 Levels in
Bronchoalveolar Lavage Fluid of Patients with
Acute Respiratory Distress Syndrome
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ABSTRACT |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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The acute respiratory distress syndrome (ARDS) frequently results in a fibroproliferative response
that precludes effective alveolar repair. Transforming growth factor-
(TGF-), a potent epithelial
and mesenchymal cell mitogen, may modulate the response to lung injury. In this study, we determined whether bronchoalveolar lavage fluid (BALF) concentrations of TGF- are increased during the
first 2 wk after the onset of ARDS and, if so, whether increased TGF- levels in lavage fluid are associated with increased levels of procollagen peptide III (PCP III), a biological marker of fibroproliferation, and with increased fatality rates. We enrolled 74 consecutive patients with ARDS prospectively
identified on admission to the intensive care unit of a tertiary care hospital, and 11 patients with
chronic interstitial lung disease. Thirteen healthy volunteers served as control subjects. TGF- concentrations were measured in BALF recovered on Days 3, 7, and 14 after the onset of ARDS (total of
130 lavage samples). TGF- was detected in the lavage fluid of 90% of patients with ARDS (67 of 74),
and in 100% of patients with idiopathic pulmonary fibrosis (IPF) (10 of 10), but in none of 13 normal
volunteers. At each day tested, the median lavage TGF- level of patients with ARDS was significantly
higher than that of normals. The overall fatality rate was 45% (33 of 74 patients). In a univariate
analysis, the median TGF- levels in nonsurvivors were 1.5-fold higher at Day 7 (p = 0.06) and 1.8-fold higher at Day 14 (p = 0.048). The fatality rate was 4 times higher (CI 1.6, 17.5) for patients with
both increased lavage TGF- and PCP III concentrations at Day 7 than for patients with low TGF- and PCP III values, indicating a synergistic relationship between TGF- and PCP III. We conclude that
increased levels of TGF- in BALF are common in patients with ARDS and that lavage TGF- is associated with a marker of the fibroproliferative response in sustained ARDS.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The acute respiratory distress syndrome (ARDS) frequently results in a fibroproliferative response that is characterized by mesenchymal cell proliferation and extracellular matrix accumulation within the alveolar and interstitial compartments of the injured lung. Autopsy series of patients with ARDS identify pulmonary fibrosis as a common feature (1, 2). Pulmonary collagen content is increased in those patients dying later than 10 d after the onset of this syndrome (2). Analysis of lung tissue obtained by open lung (3) and transbronchial biopsies (4) suggests an association between mortality and pulmonary fibrosis in established ARDS. Pulmonary edema fluid and bronchoalveolar lavage (BAL) levels of procollagen III peptide (PCP III), a marker of collagen synthesis, are also elevated in ARDS and associated with increased mortality (5, 6).
Mesenchymal cell proliferation following tissue injury results, at least in part, from the increased expression of growth factors within the tissue microenvironment. Transforming
growth factor- (TGF-), a potent mitogen and chemotactic
factor, could play a prominent role in the fibroproliferative response after acute lung injury. TGF- is a cell surface associated as well as a secreted mitogen that shares 42% homology
with human epidermal growth factor (EGF) (7) and binds to
the same cell-surface receptor as EGF, referred to as the EGF
receptor (8). TGF- stimulates the proliferation of cultured
epithelial cells (9), fibroblasts (10), and endothelial cells (11).
In addition, activation of the EGF receptor stimulates collagen and glycosaminoglycan synthesis by mesenchymal cells,
and induces the expression of matrix metalloproteinases and
tissue inhibitors of metalloproteinases in vitro (12).
Increasing evidence implicates TGF- in the fibroproliferative response to acute lung injury. Animal models demonstrate the induction of TGF- expression within the injured
lung (17). Overexpression of TGF- by respiratory epithelial cells in transgenic mice induces pulmonary fibrosis characterized by fibroblast proliferation and collagen deposition
(20). Bronchoalveolar lavage fluid (BALF) recovered within
3 d of the onset of ARDS contains TGF--like growth promoting bioactivity for cultured lung fibroblasts (21). Furthermore,
TGF- immunoreactive protein is present in pulmonary edema
fluid recovered from patients within the first 24 h of onset of
acute lung injury (6). However, TGF- concentrations in BALF
recovered from patients with established ARDS, and the association of lavage TGF- levels with biological markers of lung
injury or fibrosis, or with clinical outcome in ARDS have not
been examined.
