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C3A DES ARG and elastase-alpha 1-protease inhibitor levels in lavage fluid from patients with adult respiratory distress syndrome
ORIGINAL
INVESTIGATIONS
C3A DES ARG and Elastase-Alpha 1-Protease in Lavage Fluid From Patients With Adult Distress Syndrome Lynne
M. Zheutlin,
Eugene
J-M.A.
Thonar, Robert
Susan A,
Lemley-Gillespie,
Balk,
and
Roger
Elizabeth
Inhibitor Levels Respiratory R. Jacobs,
Michael
E. Hanley,
C. Bone
Complement activation and neutrophil release of proteolytic enzymes are thought to be important in the pathogenesis of the adult respiratory distress syndrome (ARDS). Levels of both C3a and elastolytic activity are reported to be elevated in bronchoalveolar lavage (BALI samples from patients with ARDS. The relative specificity of elevated BAL levels of complement fragments and elastase to ARDS is unknown. We compared levels of these mediators of inflammation in the BAL of patients with ARDS to those of control populations including patients with pneumonia, sepsis, and other lung diseases such as sarcoidosis and bronchitis. C3a was measured as
C3a DES ARG by radioimmunoassay. Elastase levels were measured by an ELISA which detects elastase bound to its inhibitor, alpha 1-protease inhibitor, and corrected for total protein concentration. BAL levels of elastase-alpha 1 -protease inhibitor complex were elevated in patients with ARDS, and in patients who are at risk for ARDS, such as those with sepsis or pneumonia. Elevated levels of C3a DES ARG were detected in some patients with ARDS, but this increase was not consistently observed when compared with the other patient groups. @ 7987 by Grune & Stratton, Inc.
V
metabolized to biologically inactive C3a DES ARG in vivo by the enzyme carboxypeptidase. A quantitative asssay for the C3a DES ARG content of biologic fluids allows one to assess the degree of complement activation.7 Polymorphonuclear leukocytes release neutral proteases extracellularly when exposed to chemotactic or phagocytic stimuli.8v9 One of these, neutrophil elastase, can degrade elastin, collagen, proteoglycans, and basement membrane,” all of which are structural components of lung and its vasculature. In vivo free elastase is rapidly inactivated by complex formation with inhibitors. The major serum and bronchoalveolar fluid inhibitor of neutrophil elastase is alpha 1-protease inhibitor.” The recent development of an assay for elastase in complex with its inhibitor alpha 1-protease inhibitor’ has made it possible to assesslevels of these complexes in BAL. These levels reflect local production and release of elastase by cells in the alveolar space. The relative specificity of elevated BAL levels of complement fragments and elastase to ARDS is unknown. In this study, we compared levels of both C3a DES ARG and elastase complex in the BAL of patients with ARDS to those of control populations including patients with pneumonia, sepsis, and other lung diseases such as sarcoidosis and chronic bronchitis. We sought to determine the sensitivity and specificity of levels of these mediators as markers of lung injury.
ARIOUS HYPOTHESES have been proposed to account for the pathologic changes that characterize the adult respiratory distress syndrome (ARDS). One hypothesis suggests that complement activation induced by the underlying clinical process leads to neutrophil aggregation and stasis in the pulmonary microvasculature. This is followed by neutrophil activation and release of neutral and acid proteases such as elastase, as well as toxic oxygen radicals.’ The release of these inflammatory mediators results in tissue damage and activation of other inflammatory pathways. Complement activation by either the classical or alternative pathway results in the cleavage of C3 into its fragments C3a and C3b. C3a causes histamine release from human basophils and mast cells,2,3 increases vascular permeability,4 and induces both smooth muscle contractions and lymphocyte suppression.6 C3a is rapidly From the Departments of Internal Medicine, Immunology, and Biochemistry, Rush Medical College, Chicago. Supported in part by a developmental grant from the Ofice of Consolidated Laboratory Services, Rush-Presbyterian-St Luke’s Medical Center, and by the Upjohn Company. Address reprint requests to Elizabeth R. Jacobs, MD. Department of Internal Medicine, Rush-Presbyterian-St Luke’s Medical Center, 17.53 W Congress Pkwy, Chicago, IL 60612.
o 1987 by Grune & Stratton, 0883-9441/87/0202-0001$05.00/0
86
Inc.
Journal
of Critical
Care, Vol 2, No 2 (June),
1987:
pp 86-92
C3A
AND
ELASTASE
INHIBITOR
MATERIALS
LEVELS
a7
IN BAL
AND METHODS
These protocols were approved by the Human Investigation Committee. Informed consent was obtained for BAL from patients undergoing diagnostic or therapeutic bronchoscopy. Bronchoalveolar lavage was performed on those ARDS patients who were enrolled in an ongoing therapeutic trial prior to the initiation of specific therapy. Control specimens were obtained from uninvolved lung segments of nonsmoking patients undergoing bronchoscopy for unilateral nodules or unexplained hemoptysis. Smokers were evaluated separately, as others have reported increased elastolytic activity in BAL of these patients.‘* Patients with lung disease met standard clinical criteria for the diagnosis of sarcoidosis, acute or chronic bronchitis, or idiopathic pulmonary fibrosis. The diagnosis of pneumonia was based on clinical, laboratory, and roentgenographic criteria. Lavage was performed in the lung segment with active disease. Sepsis was defined by the appearance of systemic manifestations of infection. All patients had hypotension plus at least two of the following criteria: (1) white blood cell count greater than 12,000 or less than 3,000, (2) hyperthermia or hypothermia, (3) positive blood culture, and (4) a cultured pathogen from a suspected source or gross pus in an enclosed space. Lavage was part of the initial diagnostic evaluation and usually was performed within 24 hours of the recognition of the septic process. Since the patients were categorized later in their hospital course, none of the septic patients had ARDS, although all had evidence of pneumonitis. The ARDS group consisted of patients with acute respiratory failure requiring mechanical ventilation and in whom the PaO, to Fi02 ratio was less than 200 with posititve end expiratory pressrue (PEEP) or less than 150 without PEEP. All had diffuse bilateral infiltrates on chest x-ray and all had a pulmonary capillary wedge pressure < 18 mm HG. Lavage was performed within 24 hours after the patient fulfilled the clinical criteria for ARDS. After standard preoperative preparation and local airway anesthesia,” BAL specimens were collected from the right middle lobe or lingula. Sterile saline was instilled and aspirated in six 20-mL aliquots. Lavage fluid was placed on ice and immediately transported to the research laboratory. The fluid was passed through a nylon mesh filter to exclude mucus and particles greater than 60 microns in diameter. Total cell count was quantitated by use of a hemocytometer and differential counts were performed on cytospin preparations stained with Wright Giemsa stain. Cellular material was removed by centrifugation for ten minutes at 300 x g at 4°C. Additional particulate matter was removed by centrifugation at 3,600 x g at 4°C and the supernatant was quick frozen and stored at - 90°C. Levels of C3a DES ARG in BAL were measured using a competitive binding radioimmunoassay described by Hugli and Chenoweth.’ Kits containing rz51-labeled C3a DES ARG, anti-C3a DES ARG, and five standard solutions of purified C3a DES ARG were generously provided by Upjohn Diagnostics (Kalamazoo, MI). An initial precipitation step to remove C3, IgG, and other proteins was performed as described in the kit procedure manual. Duplicate samples and standards were assayed simultaneously. This assay measures C3a and C3a DES ARG at concentrations from 40 to 1,000 ng/mL.
