Neutrophil Elastase and Pulmonary Function in Subjects with Intermediate Alpha,-Antitrypsin Deficiency (MZ Phenotype)13 STEPHEN LAM,4 RAJA T. ABBOUD, MOIRA CHAN-YEUNG, and JEAN-MARIE RUSHTON
SUMMARY We studied the relationships among neutrophil elastase, smoking, and pulmonary function in 32 subjects with intermediate alphax-antitrypsin deficiency (Pi MZ phenotype). Nineteen subjects were detected in population studies, and 13 were detected in a family study of patients with chronic airway obstruction. Of the 32 subjects, 10 less than 30 years of age and 12 more than 30 years of age had normal lung function. The remaining 10 subjects, all more than 30 years of age, had abnormal lung function as determined by one or more of the following indices: maximal expiratory flow, specific conductance, closing capacity, slope of phase III of the single-breath N 2 test, lung elastic recoil, or upstream resistance. The neutrophil elastase content of all Pi MZ subjects as a group was not significantly different from that of a control group of 26 subjects of Pi M phenotype. However, in Pi MZ subjects more than 30 years of age, those with abnormal lung function had greater neutrophil elastase content than did those with normal lung function. The 12 MZ subjects more than 30 years of age with normal lung function had less neutrophil elastase content as compared to the 20 M control subjects more than 30 years of age. In Pi MZ subjects more than 30 years of age, neutrophil elastase content correlated significantly with tests of maximal expiratory flow, airway conductance, closing capacity, and slope of phase III, whereas smoking showed a correlation with the latter 4 measurements, but not with tests of maximal expiratory flow. Neutrophil elastase and smoking interacted as significant factors related to abnormal lung function in Pi MZ subjects more than 30 years of age. We conclude that in MZ antitrypsin-deficient subjects neutrophil elastase content is a significant risk factor related to lung function abnormalities and that neutrophil elastase and smoking interact in producing abnormal lung function in Pi MZ subjects.
Introduction T h e association of alphaj-antitrypsin deficiency with emphysema and the induction of emphysema in laboratory animals by leukocyte extracts led to the hypothesis that emphysema may result (Received in original form November in revised form March 13,1979)
14, 1978 and
iFrom the Respiratory Division, Department of Medicine, University of British Columbia, Vancouver General Hospital, Vancouver, B. C. V5Z 1L6, Canada.
from relatively uninhibited digestion of lung tissue by elastolytic enzymes released by leukocytes (1). Accordingly, one would expect that lung damage in antitrypsin deficiency may be related to concentrations of leukocyte elastase. Galdston
2 Supported by Grant No. MA 5523 from the Medical Research Council of Canada, and by the Canadian Lung Association and the British Columbia Lung Association. 3 Presented in part at the annual meeting of the American Thoracic Society, Boston, Mass., May 1978. 4 Canadian Lung Association Fellow.
AMERICAN REVIEW OF RESPIRATORY DISEASE, VOLUME 119, 1979
941
942
LAM, ABBOUD, CHAN-YEUNG, AND RUSHTON
a n d co-workers (2) suggested that persons with i n t e r m e d i a t e alphaj-antitrypsin deficiency m i g h t be less likely to develop emphysema if they had small concentrations of leukocyte proteases; however, Klayton a n d co-workers (3), in a study of elderly subjects with i n t e r m e d i a t e antitrypsin deficiency, found that airway obstruction was related to smoking b u t n o t to leukocyte proteolytic enzymes. Recently, Kidokoro a n d co-workers (4) found that the degree of l u n g function abnormality in p a t i e n t s with severe alphaj-antitrypsin deficiency was related to leukocyte elastase concentration. T h e p u r p o s e of o u r study was to evaluate the relationships of n e u t r o p h i l elastase concentrations to i m p a i r m e n t of p u l m o n a r y function in subjects with i n t e r m e d i a t e a l p h a ^ a n t i t r y p s i n deficiency of the Pi MZ p h e n o t y p e , a n d to determ i n e w h e t h e r smoking a n d n e u t r o p h i l elastase interact as risk factors in Pi MZ subjects.