In this study our goals were to test the following hypotheses: (1) that TGF- concentrations are increased in the BALF
of patients during the first 2 wk of ARDS; (2) that increased
TGF- levels are associated with increased fatality rates in established ARDS; and (3) that the effect of TGF- on fatality
rates is independent of the severity of lung injury and the concentration of lavage PCP III.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Patients
All patients between the ages of 18 and 72 yr who were admitted to the intensive care units at Harborview Medical Center (Seattle, Washington) between January 1, 1988 and April 30, 1991 were screened prospectively for the onset of ARDS. Patients were screened using the following criteria: (1) critical hypoxemia defined as a ratio of arterial oxygen pressure to fraction of inspired oxygen (PaO2/FIO2) of 150 mm Hg or less or a PaO2/FIO2 ratio of 200 mm Hg or less using 5 cm H2O or more of positive end-expiratory pressure; (2) diffuse parenchymal infiltrates involving at least three quadrants on chest radiographs; (3) pulmonary artery wedge pressure of 18 mm Hg or less in patients with pulmonary artery catheters or no clinical evidence of congestive heart failure; and (4) no other explanation for these findings. Patients were excluded for safety reasons if they met any of the following criteria: (1) PaO2 of less than 80 mm Hg with FIO2 of 1.0; (2) evidence of acute ischemic heart disease; (3) severe hypotension (systolic blood pressure < 90 mm Hg); (4) cardiac dysrhythmias (heart rate > 140 beats/min or complex ventricular ectopy); (5) sustained increased intracranial pressure greater than 20 mm Hg; and (6) endotracheal tube internal diameter less than 7.0 mm (22). Patients were not excluded because of high minute ventilation, high levels of positive end-expiratory pressure, or presence of barotrauma. Informed consent was obtained from either the patient or legal surrogate. The study was approved by the University of Washington Institutional Review Committee.
Before BAL was performed, the levels of FIO2, PaO2, static compliance, and positive end-expiratory pressure were recorded. These data were used to calculate a modified lung injury score for ARDS, as described by Murray and colleagues (23), with the exception that a chest radiograph score was not included. All patients had alveolar infiltrates in three or four quadrants, therefore, the Murray acute lung injury score would be 0.75 to 1.0 points greater than our modified score. Patients in this study with lung injury scores of 2.0 or more met Murray's criteria for severe lung injury.
Risk factors associated with the development of ARDS were defined as previously described (24) and identified prospectively when
the patient entered the study. For this analysis, three risk categories
were included: sepsis syndrome, trauma, and "other risks." Trauma
risk was defined as the presence of multiple long bone or pelvic fractures, pulmonary contusion, or trauma-associated multiple transfusions (
15 units in 24 h of emergency resuscitation). The category entitled "other risks" included aspiration of gastric contents, drug
overdose, and multiple transfusions. We attempted to perform lavage
serially at Days 3, 7, and 14 after the onset of ARDS unless the patient
died, was extubated or become too unstable to tolerate the procedure,
as indicated by the above criteria. Patients were followed until death
or hospital discharge. Survival was defined as discharge from the hospital.
A convenience sample of BAL specimens collected from patients with chronic interstitial lung disease was analyzed for comparison purposes.
BAL and Analysis
All patients were intubated at the time of BAL which was performed
as previously reported (5). Eleven patients with chronic interstitial
lung disease, and 13 normal volunteers underwent lavage using a similar technique. Serum samples also were obtained from patients and
normal volunteers at the time of lavage and were stored at 
70° C. Lavage samples were evaluated for total cell counts, differential cell
counts, and total protein as previously described (5, 25, 26).