Elastase bound to its inhibitor alpha I-protease inhibitor was measured by an enzyme-linked immunoassay9~‘4 kindly provided by Merck (Darmstadt, West Germany). Rriefly, samples in various dilutions were incubated in tubes precoated with antibody to neutrophil elastase. The complex of elastase and alpha I-protease inhibitor present in the sample binds to this antibody via the elastase portion of the complex. After washing, alkaline phosphatase labeled antibody directed against alpha I-protease inhibitor was allowed to interact with complexes that had attached to the surface. Substrate for the alkaline phosphatase, p-nitrophenyl phosphate was added and the amount of p-nitrophenol produced was measured as absorbance at 405 nm. The optical density was directed proportional to the amount of elastase present in the range of 1 to 8 ng/mL. The concentration of elastase present was calculated by comparing the absorbance values to values generated from known concentrations of elastasealpha 1-protease inhibitor. Elastase levels were normalized per milligram total protein to correct for leakage of plasma alpha I-protease inhibitor into the alveolar space. An increase in alpha l-protease inhibitor concentration has been reported in BAL samples from patients with ARDS.” Total protein was measured by the Lowry assay.16 In order to determine if BAL from patients with ARDS contained free elastase, excess alpha I-protease inhibitor was added to these samples. Normal human plasma, diluted 1:5 with test buffer diluent, served as the source of alpha 1-protease inhibitor. Preliminary experiments had established that this amount of normal human plasma contained enough alpha 1-protease inhibitor to totally inhibit the enzyme activity of a solution containing 3,000 ng/mL purified free elastase. Equal volumes of BAL and diluted plasma or BAL and test buffer diluent were incubated for one hour at room temperature and then assayed simultaneously for elastase-alpha I-protease inhibitor complex as described above. The amount of elastase-alpha I-protease inhibitor complex present in diluted plasma alone (~8 ng/mL) was subtracted from the results. Individual values for patients in each group are given. Values for patient groups were compared using a nonparametric test (Friedman’s two-way test). Correlation was calculated by linear regression analysis.” A P value of c.05 was considered significant.
RESULTS
The ARDS group consisted of 17 patients who met the clinical criteria for the syndrome. They ranged in age from 43 to 88 years of age; 6 were male and 11 were female. Clinical data on these patients are presented in Table 1. The majority of these patients had sepsis as the underlying cause of ARDS. Eight patients survived more than thirty days. Seven septic patients with pneumonia or pneumonitis were evaluated. Since the categorization of the patients took place after enrollment in the study, none of these patients went on to develop ARDS. Five of the seven were intubated and
ZHEUTLIN
88
Table
1. Clinical
Information
Age
for
Patients
Survival (d)
Patient
Iv)
sex
Sepsis
Smoker
RD
63
M
D8
48
F
NO Yes
Yes Yes
JP
74 55 66
M
No NO
No Yes Yes
87 65 52
279 293
68 76
M
No
45
Yes Yes
No Yes
33 32
248 222 227
Yes NO
No YC3.S No
17 14 12
No Yes NO
10
No
2
400 331 597
No No
2 1
457 362
LK JF DA AP
F
Yes Yes
F F
MR
79
M
BR
43
F
MMA
63
F
AA
82
M
MM GN
65 68
F
NS
88
F
BL
84
F
JCR
77
M
DP
57
F
Yes Yes
Abbreviation:
A-a
gradient,
Yes Yes Yes
F
Yes Yes
A-a Gradient
>365 1365
alveolar-arterial
240 356
400
207 282 256 283
2 2
oxygen
tension
difference.
supported with mechanical ventilation. Nineteen patients with pneumonia, but without sepsis or ARDS, were also evaluated. Five of these patients were intubated and treated with mechanical ventilation. Six of the patients had aspiration pneumonia and four had postobstructive pneumonia. The lung disease group was comprised of 11 patients, none of whom were intubated. These were 4 patients with sarcoidosis, 6 patients with acute or chronic bronchitis or Table
Patient Group
Number of Patients
Nonsmoker Mean
Lavage
Cells
Cells x lO’/mL
% Macrophage
% Neutrophil
% Lymphocyte
8.2 7.6
83.6 18.2
3.9
10.0
6.0
11.4
6.6* 5.3
92.8* 4.9
1.9*
4.6%
1.9
4.3
82.1* 12.5
5.5’ 8.3
11.0* 8.4
7.9
23.9t
71.3t
4.5
7.4
24.0
27.1
9.1
9.2
68.5 26.9
28.7t 30.5
6.7 6.2
17.8
35.7t
19.6
27.4
57.3t 33.0
1.7 1.4
18
Smoker Mean SD Lung disease
11
14.3
Mean SD
26.9
ARDS Mean
17
SD Pneumonia Mean
19
SD
11.6
Sepsis Mean
7
SD
tP < .05.