Materials and Methods Subjects. Thirty-two subjects with intermediate alphaj-antitrypsin deficiency (Pi MZ phenotype) were studied. They represent 32 of 35 Pi MZ subjects detected from 2 sources: 19 were detected in an epidemiologic health survey of 734 subjects in the Greater Vancouver area, and 13 were from a study of 211 immediate relatives of patients with chronic airflow obstruction who attended the University of British Columbia Respiratory Clinic at the Vancouver General Hospital. T h e concentration of alpha^antitrypsin was determined by radial immunodiffusion using commercially available plates and standards (Behring Diagnostics), and phenotyping was done using acid starch gel electrophoresis with subsequent antigen-antibody crossed electrophoresis (5) or immunofixation (6). All subjects completed the British Medical Research Council Respiratory Questionnaire (7); all regarded themselves as healthy, had not sought medical advice for cardiopulmonary complaints, and were not taking beta-adrenergic agents or corticosteroids. Pulmonary function studies. Forced expiratory volume in one second (FEV^, maximal mid-expiratory flow (MMEF), and forced vital capacity (FVC) were measured with a 13.5-liter Collins spirometer. Functional residual capacity, airway resistance, and thoracic gas volume were measured in a constantvolume body plethysmograph (W. E. Collins, Inc.), according to the techniques of DuBois and associates (8, 9), using an Electronics for Medicine DR8 recorder. Airway resistance was converted to its reciprocal, conductance, which was divided by thoracic gas volume to obtain specific airway conductance (SGaw). Total lung capacity (TLC) was calculated by adding functional residual capacity and inspira-
tory capacity. Residual volume was obtained by subtracting FVC from TLC. Carbon monoxide diffusing capacity (DLCO) was determined by the steady-state method (10). Closing volume was determined by a modification of the technique of Anthonisen and co-workers (11) as described previously (12). T h e slope of the alveolar plateau expressed as per cent of N 2 (AN2) per liter of volume was measured as described by Buist and Ross (13). Closing capacity (CC) was obtained by adding closing volume and plethysmographic residual volume and was expressed as a percentage of TLC (CC/TLC). Maximal expiratory flow volume (MEFV) curves were obtained using a wedge spirometer (Med Science Model 570) and a rapid-response XY recorder (Hewlett Packard Model 7046 A) with the subject breathing air and were then repeated after the subjects had taken 3 vital capacity breaths of a mixture of 80 per cent helium and 20 per cent oxygen, followed by 3-min breathing of the mixture (14). T h e difference in flow between air and helium at 50 per cent of vital capacity (AVmax 50 ) and the volume at which the flow on air and helium were equal, the volume of isoflow (Viso^), were determined as described by Dosman and co-workers (15) and Hutcheon and associates (16). Static deflation pressure volume curves were determined using an esophageal balloon according to the technique of Milic-£mili and co-workers (17) and were expressed as static elastic recoil pressures at various percentages of observed TLC (18). Upstream resistance (19) was calculated at 60 per cent of TLC by dividing elastic recoil at that volume by the corresponding maximal expiratory flow from the flow-volume curve. Predicted values for pulmonary function tests were obtained from the following sources: YEV1 and MMEF from Morris and co-workers (20), lung volumes from Goldman and Becklake (21), AN 2 per liter and closing volume from Buist and Ross (13, 22), Vmax 5Q from Cherniack and Raber (23), Viso^ from Gelb and co-workers (24), SGaw from Pelzer and Thomson (25), steady-state diffusing capacity from Bates and co-workers (26), transpulmonary pressure in males from Turner and