TGF- Analysis
The concentration of biologically active TGF- in BALF specimens
or serum was determined by ELISA (Oncogene Research Products, Cambridge, MA) according to the manufacturer's instructions. The lavage fluids were concentrated 22 to 35 times by ultrafiltration (Centricon-3 Concentrator, molecular weight cutoff = 3,000; Amicon, Beverly, MA) as follows. A precisely measured volume of unconcentrated lavage fluid was concentrated to a "deadstop" volume of 40 µl. The concentrated lavage fluid was quantitatively removed from the ultrafiltration retentate chamber and the retentate chamber and ultrafiltration
membrane were rinsed with 50 µl of 0.9% NaCl. The rinse was combined with the concentrated lavage fluid and the final volume was adjusted to 115 µl using 0.9% NaCl. The TGF- immunoreactive protein
concentration in each sample was measured in duplicate. The ELISA is
sensitive to 10 pg/ml of human TGF- standard, is linear over the
range of 10 to 1,000 pg/ml and is highly specific for the biologically active forms of TGF-. There was no measurable cross reactivity observed when human EGF was assayed at a concentration of 100 ng/ml.
Samples in which TGF- levels were less than the lower detection
limit were assigned a value of 0.4 pg/ml, which represents the lower
limit of detection for the TGF- ELISA, for subsequent data analysis.
To validate the ultrafiltration procedure, known quantities of human TGF- standard were added to 3-ml aliquots of lavage fluid recovered from normal subjects. These TGF--supplemented lavage
specimens were concentrated as previously described and the TGF-
levels of the volume-adjusted concentrates were measured by ELISA.
In these control experiments the quantitative recovery of added TGF-
was 100%. Serum samples were diluted 20 times prior to TGF- immunoreactive protein determination, in accordance with the manufacturer's instructions. The results are expressed as TGF- levels in the
unconcentrated lavage fluid or serum.
Type III Procollagen Peptide Analysis
The concentrations of type III procollagen peptide (PCP III) in BALF specimens were determined by radioimmunoassay (RIAgnost PAP; Behringwerke, Marburg, Germany) as previously reported (5).
Statistical Analyses
Lavage fluid TGF- concentrations in survivors and nonsurvivors
were compared initially using the nonparametric Wilcoxon rank sum
test (27). The relative risk (RR) for fatality in patients with a lavage
fluid TGF- level of 1.08 pg/ml (the median value) or more was compared with those with values less than 1.08 pg/ml. Because of the small
sample size the 95% confidence intervals (CI) for the relative risks
were estimated using a boot strap approach (28). We also performed a
stratified analysis to determine if the relation between TGF- concentration and fatality differed as a function of lavage fluid PCP III concentration or by degree of lung injury, using the lung injury score.
Multivariate analysis using logistic regression was performed to
determine the effect of TGF- levels on the risk of death while controlling for the potential confounding effects of demographic, physiologic, and biochemical markers. Variables were included in the multivariate model based on their univariate association or because of their
potential behavior as confounders. Age, gender, lung injury score,
BAL neutrophil, macrophage, protein, and PCP III concentrations were
included together and the effect on the TGF- coefficient was noted.
Lavage fluid cell and protein concentrations were analyzed using logarithmic transformations (log10). To explore whether the effect of TGF-
changed at various levels of lung injury severity or lavage PCP-III and
to supplement the stratified analysis, appropriate interaction terms
were entered into a model containing TGF-.