Bronchoalveolar
bronchiectasis, and 1 patient with idiopathic pulmonary fibrosis. There were 13 nonsmoking and 18 smoking patients in the control groups. None of these patients were intubated. All of the patients in the control and smoking groups had lavage performed in a bronchopulmonary segment that was free of disease or inflammation. Mean total cell counts and differentials of BAL samples are shown in Table 2. There was a striking increase in the percentage of neutrophils in BAL of patients with ARDS and sepsis compared to nonsmoking and smoking controls. The patients with pneumonia also showed a significant increase in the percentage of neutrophils. Individual values of C3a for each patient group are shown in Fig 1. The amount of C3a DES ARG present in samples from 12 control patients, 17 smokers, and 10 lung disease patients is not different between groups. In addition, the levels of C3a DES ARC in 16 patients with pneumonia, 6 patients with sepsis, and 16 patients with ARDS are not significantly different from levels obtained for the control group (P > .0.5). The highest levels of C3a DES ARG, however, were detected in the ARDS population. Four patients with ARDS had C3a values greater than 500; elevations of this magnitude were not seen in other patient groups. Figure 2 shows the levels of elastase-alpha 1-protease inhibitor complex in BAL, corrected
13
SD
*Results
2.
of one patient
were
not recorded.
ET AL
C3A
AND
ELASTASE
INHIBITOR
LEVELS
IN BAL
89
.
l
.
Fig 2. BAL. The corrected by points. Fig 1. C3a levels in BAL fluid. Individual values for C3a DES ARG concentration in lavage fluid of nonsmoking patients, smokers, patients with lung disease, ARDS, pneumonia, and sepsis are represented.
between the values obtained for samples preincubated with or without normal human plasma (P = .67). These results strongly suggest that BAL from patients with ARDS does not contain any significant amount of free elastase. Neither the level of elastase-alpha I-protease inhibitor complex nor C3a DES ARG present in the BAL of patients with ARDS correlated with survival (r = - .25 and r = - .l 1 respectively). The extent of C3a elevation did not correlate with the alveolar arterial oxygen gradient. The correlation coefficient for the level of elastase complex in BAL and alveolar-arterial oxygen tension difference was significantly different from zero (r = .53). However, this relationship became insignificant when the single outlier with an alveolar-arterial oxygen gradient greater than 500 was excluded (Y = - .03). There was no correlation between levels of C3a DES ARG and levels of elastase-alpha 1-protease inhibitor (r = - .2).
for milligrams of protein. Levels detected in the control group (n = 13), smokers (n = 18), and lung disease (n = 11) are all in the same range. Levels of elastase-alpha 1-protease inhibitor complex in BAL of patients with ARDS (n = 17), pneumonia (n = 19), and sepsis (n = 7) are an order of magnitude greater than the control values. While the results from these three groups are significantly different from control group values (P = .006, .03, and .003, respectively), there is no statistical difference between results in ARDS, pneumonia, or sepsis. When elastase-alpha 1-protease inhibitor levels were not corrected for total protein, the results showed the same distribution (Table 3). The addition of normal human plasma containing a large excess of free alpha 1-protease inhibitor did not result in any change in the amount of elastase-alpha 1-protease inhibitor complex detected in nine BAL samples from patients with ARDS. Statistical analysis (paired t-test) showed that there were no differences Table Patient Group
Elastase-Alpha-l
DISCUSSION
While there are experimental data to suggest that complement activation plays a pathogenetic Protease
Nonsmoker
Smoker
Lung Disease
N
13
Mean
22.5
18 16.6
11 37.5
k SD Values
3.
are given
as ng/mL.
17.5
11.8
Elastase-inhibitor
complex
Inhibitor
73.7 levels
Elastase-alpha I-protease inhibitor levels in individual levels of elastase-inhibitor complex, for milligrams of total protein, are represented
were
not corrected
Levels
In BAL
ARDS
17
19
1582.1 2206.3 for total
Pneumonia
protein.
876.5 2801.9
Sepsis
7 8027.4 13380.4
90
role in ARDS,l determination of plasma levels of C3a DES ARG does not distinguish patients with ARDS from those patients at risk for developing ARDS.18-20 Others have reported elevated levels of C3a DES ARG in the BAL of patients with ARDS compared to normal controls and patients with pulmonary fibrosis.2’ We found elevated levels of C3a in BAL of some patients with ARDS, but an increase in complement fragments was not observed in the majority of patients with the syndrome, nor was this elevation significant when compared to levels in the other patient groups evaluated. Thus, C3a DES ARG levels do not distinguish patients with ARDS from thsoe with other inflammatory processes,such as pneumonia or sepsis. It remains to be seen if levels of C3a DES ARG greated than 400 are specific for ARDS. Elevations of C3a DES ARG levels may be due to complement activation in the alveolar space. Alternatively, neutral proteases such as elastase may digest the C3 molecule, leading to the generation of C3a that is immunologically identical to that produced by complement activation. However, there was no correlation between levels of C3a and levels of elastase-alpha 1-protease inhibitor. We were unable to show any correlation between the extent of C3a generation and the alveolararterial oxygen tension difference at the time of lavage. C3a levels were also not helpful in predicting long-term survival. Increased levels of elastin degradation products have been found in the BAL of patients with ARDS,‘* suggesting the presence of active elastase in the alveolar space. Elevated elastase levels, as determined by elastolytic activity and by immunologic identification, have been reported in the BAL of some patients with ARDS.23-25 Because inhibitors of elastase are present in excess, body fluids usually contain little if any free enzyme, even in severe inflammatory conditions.14 When there is a local imbalance between proteases and available inhibitors, however, proteolysis may occur and cause tissue injury. Lee et al found normal levels of alpha 1-protease inhibitor measured by immunoelectrophoresis in BAL of patients with ARDS.23 As these investigators were able to detect enzyme activity, they speculated that the protease inhibitor which was present was inactivated. McGuire et al have reported
