAL sample several sections from at least two EM blocks were used for evaluation. Cells in 20 copper grid squares (1 square = 100 µm2) randomly selected from the whole grid area covered by the sections were counted. Macrophages were evaluated as follows: (1) macrophages without identifiable PMN debris; (2) macrophages containing identifiable PMN debris; (3) macrophages containing distinguishable PMN with nuclei. PMN were evaluated as follows: (1) normal; (2) apoptotic (dilated endoplasmic reticulum and/or dilated nuclear envelope); (3) necrotic (deteriorated plasma membrane, pyknotic nuclei). All BAL cells were analyzed without knowledge of the patient's status.
Immunodepletion Experiments
In order to determine which factors in the BAL fluid from patients
with ARDS might be modulating apoptosis of PMN, we immunodepleted BAL fluid of each of the following cytokines: G-CSF, GM-CSF, IL-6, and IFN-. Neutralizing polyclonal antibodies directed
against human G-CSF, GM-CSF, IL-6 and IFN- (R&D Systems Co.,
Minneapolis, MN) were added to ARDS BAL, normal BAL and
complete media at the following concentrations: GM-CSF, 60 µg/ml;
G-CSF, 10 µg/ml; IL-6, 10 µg/ml and IFN-, 10 µg/ml. These concentrations were chosen to achieve > 90% neutralization of each cytokine, based on neutralization assays performed by the manufacturer.
The samples were then incubated overnight at 4° C, and then spun at
10,000 × g for 45 min. The supernatants were diluted to a 50% concentration in complete media containing 1 × 10 6 PMN/ml, isolated as
described above from normal human peripheral blood. The PMN
were incubated for 18 h at 37° C and 5%CO2 and the percentage of
apoptotic PMN was determined using flow cytometry and Annexin V
binding as described above.
Measurement of G-CSF, GM-CSF, IL-6, and IFN- in BAL
The concentrations of G-CSF, GM-CSF, IL-6, and IFN- in BAL
fluid from patients on Day 1 of ARDS and from normal volunteers were measured by immunoassay (R&D Systems).
Statistical Analyses
The apoptosis data were not normally distributed, so the data are
shown as medians and ranges. Comparisons between two groups were
performed using the Mann-Whitney U test; multiple group comparisons were performed using the Kruskall-Wallis test. All data from
PMN incubation studies are presented as means ± SE. Comparisons
between two groups were made using the Student's t test and comparisons among multiple groups were performed using one-way ANOVA
with Fisher's post-hoc test. A p value 
0.05 was considered to be statistically significant.
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INTRODUCTION
METHODS
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DISCUSSION
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Demographic Data
Between February 1994 and April 1996, 45 patients were enrolled in the study. Thirteen of these patients met criteria for ARDS-at risk; two of these patients subsequently developed ARDS. Thirty-four of the patients met criteria for ARDS. All were studied within 24 h of the onset of ARDS, then serially on Days 3, 7, 14, and 21 as long as they remained intubated. The demographic characteristics of these patients are shown in Table 1. The results of the BAL fluid cell counts, differentials and total proteins are shown in Table 2.
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Validation of Morphologic Criteria for PMN Apoptosis
In order to test the validity of morphologic criteria for PMN apoptosis on cytospin preparations, we studied normal human PMN and compared measurements of apoptosis by morphology, flow cytometry using Annexin V binding, and DNA electrophoresis.
First, normal PMN were incubated in complete media for 0, 4, 6, 18, and 24 h, and the percentage of apoptotic PMN was determined using morphologic criteria (Figure 1). Parallel aliquots of the same PMN were analyzed for Annexin V binding using flow cytometry. There was excellent agreement between the two different methods (Figure 2). Additional aliquots of the same PMN were used for DNA extraction. The DNA ladders were visible beginning at 6 h, and the signal increased with time, reaching a maximum at 24 h (Figure 3). The appearance of DNA laddering paralleled the increase in Annexin V binding, and the appearance of morphologic criteria for apoptosis (Figure 2).