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RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Patients
Lavage fluid samples were available from 74 of 306 (24%) patients prospectively identified as having ARDS between January 1, 1988 and April 30, 1991. Patients enrolled in this study
were similar to the 232 individuals who were not enrolled according to risk group, race, and sex distribution, but they were
slightly younger (median age, 41 yr compared with 43 yr; p = 0.0123) and had a lower fatality rate (45% compared with
57%; p = 0.07). The latter difference may be a consequence of
the entry criteria which excluded unstable patients and patients who died before Day 3. In the subset of trauma patients,
injury severity scores (29) were nearly identical to those of
trauma victims with ARDS who were not entered into the
study (mean score, 26). Reasons for exclusion from the study
included: patient unstable, n = 27 (11.5%); age less than 18 or
greater than 72 yr, n = 37 (16%); died before Day 3, n = 10 (4.5%); unable to consent, n = 30 (13%); consent refused, n = 6 (2.5%); investigator unavailable, n = 15 (6.5%); enrolled in
another study, n = 12 (5%); patient "missed," n = 12 (5%);
severe chronic health problems, n = 6 (2.5%); insufficient lavage specimen, n = 31 (13.5%) or other, n = 20 (9%) (30). As
discussed previously, the selection tended to eliminate the most severely ill patients as well as those who recovered rapidly (5). Of 105 patients who underwent lavage during this period, 74 (70%) had sufficient lavage specimen available for
measurement of TGF-.
A total of 130 BAL procedures were performed in 74 patients. Fifty-five were done 3 d after the onset of ARDS, 49 lavages were done 7 d after onset, and 26 lavages were done 14 d after onset. Thirty-two patients had one lavage procedure, 28 had two lavage procedures, and 14 had three serial lavage procedures on Days 3, 7, and 14 after the onset of ARDS. Eighteen patients had lavage on Days 3 and 7 only, three patients had lavage on Days 3 and 14 only, and seven patients had lavage on Days 7 and 14 only. Of 55 patients who had lavage on Day 3, 32 patients also had lavage on Day 7. Of 23 patients who dropped out, three died, 13 were extubated, four were "missed" because of insufficient lavage fluid for analysis, and three were missed for other reasons. On Day 7, the 49 patients who had lavage included 17 new patients who met eligibility and safety criteria.
Clinical characteristics of the study population are shown in Table 1. Sepsis and major trauma were risk factors for ARDS in most of our study patients (n = 49). Other risk factors (gastric aspiration, drug overdose, and massive transfusion) were present in 25 patients. Impairment of gas exchange and lung mechanics was severe as indicated by PaO2/FIO2 ratios and modified lung injury scores. Lavage macrophage concentration at Day 3 (p = 0.003), and the PaO2/FIO2 ratio measured at Day 7 (p = 0.001) were higher in survivors. Lavage protein concentration at Day 3 (p = 0.03), lavage PCP III concentrations at Day 3 (p = 0.0029) and Day 7 (p = 0.0014), and modified lung injury score at Day 7 (p = 0.002) were higher in nonsurvivors. Survivors and nonsurvivors were similar with respect to the modified lung injury scores measured at Days 3 and 14, lavage PCP III levels at Day 14, lavage macrophage and protein concentrations measured at Days 7 and 14, and lavage neutrophil concentration at Days 3, 7, and 14.
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Univariate Analysis
TGF- was detected in the BAL in 67 of 74 (90%) patients
within the first 14 d after the onset of ARDS. The median
TGF- value for the entire population of patients with ARDS
was 1.08 pg/ml (range, 0.4 to 6.44 pg/ml). TGF- was also detected in lavage samples from 10 of 10 (100%) patients with
idiopathic pulmonary fibrosis (IPF) with a median TGF-
concentration of 0.85 pg/ml (range, 0.46 to 4.47 pg/ml). In contrast, TGF- was undetectable in lavage samples from 13 normal volunteers and one patient with hypersensitivity pneumonitis. For the purposes of statistical analysis, the lavage
TGF- concentrations for the normal subjects were assigned a
value of 0.4 pg/ml which represents the lower limit of detection for the assay. At each day tested, the median lavage TGF-
level of patients with ARDS was significantly higher than that
of normals (p = 0.001) (Figure 1). Likewise, the median lavage
TGF- level of patients with IPF was significantly higher than
that of normals (p = 0.0001). There was no significant difference in median lavage TGF- levels among the three ARDS
risk groups at each day. Similarly, there was no significant difference in median lavage TGF- levels between patients with
IPF compared with patients with ARDS for the three risk groups
at each day. The association between lavage TGF- levels and
outcome was analyzed at each day (Figure 2). At 14 d after the
onset of ARDS, the median TGF- level was 1.8-fold higher in
nonsurvivors (1.27 compared with 0.7, p = 0.048). Similarly, at
7 d after the onset of ARDS there was a trend toward a higher
median lavage TGF- level in patients who died (1.3 compared
with 0.87, p = 0.06).