ZHEUTLIN
ET AL
the presence of alpha 1-protease inhibitor bound to elastase in some patients with ARDS, while in others the alpha I-protease inhibitor was inactivated by oxidant activity.24*26 Although there may be a reduction in the amount of functional alpha 1-protease inhibitor present in the alveoli of patients with ARDS, elevated levels of elastase-alpha I-protease inhibitor complex in BAL are an indication of increased extracellular elastase release.i4 In our experiments, the addition of excess alpha 1-protease inhibitor to BAL samples from patients with ARDS did not result in a significant change in the amount of elastasealpha 1-protease inhibitor complex detected. We found markedly elevated levels of elastase bound to to alpha I-protease inhibitor in the BAL of patients with ARDS, as well as patients with pneumonia and sepsis, conditions which are recognized risk factors for the development of ARDS. We found that the mean amount of elastase inhibitor complex in BAL of patients with ARDS was two orders of magnitude greater than the mean for both nonsmoking and smoking control populations. Our results are similar to those reported by Idell et al, even though they used an assay that measured both free and bound elastase.25 In contrast to their results, however, we did not find a correlation between the level of elastase in BAL and the alveolar-arterial oxygen tension difference. Our results demonstrate that while elevated levels of elastase-alpha I-protease inhibitor complex are a very sensitive marker of lung injury, they are not diagnostic for ARDS. Others have also reported the presence of elastolytic activity and elastin-derived peptides in the BAL of some patients with pneumonia.27 We found that the levels of complex measured in BAL of smokers were not significantly different from those in nonsmokers. Other studies have shown that smokers have higher levels of free elastolytic activity in BAL than do nonsmokers.” However, these investigators presented evidence to suggest that the elastase detected was of macrophage origin. It is also possible that some of the inhibitor present in BAL of smokers has been inactivated by the oxidant activity of cigarette smoke, and thus would not form complexes with neutrophi1 elastase. Recently, Jochum et al reported that smokers and nonsmokers have similar levels of elastase-alpha I-protease complex in concen-
C3A AND
ELASTASE
INHIBITOR
LEVELS
IN BAL
91
trated BAL.*’ We also found no difference in elastase-alpha 1-protease inhibitor levels between patients with lung malignancies and those in whom no pathology could be found. The presence of increased levels of elastasealpha 1-protease inhibitor complex in BAL fluid suggests the possibility that elastase release may be involved in the pathogenesis of ARDS. However, elastase-alpha 1-protease inhibitor complex elevation does not appear to be specific for ARDS, as it is found in other inflammatory conditions associated with an influx of neutrophils into the alevolar space. The highest levels of C3a DES ARG were observed in patients with
ARDS, which raises the possibility that levels of C3a DES ARG above a threshold value (such as 500 to 600 ng/mL) may be specific for patients with ARDS. This hypothesis cannot be tested with our data, but must be evaluated prospectively in another study. Further, marked elevations in C3a DES ARC are not observed in the majority of these patients with ARDS, and do not appear to be a sensitive marker of the syndrome. ACKNOWLEDGMENT The authors would like to thank excellent technical assistance.
Mary
Ellen
Lenz
for her
REFERENCES 1. Jacob HS, Craddock PR, Hammerschmidt DE, et al: Complement-induced granulocyte aggregation, an unsuspected mechanism of disease. N Engl J Med 302:789-794, 1980 2. Grant JA, Settle L, Whorton EB, et al: Complementmediated release of histamine from human basophils. J Immunol 117:450-456, 1976 3. Johnson AR, Hugh TE, Muller-Eberhard HJ: Release of histamine from rat mast cells by the complement peptides C3a and C5a. Immunology 28:1067-1080, 1975 4. Bjork J, Hugh TE, Smedegard G: Microvascular effects of anaphylatoxins C3a and C5a. J Immunol 134: 1115-1119,1985
5. Cochrane CG, Muller-Eberhard HJ: The derivation of two distinct anaphylatoxin activities from the third and fifth components of human complement. J Exp Med 127:371-386, 1968 6. Weigle WO, Morgan EL, Goodman MG, et al: Modulation of the immune response by anaphylatoxin in the microenvironment of the interacting cells. Fed Proc 41:30993103, 1982 7. Hugh TE, Chenoweth DE: Biologically active peptides of complement: Techniques and significance of C3a and C5a measurements, in Nakamura RM, Dito WR, Tucker ES (eds): Immunoassays: Clinical Laboratory Techniques for the 1980’s. New York, Liss, 1980, pp 443-460 8. Wright DG, Gallin JI: Secretory responses of human neutrophils: Exocytosis of specific (secondary) granules by human neutrophils during adherence in vitro and during exudation in vivo. J Immunol 123:285-294, 1979 9. Neumann S, Hennrich N, Gunzer G, et al: Enzymelinked immunoassay for human granulocyte elastase in complex with alpha,-protease inhibitor, in Horl WH, Heidland A (eds): Proteases. New York, Plenum, 1984, pp 379-390 10. Janoff A, White R, Carp H, et al: Lung injury induced by leukocytic proteases. Am J Pathol97:111-136, 1979 11. Gadek JE, Fells GA, Zimmerman RL, et al: Antielastases of the human alveolar structures. Implications for the protease-antiprotease theory of emphysema. J Clin Invest 68889-898, 1981 12. Neiderman MS, Fritts LL, Merrill WW, et al: Demonstration of a free elastolytic metalloenzyme in human lung