co-workers (18), and in females from Gibson and Pride (27). A value was considered abnormal if it was different from the mean predicted value by more than 1.645 times the SD, the value within which 95 per cent of the normal population would fall (28). Values for DLCO and elastic recoil at 60 per cent of TLC (P 60 ) were arbitrarily taken as abnormal if they were less than 70 per cent of predicted. Neutrophil lysosomal elastase determination. T h e method followed the procedure of Janoff and Scherer (29) with slight modifications and has been described in detail in a separate publication (30). Neutrophils were isolated from 30 ml of peripheral venous blood by Hypaque-Ficoll centrifugation followed by Dextran sedimentation according to the method of Boyum (31); the resulting suspension
NEUTROPHIL ELASTASE AND LUNG FUNCTION IN PiMZ
averaged 98 per cent neutrophils. Contaminating erythrocytes were disrupted by hypotonic lysis (29), and neutrophils were disrupted by sonification (32); lysosomes were then isolated by differential centrifugation in 0.34 M sucrose and were suspended in phosphate-buffered saline (pH, 7.0). Lysosomal membranes were disrupted by freeze-thawing and were sedimented by high-speed centrifugation; the resultant supernatant was used as lysosomal extract and stored at —60° C until assayed. Enzyme assays were done using bovine ligamentum nuchea elastin (Sigma) that was tritiated, and elastase assays were determined as described by Takahashi and associates (33). With each assay, a calibration curve was made using a standard solution of porcine pancreatic elastase (chromatographically purified, NBC); elastase activity of the extract was expressed in equivalent micrograms of porcine pancreatic elastase. Protein concentration of lysosomal extract was determined by the Lowry method (34). Elastase activity was also determined by using a synthetic substrate, succinyl-trialanyl-/?-nitroanilide (SLAPN), according to the method of Bieth and co-workers (35); activity was expressed as a change in optical density at a wavelength of 410 nm. Enzyme activity was expressed per 25 /xg of lysosomal protein and per 108 neutrophils isolated and used to prepare the lysosomal extract. The coefficient of variation of duplicate extractions and elastase assays in the same subject on different days was 19.5 per cent (SO). Statistical analyses of the difference between means of different groups were done using the non-paired t test; simple linear and multiple-regression analyses were used to test the correlation between variables and the different variables of lung function (28). A P value of less than 0.05 was considered significant.
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Results A total of 10 persons, five of 19 survey subjects a n d five of 13 i m m e d i a t e relatives of patients, h a d o n e or m o r e a b n o r m a l results of l u n g function tests. Because all persons with a b n o r m a l l u n g function were m o r e t h a n 30 years of age, all Pi MZ subjects studied were divided into 3 groups: g r o u p I, persons less t h a n 30 years of age, all of w h o m h a d n o r m a l l u n g function (10 subjects); g r o u p II, 12 subjects m o r e t h a n 30 years of age with n o r m a l l u n g function; g r o u p III, 10 subjects older t h a n 30 years with abnorm a l l u n g function. T h e clinical a n d leukocyte d a t a are summarized in table 1. T h e m e a n age ( ± SD) of subjects in g r o u p II, 47.2 ± 10.7 years, was n o t significantly different (P > 0.05) from that for g r o u p III, 52.7 ± 1 1 . 1 years. Subjects in g r o u p II were either ex- or nonsmokers, whereas most of the subjects in g r o u p III were c u r r e n t
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89 61 74 65 45 27 46 72 36 54 57 ± 15
106 ± 18
101±24
MMEF*
0.175 0.175 0.209 0.157 7 ±0.056 ±0.056