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Lavage TGF- concentration was analyzed as a dichotomous variable to determine the relative risk for fatality after
the onset of ARDS. The cutoff value for lavage TGF- (1.08 pg/ml) used in this categorical analysis was the median value
of our study population. At 7 and 14 d after onset, the relative
risk for fatality was approximately 1.5 times greater in patients
with elevated lavage TGF- concentrations of 1.08 pg/ml or
more compared with those with concentrations less than 1.08 pg/ml (Figure 3).
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To investigate the relationship between lavage TGF- levels and severity of lung injury, the patient populations were
stratified on each day by lung injury score (< 2 compared with
2) (Figure 4). At 7 d after onset, patients with increased
lung injury scores and lavage TGF- levels had a fatality rate
of 78% compared with 53% for patients with increased lung
injury scores alone, representing a relative risk for fatality of
approximately 1.47 (CI 0.9, 2.4). Of note, the fatality rate was
5.4 times greater (CI 1.7, 12.5) for patients with both elevated
lung injury scores and elevated lavage TGF- levels than for
patients with low lung injury scores and lavage TGF- concentrations at 7 d. Similarly, at 14 d after onset, patients with elevated lung injury scores and lavage TGF- concentrations had
an approximately 1.8-fold increase (CI 0.6, 8.4) in the relative
risk for fatality compared with patients with increased lung injury scores alone.
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We previously reported that lavage PCP III concentrations
were associated with an increased risk for fatality in 117 patients with ARDS (5). Because the present patient population
is a subset from that study, patients were stratified on each day
by lavage PCP III levels (< 1.75 U/ml compared with 1.75 U/ml) (Figure 5). At 7 d after onset, patients with increased lavage PCP III and TGF- levels had a fatality rate of 80% compared with 56% for patients with increased procollagen levels
alone, which represents a relative risk of 1.4 (CI 0.9, 2.6). The
fatality rate was 4 times higher (CI 1.6, 17.5) for patients with
both increased lavage PCP III and elevated TGF- concentrations than for patients with low PCP III and TGF- values.
Fourteen days after onset, the relative risk for fatality was 1.8 (CI 0.6, 8.6) for patients with elevated PCP III and TGF- levels compared with patients with elevated procollagen levels
alone.
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Multivariate Analysis
We performed a multivariate analysis that included variables
that might be associated independently with increased risk for fatality or might account independently for the increased lavage TGF- concentration. To simplify the interpretation of
the coefficients and interaction relationships, we coded lung
injury score ( 2 compared with < 2) and lavage PCP III level
( 1.75 Units/ml compared with < 1.75 Units/ml) as categorical variables using cutoff values identical to the ones we previously reported (5). Similarly, lavage TGF- level was coded as
a categorical variable ( 1.08 pg/ml compared with < 1.08 pg/
ml) based on the median lavage TGF- concentration of our
study population. The observed effect on fatality rate of increased lavage TGF- concentration (odds ratio 2.3, CI 0.7 to
7.0) on Day 7 was independent of the potentially confounding
variables of patient age, Day 7 PCP III levels, neutrophil or
protein concentration, or Day 7 lung injury score. Furthermore, interaction terms for lavage PCP III level and lung injury score were not statistically significant suggesting that the
effect of TGF- was constant across different levels of these
variables.
Serum TGF- Levels
To determine whether increased BAL TGF- concentrations
might be a reflection of increased serum TGF- levels, we analyzed TGF- values in serum collected concurrently with
BAL at Day 7 after the onset of ARDS (n = 16). TGF- was
undetectable in the patients' serum despite lavage levels of
0.82 to 5.83 pg/ml. TGF- was not detected in the serum of
normal subjects (n = 4).