lavage fluid and its relationship to alpha,-antiprotease. Am Rev Respir Dis 129:943-947, 1984 13. Hunninghake GW, Gadek JE, Kawanami 0, et al: Inflammatory and immune processes in the human lung in health and disease: evaluation by bronchoalveolar lavage. Am J Pathol 97:147-160, 1979 14. Jochum M, Duswald KH, Neumann S, et al: Proteinases and their inhibitors in septicemia-basic concepts and clinical impressions, in Horl WH, Heidland A (eds): Proteases. New York, Plenum, 1984, pp 391-404 15. Fowler AA, Walchak S, Giclas PC, et al: Characterization of antiprotease activity in the adult respiratory distress syndrome. Chest 5:5OS-51S, 1982 16. Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurement with the Folin reagent. J Biol Chem 193:265275,195l 17. Snedecor GW, Cochran WG: Statistical Methods (ed 7). Ames, IA, Iowa State University, 1980 18. Maunder RJ, Harlan JM, Talucci RC, et al: Measurement of C3a and C5a in high-risk patients does not predict ARDS. Am Rev Respir Dis 129:A104, 1984 (abstr) 19. Duchateau J, Haas M, Schreyen H, et al: Complement activation in patients at risk of developing the adult respiratory distress syndrome. Am Rev Respir Dis 130:10581064,1984 20. Weinberg PF, Matthay MA, Webster RO, et al: Biologically active products of complement and acute lung injury in patients with the sepsis syndrome. Am Rev Respir Dis 130:791-796, 1984 21. Idell S, Fein A, Koch S, et al: Anaphylatoxins C3a and C5a in bronchoalveolar lavage in the adult respiratory distress syndrome. Am Rev Respir Dis 129:A104, 1984 (abstr) 22. Morgan L, Kucich U, Dershaw B, et al: Elastin degradation in the adult respiratory distress syndrome. Am Rev Respir Dis 123:93, 198 1 (suppl) (abstr) 23. Lee CT, Fein AM, Lippmann M, et al: Elastolytic activity in pulmonary lavage fluid from patients with adult respiratory distress syndrome. N Engl J Med 304:192-196, 1981 24. McGuire WW, Spragg RG, Cohen AB, et al: Studies on the pathogenesis of the adult respiratory distress syndrome. J Clin Invest 69:543-553, 1982
92
25. Idell S, Kucich U, Fein A, et al: Neutorphil elastasereleasing factors in bronchoalveolar lavage from patients with the adult respiratory distress syndrome. Am Rev Respir Dis 132:1098-1105, 1985 26. Cochrane CG, Spragg R, Revak SD: Pathogenesis of the adult respiratory distress syndrome: Evidence of oxidant activity in bronchoalveolar lavage fluid. J Clin Invest 71:754761, 1983
ZHEUTLIN
ET AL
27. Abrams WR, Fein AM, Kucich U, et al: Proteinase inhibitory function in inflammatory lung disease. Am Rev Respir Dis 129:735-741, 1984 28. Jochum M, Pelletier A, Boudier C, et al: The concentration of leukocyte elastase-alpha,-proteinase inhibitor complex in bronchoalveolar lavage fluids from healthy human subjects. Am Rev Respir Dis 132:913-914, 1985
INVESTIGATIONS
C3A DES ARG and Elastase-Alpha 1-Protease in Lavage Fluid From Patients With Adult Distress Syndrome Lynne
M. Zheutlin,
Eugene
J-M.A.
Thonar, Robert
Susan A,
Lemley-Gillespie,
Balk,
and
Roger
Elizabeth
Inhibitor Levels Respiratory R. Jacobs,
Michael
E. Hanley,
C. Bone
Complement activation and neutrophil release of proteolytic enzymes are thought to be important in the pathogenesis of the adult respiratory distress syndrome (ARDS). Levels of both C3a and elastolytic activity are reported to be elevated in bronchoalveolar lavage (BALI samples from patients with ARDS. The relative specificity of elevated BAL levels of complement fragments and elastase to ARDS is unknown. We compared levels of these mediators of inflammation in the BAL of patients with ARDS to those of control populations including patients with pneumonia, sepsis, and other lung diseases such as sarcoidosis and bronchitis. C3a was measured as
C3a DES ARG by radioimmunoassay. Elastase levels were measured by an ELISA which detects elastase bound to its inhibitor, alpha 1-protease inhibitor, and corrected for total protein concentration. BAL levels of elastase-alpha 1 -protease inhibitor complex were elevated in patients with ARDS, and in patients who are at risk for ARDS, such as those with sepsis or pneumonia. Elevated levels of C3a DES ARG were detected in some patients with ARDS, but this increase was not consistently observed when compared with the other patient groups. @ 7987 by Grune & Stratton, Inc.
V
metabolized to biologically inactive C3a DES ARG in vivo by the enzyme carboxypeptidase. A quantitative asssay for the C3a DES ARG content of biologic fluids allows one to assess the degree of complement activation.7 Polymorphonuclear leukocytes release neutral proteases extracellularly when exposed to chemotactic or phagocytic stimuli.8v9 One of these, neutrophil elastase, can degrade elastin, collagen, proteoglycans, and basement membrane,” all of which are structural components of lung and its vasculature. In vivo free elastase is rapidly inactivated by complex formation with inhibitors. The major serum and bronchoalveolar fluid inhibitor of neutrophil elastase is alpha 1-protease inhibitor.” The recent development of an assay for elastase in complex with its inhibitor alpha 1-protease inhibitor’ has made it possible to assesslevels of these complexes in BAL. These levels reflect local production and release of elastase by cells in the alveolar space. The relative specificity of elevated BAL levels of complement fragments and elastase to ARDS is unknown. In this study, we compared levels of both C3a DES ARG and elastase complex in the BAL of patients with ARDS to those of control populations including patients with pneumonia, sepsis, and other lung diseases such as sarcoidosis and chronic bronchitis. We sought to determine the sensitivity and specificity of levels of these mediators as markers of lung injury.
ARIOUS HYPOTHESES have been proposed to account for the pathologic changes that characterize the adult respiratory distress syndrome (ARDS). One hypothesis suggests that complement activation induced by the underlying clinical process leads to neutrophil aggregation and stasis in the pulmonary microvasculature. This is followed by neutrophil activation and release of neutral and acid proteases such as elastase, as well as toxic oxygen radicals.’ The release of these inflammatory mediators results in tissue damage and activation of other inflammatory pathways. Complement activation by either the classical or alternative pathway results in the cleavage of C3 into its fragments C3a and C3b. C3a causes histamine release from human basophils and mast cells,2,3 increases vascular permeability,4 and induces both smooth muscle contractions and lymphocyte suppression.6 C3a is rapidly From the Departments of Internal Medicine, Immunology, and Biochemistry, Rush Medical College, Chicago. Supported in part by a developmental grant from the Ofice of Consolidated Laboratory Services, Rush-Presbyterian-St Luke’s Medical Center, and by the Upjohn Company. Address reprint requests to Elizabeth R. Jacobs, MD. Department of Internal Medicine, Rush-Presbyterian-St Luke’s Medical Center, 17.53 W Congress Pkwy, Chicago, IL 60612.
o 1987 by Grune & Stratton, 0883-9441/87/0202-0001$05.00/0
86
Inc.