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Apoptotic PMN in BAL from Patients with ARDS and at Risk for ARDS
The percentages of apoptotic PMN in the BAL fluid of patients with ARDS and at risk for ARDS are shown in Figure 4. On the first day of ARDS, the median percentage of apoptotic PMN in the BAL fluid was 1.6% (range 0-8.3%, n = 34). The percentage of apoptotic PMN remained low throughout the course of illness. In patients at risk for ARDS the median percentage of apoptotic PMN was 0.5% on Day 1 (range 0-6.3%, n = 13) and 1.6% on Day 3 (range 0-19%, n = 11). There were no significant differences in the median percentage of apoptotic PMN during the course of ARDS or between patients at risk for ARDS and patients with established disease.
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In order to determine whether the proportion of apoptotic PMN changed over time in the BAL fluid of individual patients, we compared the percentage of apoptotic PMN on Day 1 of ARDS with the day of the last BAL for each patient. The median percentage of apoptotic PMN was 1.5% on Day 1 of ARDS (range 0-13.5) and 3% on the day of the last BAL (range 0-14.5) (p > 0.05).
Apoptosis and patient survival. Of the patients with ARDS, 27 (70.1%) survived to hospital discharge and eight died (29.9%). On Day 1 of ARDS, the median pecentage of apoptotic PMN in BAL fluid of patients who survived was 2.4% (range 0-13.5), versus 1.8% (range 0-7.3%) in patients who died. There was no significant difference between these two groups. On the day of the last BAL, the median percentage of apoptotic PMN in survivors was 2.5% (range 0.7-11) and 3% (range 0-7.3%) in patients who died (p > 0.05).
PMN Apoptosis in BAL Fluid Detected by Annexin V Labeling and Electron Microscopy
In order to provide further validation for the morphologic measurement of apoptotic PMN in the patients with ARDS, we measured apoptotic PMN in BAL fluid of three patients on Day 1 of ARDS prospectively, using Annexin V binding and electron microscopy. By morphologic criteria there were 0.5% ± 0.5% apoptotic PMN, by Annexin V labeling and flow cytometry there were 2.1% ± 1.1% apoptotic PMN, showing excellent agreement between the two methods. More PMN met criteria for apoptosis by electron microscopy (14.6% ± 1.6%). The percentage of alveolar macrophages that contained ultrastructural evidence of intracellular PMN debris was 30% ± 2%, whereas 8 ± 4.6% of the alveolar macrophages contained recognizable PMN.
Effect of ARDS BAL Fluid on Apoptosis of Normal PMN
Because the percentage of apoptotic PMN was low in patients with ARDS, we studied whether BAL fluid from patients with ARDS contains factors that inhibit PMN apoptosis. We incubated normal peripheral blood PMN in complete media supplemented with BAL fluid from either patients with ARDS or healthy volunteers. To control for the possible effects of BAL fluid protein concentration on PMN survival or on the concentration of any potential soluble mediators that affect apoptosis, we used BAL fluids containing either high protein concentration (approximately 1,000 µg/ml) or low protein concentration (100 to 200 µg/ml). The PMN were incubated for 18 h, then labeled with Annexin V and propidium iodide, and evaluated by flow cytometry. After 18 h in complete media supplemented with 50% BAL fluid from normal subjects, the proportion of apoptotic PMN was 76 ± 7% (Figure 5). This value was not significantly different from that of PMN incubated in complete media supplemented with 0.9% NaCl (81 ± 4%). When normal PMN were incubated in complete media supplemented with 50% BAL fluid from patients with ARDS, the proportion of apoptotic PMN was significantly lower (50 ± 4% for the low protein BAL, p < 0.05, and 48 ± 9% for the high protein BAL, p < 0.05). The protein concentration of the BAL fluid did not affect the percentage of apoptotic PMN. There was no change in the anti-apoptotic activity when ARDS BAL was diluted 1:2 or 1:4.