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DISCUSSION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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The major goal of this study was to investigate the relationship
between TGF- concentrations in alveolar lining fluids and the course of patients with established ARDS. Our major interest was to determine whether TGF- was associated with
other markers of the fibroproliferative response in sustained
ARDS. We found that TGF- levels in BALF are significantly
elevated in most patients with established ARDS. At Days 3, 7, and 14 after onset, the median lavage TGF- levels of patients with ARDS are significantly higher than that of normals. In patients with sustained ARDS, there is a trend toward increased fatality when lavage TGF- levels are elevated
at Days 7 and 14. Furthermore, we observed that the fatality rate was 4 times higher for patients with both increased lavage TGF- and PCP III concentrations at Day 7 than for patients
with low TGF- and PCP III values, indicating a synergistic
relationship between TGF- and PCP III.
Our study is the first to demonstrate that TGF- levels in
BALF are elevated in the vast majority of a large cohort of patients with established ARDS and in patients with IPF compared with normal subjects. The presence of TGF- in the
lavage of patients with ARDS supports the hypothesis that
TGF- may modulate the fibroproliferative response following acute lung injury in humans. TGF- in the alveolar microenvironment could contribute to the pulmonary fibrosis
found in patients with delayed resolution of ARDS (2) and, in
part, account for the restrictive pulmonary impairment observed in many survivors of ARDS (31). The presence of
TGF- in the alveolar lining fluid recovered from patients
with IPF provides additional support to a possible role for
TGF- in the pathogenesis of lung fibrosis.
The results suggest that elevated lavage TGF- concentrations may be associated with an increased fatality rate in patients with delayed resolution of ARDS. Furthermore, our
formal multivariate analysis suggests that TGF-, lung injury
score, and PCP III exert their effect independent of each
other. In addition, the fatality risk associated with TGF- concentrations was independent of other putative lavage markers
of disease activity such as total protein concentration, and
neutrophil or alveolar macrophage concentration. It would
have been interesting to perform serial analysis of lavage TGF- levels and compare the outcome of patients with persistent elevations in TGF- with patients in whom TGF- levels fell with time. However, only 58% of the patients who underwent lavage at Day 3 also had available lavage specimens
at Day 7, and only 19% of study patients had lavage performed at Days 3, 7, and 14 after the onset of ARDS. This loss
of study patients at the later time points limited serial analysis
because the dataset was too small.
Patients at even higher risk for fatality were identified by
considering the lavage TGF- concentration together with the lung injury score or lavage PCP III concentration. Patients
with elevated TGF- concentrations and either elevated lung
injury scores or increased PCP III levels on Day 7 had mortality rates in excess of 75%. The identification of patients at
high risk for fatality as demonstrated in this cohort could be
useful in the design and interpretation of future intervention
studies. The cutoff value for lavage TGF- (1.08 pg/ml) used
in this categorical analysis was the median value in our study
population. The performance of this value in other populations remains to be validated. In the present study, lavage PCP
III levels at Day 14 failed to attain statistical significance for
fatality compared with our previous report. The reason for
this difference is because the present data set is a subset of
that previously described and includes fewer patients at Day 14.
We observed a trend toward improved survival in patients
with elevated lavage TGF- levels at Day 3 after onset of
ARDS. This observation is consistent with the study by Chesnutt and coworkers in which elevated TGF- levels in pulmonary edema fluid on Day 1 after onset of ARDS were not associated with an increased fatality rate (6). Early in the course
of acute lung injury, elevated TGF- levels could promote
processes that restore normal lung architecture. TGF- has
been shown to increase alveolar liquid clearance in ventilated
rats (32), stimulate cultured alveolar epithelial cell proliferation (33), and promote epithelial cell migration and spreading
in vitro (33, 34). However, when resolution of ARDS is delayed, increased lavage TGF- levels may act in concert with
other profibrotic cytokines such as platelet-derived growth factor and transforming growth factor-
to orchestrate disordered lung repair. In support of this fibrogenic capacity, TGF-
stimulates the proliferation of fibroblasts in vitro (10) and induces cultured fibroblast secretion of tissue inhibitors of matrix metalloproteinases capable of inhibiting degradation of
newly deposited collagen within areas of lung injury (16). In
transgenic mice, overexpression of TGF- has been shown to
induce lung fibrosis (20).