Journal
of Critical
Care, Vol 2, No 2 (June),
1987:
pp 86-92
C3A
AND
ELASTASE
INHIBITOR
MATERIALS
LEVELS
a7
IN BAL
AND METHODS
These protocols were approved by the Human Investigation Committee. Informed consent was obtained for BAL from patients undergoing diagnostic or therapeutic bronchoscopy. Bronchoalveolar lavage was performed on those ARDS patients who were enrolled in an ongoing therapeutic trial prior to the initiation of specific therapy. Control specimens were obtained from uninvolved lung segments of nonsmoking patients undergoing bronchoscopy for unilateral nodules or unexplained hemoptysis. Smokers were evaluated separately, as others have reported increased elastolytic activity in BAL of these patients.‘* Patients with lung disease met standard clinical criteria for the diagnosis of sarcoidosis, acute or chronic bronchitis, or idiopathic pulmonary fibrosis. The diagnosis of pneumonia was based on clinical, laboratory, and roentgenographic criteria. Lavage was performed in the lung segment with active disease. Sepsis was defined by the appearance of systemic manifestations of infection. All patients had hypotension plus at least two of the following criteria: (1) white blood cell count greater than 12,000 or less than 3,000, (2) hyperthermia or hypothermia, (3) positive blood culture, and (4) a cultured pathogen from a suspected source or gross pus in an enclosed space. Lavage was part of the initial diagnostic evaluation and usually was performed within 24 hours of the recognition of the septic process. Since the patients were categorized later in their hospital course, none of the septic patients had ARDS, although all had evidence of pneumonitis. The ARDS group consisted of patients with acute respiratory failure requiring mechanical ventilation and in whom the PaO, to Fi02 ratio was less than 200 with posititve end expiratory pressrue (PEEP) or less than 150 without PEEP. All had diffuse bilateral infiltrates on chest x-ray and all had a pulmonary capillary wedge pressure < 18 mm HG. Lavage was performed within 24 hours after the patient fulfilled the clinical criteria for ARDS. After standard preoperative preparation and local airway anesthesia,” BAL specimens were collected from the right middle lobe or lingula. Sterile saline was instilled and aspirated in six 20-mL aliquots. Lavage fluid was placed on ice and immediately transported to the research laboratory. The fluid was passed through a nylon mesh filter to exclude mucus and particles greater than 60 microns in diameter. Total cell count was quantitated by use of a hemocytometer and differential counts were performed on cytospin preparations stained with Wright Giemsa stain. Cellular material was removed by centrifugation for ten minutes at 300 x g at 4°C. Additional particulate matter was removed by centrifugation at 3,600 x g at 4°C and the supernatant was quick frozen and stored at - 90°C. Levels of C3a DES ARG in BAL were measured using a competitive binding radioimmunoassay described by Hugli and Chenoweth.’ Kits containing rz51-labeled C3a DES ARG, anti-C3a DES ARG, and five standard solutions of purified C3a DES ARG were generously provided by Upjohn Diagnostics (Kalamazoo, MI). An initial precipitation step to remove C3, IgG, and other proteins was performed as described in the kit procedure manual. Duplicate samples and standards were assayed simultaneously. This assay measures C3a and C3a DES ARG at concentrations from 40 to 1,000 ng/mL.
Elastase bound to its inhibitor alpha I-protease inhibitor was measured by an enzyme-linked immunoassay9~‘4 kindly provided by Merck (Darmstadt, West Germany). Rriefly, samples in various dilutions were incubated in tubes precoated with antibody to neutrophil elastase. The complex of elastase and alpha I-protease inhibitor present in the sample binds to this antibody via the elastase portion of the complex. After washing, alkaline phosphatase labeled antibody directed against alpha I-protease inhibitor was allowed to interact with complexes that had attached to the surface. Substrate for the alkaline phosphatase, p-nitrophenyl phosphate was added and the amount of p-nitrophenol produced was measured as absorbance at 405 nm. The optical density was directed proportional to the amount of elastase present in the range of 1 to 8 ng/mL. The concentration of elastase present was calculated by comparing the absorbance values to values generated from known concentrations of elastasealpha 1-protease inhibitor. Elastase levels were normalized per milligram total protein to correct for leakage of plasma alpha I-protease inhibitor into the alveolar space. An increase in alpha l-protease inhibitor concentration has been reported in BAL samples from patients with ARDS.” Total protein was measured by the Lowry assay.16 In order to determine if BAL from patients with ARDS contained free elastase, excess alpha I-protease inhibitor was added to these samples. Normal human plasma, diluted 1:5 with test buffer diluent, served as the source of alpha 1-protease inhibitor. Preliminary experiments had established that this amount of normal human plasma contained enough alpha 1-protease inhibitor to totally inhibit the enzyme activity of a solution containing 3,000 ng/mL purified free elastase. Equal volumes of BAL and diluted plasma or BAL and test buffer diluent were incubated for one hour at room temperature and then assayed simultaneously for elastase-alpha I-protease inhibitor complex as described above. The amount of elastase-alpha I-protease inhibitor complex present in diluted plasma alone (~8 ng/mL) was subtracted from the results. Individual values for patients in each group are given. Values for patient groups were compared using a nonparametric test (Friedman’s two-way test). Correlation was calculated by linear regression analysis.” A P value of c.05 was considered significant.