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Immunodepletion Studies
In order to identify the factors responsible for the antiapoptotic effect of ARDS BAL, we immunodepleted BAL from
either patients or normal volunteers of G-CSF, GM-CSF,
IFN-, and IL-6. Then we incubated normal human PMN for
18 h in the immunodepleted BAL at a 50% final concentration in complete media and measured the percentage of apoptotic PMN using Annexin V binding and flow cytometry. The
percentage of apoptotic PMN was significantly lower after incubation in BAL fluid from patients with ARDS (45 ± 4%)
than after incubation in normal BAL (79 ± 4%) (p < 0.05)
(Figure 6). Depleting ARDS BAL of G-CSF or GM-CSF increased the number of apoptotic PMN (74 ± 6% and 62 ± 8%, respectively) (p < 0.05), but depleting IL-6 or IFN-, or
using nonimmune IgG, had no significant effect (55 ± 4%,
57 ± 9%, 54 ± 2.8%, respectively, for anti IL-6, anti-IFN-,
and nonimmune IgG). Depleting normal BAL of G-CSF, GM-CSF, IL-6, and IFN- had no effect on PMN apoptosis. Depletion of both G-CSF and GM-CSF completely abolished the antiapoptotic effect of ARDS BAL (Figure 7).
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Measurement of G-CSF, GM-CSF, IL-6, and IFN- in BAL
We measured the concentrations of G-CSF, GM-CSF, IL-6,
and IFN- on BAL fluid from patients on Day 1 of ARDS and
normal volunteers. The median concentration of G-CSF was
238 pg/ml (range 19.5-5,241); GM-CSF 15 pg/ml (range 3.9-
159.91) and IL-6 1,132 pg/ml (range 6.25-12,495). Interferon-
was undetectable. The mean values for normal BAL were:
G-CSF, 25 ± 20 pg/ml; GM-CSF, 4 ± 0.4 pg/ml. IL-6 and
IFN- were undetectable in normal BAL.
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DISCUSSION |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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The major goal of this study was to determine the proportion of alveolar PMN that are undergoing apoptosis at different times during the course of ARDS. First, we determined whether PMN apoptosis can be measured reliably using morphologic criteria on BAL fluid cytospins. Then we investigated the proportion of apoptotic PMN in BAL fluid from patients with ARDS and determined whether this proportion changes during the course of the disease. We also investigated whether the proportion of apoptotic PMN is different before or after the onset of ARDS or in patients who lived versus those who died. Finally, we determined whether BAL fluid from patients with ARDS affects apoptosis of normal PMN in vitro.
We found that the percentage of apoptotic PMN can be measured reliably using morphologic criteria on stored BAL cytospin preparations. Using these criteria we found that in contrast to peripheral blood PMN, which rapidly became apoptotic in vitro, the proportion of apoptotic PMN in BAL fluid from patients with ARDS was remarkably low. This low proportion of apoptotic PMN remained constant throughout the course of ARDS. Patients at risk for ARDS also had very low proportions of apoptotic PMN in their BAL fluid. There was no significant difference in the percentage of apoptotic PMN between patients with ARDS who lived and patients who died. We also found that BAL fluid from patients with ARDS inhibits apoptotis of normal PMN incubated in vitro regardless of the BAL fluid total protein concentration. This effect is partially mediated by G-CSF and GM-CSF.
There have been few studies of PMN apoptosis during inflammatory responses in vivo. Initial studies by Lee and coworkers showed few apoptotic PMN in lung sections of rabbits with pneumonitis (6). Grigg and coworkers found evidence of apoptotic PMN in BAL from infants with neonatal distress syndrome (24). Tsuchida noted that PMN isolated from the peritoneal cavity of rats following an intraperitoneal injection of proteose peptone appear to have prolonged survival in vitro as compared with normal peripheral blood PMN (11). In contrast, inflammatory synovial fluid appeared to enhance neutrophil apoptosis in vitro (25).