The relationship between TGF- and outcome might have
been understated because we were not able to study all patients with ARDS. Both the sickest and the healthiest patients
were excluded from the study (e.g., those who had died, were
clinically unstable, or who had improved and been extubated
prior to Day 3). Thus, the extremes of illness severity were not
represented in the study population. We believe that it will always be difficult in this type of study to include all patients
with ARDS, because ethical issues and safety issues prohibit
the study of patients who are the most unstable and have the
highest fatality rates, and because we felt it was not appropriate to perform BAL after extubation in patients who had improved.
The concentration of TGF- in the alveolar lining fluid is
within a biologically relevant range. BAL has been estimated
to dilute edema fluid approximately 100-fold (35). This suggests that the median TGF- concentration in the alveolar lining fluid was in the range of 100 pg/ml. This assumption is consistent with the report by Chesnutt and coworkers who found
that TGF- concentrations in undiluted pulmonary edema
fluid were in the range of 0.035 to 2.57 ng/ml within the first
day of acute lung injury (6). As TGF- has been shown to induce cultured epithelial cell proliferation at a concentration of
100 pg/ml or less, it is likely that the TGF- levels that we detected would be sufficient to stimulate cell proliferation in
vivo (19, 36).
The biological basis for increased TGF- levels in the alveolar lining fluid is most likely the result of increased TGF- release by secretory effector cells within the injured lung.
TGF- was not detected in the serum of patients with ARDS
at a time when it was identified in lavage fluid, so it seems unlikely that TGF- extravasated from the vascular space into
the alveolar compartment. Furthermore, there was no correlation between TGF- levels and total protein concentrations in
the lavage fluids. One possible source of TGF- is inflammatory cells present in the alveolar lumen because TGF- messenger RNA (mRNA) is detected by reverse transcription-
polymerase chain reaction amplification of bronchoalveolar lavage cell RNA recovered from patients with ARDS (37).
This supposition is consistent with the observation that activated human alveolar macrophages transcribe and secrete
TGF- in an inducible manner in vitro (38). Other possible
sources of TGF- in the lavage fluid of patients with ARDS
are pulmonary epithelial cells and fibroblasts (17, 18, 39). Additional studies using immunohistochemistry and in situ hybridization on cells recovered in the lavage or lung tissue obtained from patients with ARDS are needed to identify the
cellular sources of TGF- in this patient population.
The association between lavage fluid TGF- concentration
and biological markers of lung fibrosis suggests that increased
TGF- secretion beyond the first few days after lung injury
may be detrimental to effective alveolar repair in some instances. Previous reports from our group documented that ongoing respiratory failure was present in most fatal cases of
ARDS, whereas death was infrequent among patients who no
longer required ventilatory support (5, 40). It is reasonable to
speculate that increased TGF- production, in conjunction
with the release of other profibrotic cytokines, promotes mesenchymal cell proliferation, subsequent lung fibrosis, and prolonged or irreversible respiratory failure. On the basis of our
results, we conclude that TGF- secretion in the lung is increased in patients with sustained ARDS. We suggest that this growth factor plays a role in modulating the fibroproliferative response and that persistently elevated TGF- levels may contribute to disordered lung repair.
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Footnotes |
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Correspondence and requests for reprints should be addressed to David K. Madtes, M.D., Fred Hutchinson Cancer Research Center, 1124 Columbia Street M677, Seattle, WA 98104-2092.
(Received in original form November 25, 1997 and in revised form March 16, 1998).
Acknowledgments: The authors thank Margaret K. Greer and John Ruzinski for excellent technical assistance, and Ellen Caldwell for assistance in the statistical analysis.
Supported by an American Lung Association of Washington Research Training Fellowship (L.D.K.), NIH FIRST Award HL 49401 (D.K.M.), NIH SCOR Grant HL30542 (J.G.C., T.R.M., L.D.H.), and by the Medical Research Service of the Department of Veteran Affairs (T.R.M.).
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References |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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