RESULTS
The ARDS group consisted of 17 patients who met the clinical criteria for the syndrome. They ranged in age from 43 to 88 years of age; 6 were male and 11 were female. Clinical data on these patients are presented in Table 1. The majority of these patients had sepsis as the underlying cause of ARDS. Eight patients survived more than thirty days. Seven septic patients with pneumonia or pneumonitis were evaluated. Since the categorization of the patients took place after enrollment in the study, none of these patients went on to develop ARDS. Five of the seven were intubated and
ZHEUTLIN
88
Table
1. Clinical
Information
Age
for
Patients
Survival (d)
Patient
Iv)
sex
Sepsis
Smoker
RD
63
M
D8
48
F
NO Yes
Yes Yes
JP
74 55 66
M
No NO
No Yes Yes
87 65 52
279 293
68 76
M
No
45
Yes Yes
No Yes
33 32
248 222 227
Yes NO
No YC3.S No
17 14 12
No Yes NO
10
No
2
400 331 597
No No
2 1
457 362
LK JF DA AP
F
Yes Yes
F F
MR
79
M
BR
43
F
MMA
63
F
AA
82
M
MM GN
65 68
F
NS
88
F
BL
84
F
JCR
77
M
DP
57
F
Yes Yes
Abbreviation:
A-a
gradient,
Yes Yes Yes
F
Yes Yes
A-a Gradient
>365 1365
alveolar-arterial
240 356
400
207 282 256 283
2 2
oxygen
tension
difference.
supported with mechanical ventilation. Nineteen patients with pneumonia, but without sepsis or ARDS, were also evaluated. Five of these patients were intubated and treated with mechanical ventilation. Six of the patients had aspiration pneumonia and four had postobstructive pneumonia. The lung disease group was comprised of 11 patients, none of whom were intubated. These were 4 patients with sarcoidosis, 6 patients with acute or chronic bronchitis or Table
Patient Group
Number of Patients
Nonsmoker Mean
Lavage
Cells
Cells x lO’/mL
% Macrophage
% Neutrophil
% Lymphocyte
8.2 7.6
83.6 18.2
3.9
10.0
6.0
11.4
6.6* 5.3
92.8* 4.9
1.9*
4.6%
1.9
4.3
82.1* 12.5
5.5’ 8.3
11.0* 8.4
7.9
23.9t
71.3t
4.5
7.4
24.0
27.1
9.1
9.2
68.5 26.9
28.7t 30.5
6.7 6.2
17.8
35.7t
19.6
27.4
57.3t 33.0
1.7 1.4
18
Smoker Mean SD Lung disease
11
14.3
Mean SD
26.9
ARDS Mean
17
SD Pneumonia Mean
19
SD
11.6
Sepsis Mean
7
SD
tP < .05.
Bronchoalveolar
bronchiectasis, and 1 patient with idiopathic pulmonary fibrosis. There were 13 nonsmoking and 18 smoking patients in the control groups. None of these patients were intubated. All of the patients in the control and smoking groups had lavage performed in a bronchopulmonary segment that was free of disease or inflammation. Mean total cell counts and differentials of BAL samples are shown in Table 2. There was a striking increase in the percentage of neutrophils in BAL of patients with ARDS and sepsis compared to nonsmoking and smoking controls. The patients with pneumonia also showed a significant increase in the percentage of neutrophils. Individual values of C3a for each patient group are shown in Fig 1. The amount of C3a DES ARG present in samples from 12 control patients, 17 smokers, and 10 lung disease patients is not different between groups. In addition, the levels of C3a DES ARC in 16 patients with pneumonia, 6 patients with sepsis, and 16 patients with ARDS are not significantly different from levels obtained for the control group (P > .0.5). The highest levels of C3a DES ARG, however, were detected in the ARDS population. Four patients with ARDS had C3a values greater than 500; elevations of this magnitude were not seen in other patient groups. Figure 2 shows the levels of elastase-alpha 1-protease inhibitor complex in BAL, corrected
13
SD
*Results
2.
of one patient
were
not recorded.
ET AL
C3A
AND
ELASTASE
INHIBITOR
LEVELS
IN BAL
89
.
l
.
Fig 2. BAL. The corrected by points. Fig 1. C3a levels in BAL fluid. Individual values for C3a DES ARG concentration in lavage fluid of nonsmoking patients, smokers, patients with lung disease, ARDS, pneumonia, and sepsis are represented.
between the values obtained for samples preincubated with or without normal human plasma (P = .67). These results strongly suggest that BAL from patients with ARDS does not contain any significant amount of free elastase. Neither the level of elastase-alpha I-protease inhibitor complex nor C3a DES ARG present in the BAL of patients with ARDS correlated with survival (r = - .25 and r = - .l 1 respectively). The extent of C3a elevation did not correlate with the alveolar arterial oxygen gradient. The correlation coefficient for the level of elastase complex in BAL and alveolar-arterial oxygen tension difference was significantly different from zero (r = .53). However, this relationship became insignificant when the single outlier with an alveolar-arterial oxygen gradient greater than 500 was excluded (Y = - .03). There was no correlation between levels of C3a DES ARG and levels of elastase-alpha 1-protease inhibitor (r = - .2).
for milligrams of protein. Levels detected in the control group (n = 13), smokers (n = 18), and lung disease (n = 11) are all in the same range. Levels of elastase-alpha 1-protease inhibitor complex in BAL of patients with ARDS (n = 17), pneumonia (n = 19), and sepsis (n = 7) are an order of magnitude greater than the control values. While the results from these three groups are significantly different from control group values (P = .006, .03, and .003, respectively), there is no statistical difference between results in ARDS, pneumonia, or sepsis. When elastase-alpha 1-protease inhibitor levels were not corrected for total protein, the results showed the same distribution (Table 3). The addition of normal human plasma containing a large excess of free alpha 1-protease inhibitor did not result in any change in the amount of elastase-alpha 1-protease inhibitor complex detected in nine BAL samples from patients with ARDS. Statistical analysis (paired t-test) showed that there were no differences Table Patient Group
Elastase-Alpha-l
DISCUSSION
While there are experimental data to suggest that complement activation plays a pathogenetic Protease
Nonsmoker
Smoker
Lung Disease
N
13
Mean
22.5
18 16.6
11 37.5
k SD Values
3.
are given
as ng/mL.
17.5
11.8
Elastase-inhibitor
complex
Inhibitor
73.7 levels
Elastase-alpha I-protease inhibitor levels in individual levels of elastase-inhibitor complex, for milligrams of total protein, are represented
were
not corrected
Levels
In BAL
ARDS
17
19
1582.1 2206.3 for total
Pneumonia
protein.