Work from several groups has clearly established that many
inflammatory mediators inhibit apoptosis and prolong PMN
survival in vitro. These include LPS, C5a, G-CSF, GM-CSF,
IFN-, IL-2, IL-6, and LTB4 (6, 23). Some of these mediators, along with other cytokines, have been found to be elevated in BAL fluid from patients with ARDS (17, 26). We
found that the ARDS BAL fluid contains significant concentrations of G-CSF and GM-CSF, but IFN- was not detectable
in unconcentrated fluid. Taken together, these in vitro and in
vivo studies support the hypothesis that apoptosis of PMN is
inhibited during inflammation in vivo. Whether this is unique
to ARDS, or is also characteristic of other inflammatory processes remains to be determined.
Only limited information exists about the fate of PMN in the airspaces of the lungs. PMN that enter the airspaces of humans in response to chemotactic signals lose some specific granules during migration, but retain normal functional activity, including chemotactic responses and superoxide production (30). In contrast, the PMN that are recovered from the lungs of patients with ARDS are functionally impaired, with deficient migratory responses to a variety of stimuli and reduced respiratory burst activity (31). The present study suggests that the altered functional responses of alveolar PMN in ARDS are not explained by the rapid onset of either apoptosis or necrosis in the alveolar PMN. The change in functional responses must be explained by other factors in the alveolar milieu; e.g., inflammatory stimuli that may cause rapid degranulation (31).
Estimates of PMN apoptosis represent the static measurement of a dynamic process, and reflect the equilibrium between the development of apoptosis and phagocytosis of apoptotic PMN by macrophages and other cells. It seems unlikely
that differential washout characteristics of apoptotic PMN
could explain the low number of apoptotic PMN, as apoptotic
PMN lose adhesion molecules and should be less adherent (3).
It is possible that the low percentage of apoptotic PMN that
we found might be due to rapid phagocytosis of apoptotic
PMN by alveolar macrophages in the airspaces, as 38% of alveolar macrophages had evidence of either intracellular PMN
or PMN remnants by electron microscopy. In fact, Ren and
Savill found that inflammatory cytokines such as GM-CSF,
IFN-, IL-1
, TNF-, and TGF-1 enhance phagocytosis of
apoptotic PMN by human monocyte-derived macrophages
(32). This has not been tested with human alveolar macrophages. However, the finding that BAL fluid from ARDS patients reduces the proportion of apoptotic PMN in vitro supports the interpretation that constituents of the inflammatory
alveolar environment prolong PMN survival in vivo. Because
G-CSF and GM-CSF appear to be major factors that inhibit PMN apoptosis in the lungs of patients with ARDS, it is possible that in ARDS, G-CSF and GM-CSF have a dual effect on
PMN, prolonging survival of PMN by delaying apoptosis, and
at the same time enhancing phagocytosis of those PMN that
have become apoptotic.
We conclude that a low proportion of PMN recovered directly from the lungs of patients with ARDS are apoptotic and that this is true throughout the course of ARDS. The alveolar microenvironment of patients with established ARDS contains factors that prolong the survival of normal human PMN in vitro, and the growth factors G-CSF and GM-CSF play an important role. The precise role of apoptosis in the pathogenesis and resolution of alveolar inflammation in ARDS remains to be defined.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Thomas R. Martin, M.D., Seattle VA Medical Center, 151L, 1660 South Columbian Way, Seattle, WA 98108-1597. E-mail: trmartin{at}u.washington.edu
(Received in original form December 16, 1996 and in revised form April 15, 1997).
Acknowledgments: The authors thank Gordon Rubenfeld, M.D., and Ellen Caldwell, M.A., for assistance with the statistical analysis, and Venus A. Wong for expert technical assistance.
Supported in part by grants HL 30542 and AI29103 from the National Institutes of Health and by the Medical Research Service of the Department of Veterans Affairs. W.C.L. is a Pfizer Postdoctoral Fellow.
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References |
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METHODS
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DISCUSSION
REFERENCES
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1.