876.5 2801.9
Sepsis
7 8027.4 13380.4
90
role in ARDS,l determination of plasma levels of C3a DES ARG does not distinguish patients with ARDS from those patients at risk for developing ARDS.18-20 Others have reported elevated levels of C3a DES ARG in the BAL of patients with ARDS compared to normal controls and patients with pulmonary fibrosis.2’ We found elevated levels of C3a in BAL of some patients with ARDS, but an increase in complement fragments was not observed in the majority of patients with the syndrome, nor was this elevation significant when compared to levels in the other patient groups evaluated. Thus, C3a DES ARG levels do not distinguish patients with ARDS from thsoe with other inflammatory processes,such as pneumonia or sepsis. It remains to be seen if levels of C3a DES ARG greated than 400 are specific for ARDS. Elevations of C3a DES ARG levels may be due to complement activation in the alveolar space. Alternatively, neutral proteases such as elastase may digest the C3 molecule, leading to the generation of C3a that is immunologically identical to that produced by complement activation. However, there was no correlation between levels of C3a and levels of elastase-alpha 1-protease inhibitor. We were unable to show any correlation between the extent of C3a generation and the alveolararterial oxygen tension difference at the time of lavage. C3a levels were also not helpful in predicting long-term survival. Increased levels of elastin degradation products have been found in the BAL of patients with ARDS,‘* suggesting the presence of active elastase in the alveolar space. Elevated elastase levels, as determined by elastolytic activity and by immunologic identification, have been reported in the BAL of some patients with ARDS.23-25 Because inhibitors of elastase are present in excess, body fluids usually contain little if any free enzyme, even in severe inflammatory conditions.14 When there is a local imbalance between proteases and available inhibitors, however, proteolysis may occur and cause tissue injury. Lee et al found normal levels of alpha 1-protease inhibitor measured by immunoelectrophoresis in BAL of patients with ARDS.23 As these investigators were able to detect enzyme activity, they speculated that the protease inhibitor which was present was inactivated. McGuire et al have reported
ZHEUTLIN
ET AL
the presence of alpha 1-protease inhibitor bound to elastase in some patients with ARDS, while in others the alpha I-protease inhibitor was inactivated by oxidant activity.24*26 Although there may be a reduction in the amount of functional alpha 1-protease inhibitor present in the alveoli of patients with ARDS, elevated levels of elastase-alpha I-protease inhibitor complex in BAL are an indication of increased extracellular elastase release.i4 In our experiments, the addition of excess alpha 1-protease inhibitor to BAL samples from patients with ARDS did not result in a significant change in the amount of elastasealpha 1-protease inhibitor complex detected. We found markedly elevated levels of elastase bound to to alpha I-protease inhibitor in the BAL of patients with ARDS, as well as patients with pneumonia and sepsis, conditions which are recognized risk factors for the development of ARDS. We found that the mean amount of elastase inhibitor complex in BAL of patients with ARDS was two orders of magnitude greater than the mean for both nonsmoking and smoking control populations. Our results are similar to those reported by Idell et al, even though they used an assay that measured both free and bound elastase.25 In contrast to their results, however, we did not find a correlation between the level of elastase in BAL and the alveolar-arterial oxygen tension difference. Our results demonstrate that while elevated levels of elastase-alpha I-protease inhibitor complex are a very sensitive marker of lung injury, they are not diagnostic for ARDS. Others have also reported the presence of elastolytic activity and elastin-derived peptides in the BAL of some patients with pneumonia.27 We found that the levels of complex measured in BAL of smokers were not significantly different from those in nonsmokers. Other studies have shown that smokers have higher levels of free elastolytic activity in BAL than do nonsmokers.” However, these investigators presented evidence to suggest that the elastase detected was of macrophage origin. It is also possible that some of the inhibitor present in BAL of smokers has been inactivated by the oxidant activity of cigarette smoke, and thus would not form complexes with neutrophi1 elastase. Recently, Jochum et al reported that smokers and nonsmokers have similar levels of elastase-alpha I-protease complex in concen-
C3A AND
ELASTASE
INHIBITOR
LEVELS
IN BAL
91
trated BAL.*’ We also found no difference in elastase-alpha 1-protease inhibitor levels between patients with lung malignancies and those in whom no pathology could be found. The presence of increased levels of elastasealpha 1-protease inhibitor complex in BAL fluid suggests the possibility that elastase release may be involved in the pathogenesis of ARDS. However, elastase-alpha 1-protease inhibitor complex elevation does not appear to be specific for ARDS, as it is found in other inflammatory conditions associated with an influx of neutrophils into the alevolar space. The highest levels of C3a DES ARG were observed in patients with
ARDS, which raises the possibility that levels of C3a DES ARG above a threshold value (such as 500 to 600 ng/mL) may be specific for patients with ARDS. This hypothesis cannot be tested with our data, but must be evaluated prospectively in another study. Further, marked elevations in C3a DES ARC are not observed in the majority of these patients with ARDS, and do not appear to be a sensitive marker of the syndrome. ACKNOWLEDGMENT The authors would like to thank excellent technical assistance.
Mary
Ellen
Lenz
for her
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25. Idell S, Kucich U, Fein A, et al: Neutorphil elastasereleasing factors in bronchoalveolar lavage from patients with the adult respiratory distress syndrome. Am Rev Respir Dis 132:1098-1105, 1985 26. Cochrane CG, Spragg R, Revak SD: Pathogenesis of the adult respiratory distress syndrome: Evidence of oxidant activity in bronchoalveolar lavage fluid. J Clin Invest 71:754761, 1983
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ET AL
27. Abrams WR, Fein AM, Kucich U, et al: Proteinase inhibitory function in inflammatory lung disease. Am Rev Respir Dis 129:735-741, 1984 28. Jochum M, Pelletier A, Boudier C, et al: The concentration of leukocyte elastase-alpha,-proteinase inhibitor complex in bronchoalveolar lavage fluids from healthy human subjects. Am Rev Respir Dis 132:913-914, 1985