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G. S. Filippatos, N. Gangopadhyay, O. Lalude, N. Parameswaran, S. I. Said, W. Spielman, and B. D. Uhal Regulation of apoptosis by vasoactive peptides Am J Physiol Lung Cell Mol Physiol, October 1, 2001; 281(4): L749 - L761. [Abstract] [Full Text] [PDF]
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V. Durand, Y. Renaudineau, J.-O. Pers, P. Youinou, and C. Jamin Cross-Linking of Human Fc{gamma}RIIIb Induces the Production of Granulocyte Colony-Stimulating Factor and Granulocyte-Macrophage Colony-Stimulating Factor by Polymorphonuclear Neutrophils J. Immunol., October 1, 2001; 167(7): 3996 - 4007. [Abstract] [Full Text] [PDF]
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 C. OBERHOLZER, A. OBERHOLZER, M. CLARE-SALZLER, and L. L. MOLDAWER Apoptosis in sepsis: a new target for therapeutic exploration FASEB J, April 1, 2001; 15(6): 879 - 892. [Abstract] [Full Text] [PDF]
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Y. KITAMURA, S. HASHIMOTO, N. MIZUTA, A. KOBAYASHI, K. KOOGUCHI, I. FUJIWARA, and H. NAKAJIMA Fas/FasL-dependent Apoptosis of Alveolar Cells after Lipopolysaccharide-induced Lung Injury in Mice Am. J. Respir. Crit. Care Med., March 1, 2001; 163(3): 762 - 769. [Abstract] [Full Text] [PDF]
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A. KURDOWSKA, J. M. NOBLE, K. P. STEINBERG, J. T. RUZINSKI, L. D. HUDSON, and T. R. MARTIN Anti-interleukin 8 Autoantibody:Interleukin 8 Complexes in the Acute Respiratory Distress Syndrome . Relationship between the Complexes and Clinical Disease Activity Am. J. Respir. Crit. Care Med., February 1, 2001; 163(2): 463 - 468. [Abstract] [Full Text]
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C. SITTIPUNT, K. P. STEINBERG, J. T. RUZINSKI, C. MYLES, S. ZHU, R. B. GOODMAN, L. D. HUDSON, S. MATALON, and T. R. MARTIN Nitric Oxide and Nitrotyrosine in the Lungs of Patients with Acute Respiratory Distress Syndrome Am. J. Respir. Crit. Care Med., February 1, 2001; 163(2): 503 - 510. [Abstract] [Full Text]
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G. MATUTE-BELLO, C. W. FREVERT, O. KAJIKAWA, S. J. SKERRETT, R. B. GOODMAN, D. R. PARK, and T. R. MARTIN Septic Shock and Acute Lung Injury in Rabbits with Peritonitis . Failure of the Neutrophil Response to Localized Infection Am. J. Respir. Crit. Care Med., January 1, 2001; 163(1): 234 - 243. [Abstract] [Full Text]
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A. J. FISHER, S. C. DONNELLY, N. HIRANI, C. HASLETT, R. M. STRIETER, J. H. DARK, and P. A. CORRIS Elevated Levels of Interleukin-8 in Donor Lungs Is Associated with Early Graft Failure after Lung Transplantation Am. J. Respir. Crit. Care Med., January 1, 2001; 163(1): 259 - 265. [Abstract] [Full Text]
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G. Matute-Bello, R. K. Winn, M. Jonas, E. Y. Chi, T. R. Martin, and W. C. Liles Fas (CD95) Induces Alveolar Epithelial Cell Apoptosis in Vivo : Implications for Acute Pulmonary Inflammation Am. J. Pathol., January 1, 2001; 158(1): 153 - 161. [Abstract] [Full Text] [PDF]
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S. J. Leuenroth, P. S. Grutkoski, A. Ayala, and H. H. Simms The loss of Mcl-1 expression in human polymorphonuclear leukocytes promotes apoptosis J. Leukoc. Biol., July 1, 2000; 68(1): 158 - 166. [Abstract] [Full Text]
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D. Droemann, S. P. Aries, F. Hansen, M. Moellers, J. Braun, H. A. Katus, and K. Dalhoff Decreased Apoptosis and Increased Activation of Alveolar Neutrophils in Bacterial Pneumonia Chest, June 1, 2000; 117(6): 1679 - 1684. [Abstract] [Full Text] [PDF]
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M. Behnia, K. A. Robertson, and W. J. Martin II Lung Infections : Role of Apoptosis in Host Defense and Pathogenesis of Disease Chest, June 1, 2000; 117(6): 1771 - 1777. [Abstract] [Full Text] [PDF]
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 L. B. Ware and M. A. Matthay The Acute Respiratory Distress Syndrome N. Engl. J. Med., May 4, 2000; 342(18): 1334 - 1349. [Full Text] [PDF]
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K. E. GREENE, J. R. WRIGHT, K. P. STEINBERG, J. T. RUZINSKI, E. CALDWELL, W. B. WONG, W. HULL, J. A. WHITSETT, T. AKINO, Y. KUROKI, et al. Serial Changes in Surfactant-associated Proteins in Lung and Serum before and after Onset of ARDS Am. J. Respir. Crit. Care Med., December 1, 1999; 160(6): 1843 - 1850. [Abstract] [Full Text]
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 E. R. Goodman, P. Stricker, M. Velavicius, R. Fonseca, E. Kleinstein, R. Lavery, E. A. Deitch, C. J. Hauser, and H. H. Simms Role of Granulocyte-Macrophage Colony-Stimulating Factor and Its Receptor in the Genesis of Acute Respiratory Distress Syndrome Through an Effect on Neutrophil Apoptosis Arch Surg, October 1, 1999; 134(10): 1049 - 1054. [Abstract] [Full Text] [PDF]
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G. Matute-Bello, W. C. Liles, K. P. Steinberg, P. A. Kiener, S. Mongovin, E. Y. Chi, M. Jonas, and T. R. Martin Soluble Fas Ligand Induces Epithelial Cell Apoptosis in Humans with Acute Lung Injury (ARDS) J. Immunol., August 15, 1999; 163(4): 2217 - 2225. [Abstract] [Full Text] [PDF]
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T. R. Martin Lung Cytokines and ARDS: Roger S. Mitchell Lecture Chest, July 1, 1999; 116 (2009): 2S - 8S. [Full Text]
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 M. Weiss, L. L. Moldawer, and E. M. Schneider Granulocyte Colony-Stimulating Factor to Prevent the Progression of Systemic Nonresponsiveness in Systemic Inflammatory Response Syndrome and Sepsis Blood, January 15, 1999; 93(2): 425 - 439. [Full Text] [PDF]
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K. Yamashita, A. Takahashi, S. Kobayashi, H. Hirata, P. W. Mesner Jr, S. H. Kaufmann, S. Yonehara, K. Yamamoto, T. Uchiyama, and M. Sasada Caspases Mediate Tumor Necrosis Factor-alpha -Induced Neutrophil Apoptosis and Downregulation of Reactive Oxygen Production Blood, January 15, 1999; 93(2): 674 - 685. [Abstract] [Full Text] [PDF]
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E. R. Goodman, E. Kleinstein, A. M. Fusco, D. P. Quinlan, R. Lavery, D. H. Livingston, E. A. Deitch, and C. J. Hauser Role of Interleukin 8 in the Genesis of Acute Respiratory Distress Syndrome Through an Effect on Neutrophil Apoptosis Arch Surg, November 1, 1998; 133(11): 1234 - 1239. [Abstract] [Full Text] [PDF]
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R. MARSHALL, G. BELLINGAN, and G. LAURENT The acute respiratory distress syndrome: fibrosis in the fast lane Thorax, October 1, 1998; 53(10): 815 - 817. [Full Text]
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E R CHILVERS, A G ROSSI, J MURRAY, and C HASLETT Regulation of granulocyte apoptosis and implications for anti-inflammatory therapy Thorax, July 1, 1998; 53(7): 533 - 534. [Full Text]
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