0.147 0.147 0.073 0.164 0.167 0.167 0.229 0.666 0.666 0.186 0.186
0.218 ± 0 . 0 6 0
0.216±0.044
SGaw {per sec per cm H20)
115 115 136 82 107 107 104 200 200 140 140 132 132 100 100 96 121 ±33
91 ± 17
91±20
RV*
58 58 51 45 64 64 44 28 28 48 48 59 59 31 31 50 48 ± ±112
89 ± 18
92 ± 19
Vmax50
49 ± 14
55 ± 11
154 52 151 13 85 44 183 35 133 40 100 29 114 27 88 47 127 37 134 22 127 ±31 35 ±12
97 ± 30
88 ± 16
;
Visoy* A V m a x 5 0 ( % )
140 95 71 124 158 162 165 112 155 151 133 ±32
107 ± 2 1
100±32
CV/VC*
125 129 91 123 135 146 126 123 119 117 123 ± 14
107 ± 7
109 ± 13
CC/TLC*
187 187 126 104 166 166 175 210 210 171 171 168 168 167 167 164 164 ±30
82 ± 1 8
101±23
AN2 (per liter)*
217 217 59 124 ± 50
96 96 119 113 107 107 102 196 196 105 105
145 ± 4 2 (n = 8)
116±23 (n = 5)
DLco*
COMPARED T O MEAN D A T A OF MZ SUBJECTS W I T H N O R M A L LUNG F U N C T I O N
77 104 144 65 63 118 67 48 93 111 89 ±30
116 ± 2 1 (n = 9)
93 ± 25 (n = 8)
P 6 0*
1.50 5.20 4.39 2.04 3.22 22.40 2.10 5.02 7.29 7.17 4.21±2.16f
2.06 ± 0.82 (n = 9)
1.51±0.31 (n = 8)
Rus {cm H20 per liter per sec)
Definitions of abbreviations :FEV i = forced expiratory volume in one sec; MMEF = maximal mid-expiratory flow; SGaw = specific airway conductance; R V = residual volume; Vmax 5 o = maximal expiratory flow at 50% of vital capacity; Visoy= volume of isoflow; AVmax 5 o = P e r c e n t increase in Vmax 5 Q with helium 0 2 ; C V / V C = ratio of closing volume to vital capacity; CC/TLC = ratio of closing capacity to total lung capacity; A N 2 per liter = slope of alveolar plateau from single-breath N 2 washout; D L c o = CO diffusing capacity; P 6 0 = static recoil pressure at 60% of T L C ; Rus>= upstream resistance at 60% of T L C . Per cent of predicted values. * Value for Subject 6 was excluded from mean ± S D .
1 2 3 4 5 6 7 8 9 10 Mean±SD
117 93 106 97 86 70 106 100 74 91 94 ± 15
105 ± 1 4
Group 11 (mean+SD)
Group III
103±11
FEV/
Group I (mean+SD)
;
Subject
TABLE 2 P U L M O N A R Y F U N C T I O N TEST RESULTS IN 10 Pi MZ SUBJECTS W I T H A B N O R M A L LUNG F U N C T I O N
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945
NEUTROPHIL ELASTASE AND LUNG FUNCTION IN PiMZ
smokers; as expected, the average amount smoked in pack-years was significantly less in group II as compared to group III. Serum alpha^antitrypsin concentrations were very similar in groups II and III. When all Pi MZ subjects were considered, there was a negative correlation between the absolute neutrophil count and age (correlation coefficient r = —0.75, P < 0.001); however, there was no correlation between the absolute neutrophil count and smoking in pack-years or current smoking status. The absolute neutrophil count was significantly greater in group III as compared to group II, despite a slightly greater mean age (P = 0.005). Shown in table 2 are individual pulmonary function results for the 10 subjects in group III, together with mean values for groups I and II; results are expressed as per cent of predicted, except for A^max^, which is expressed as per cent of Vmax 50 , and SGaw and upstream resistance, which are expressed in units of measurement. There was no significant difference in all lung function results between groups I and II; however, as expected, all lung function values except FEVX and DLCO were significantly different in group III as compared to group II. In group III, there was no significant difference in lung function results in subjects who were immediate relatives of patients (Subjects 6 through 10), as compared to survey subjects (subjects 1 through 5). Lung function abnormality was detected most
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11 M^30yrs MZ > 30yrs Normals Normal Abnormal
Fig. 1. Comparison of the neutrophil elastase activity in the different groups. All MZ = groups I, II, and III combined. MZ ^ 30 years Normal = group II. MZ > 30 years Abnormal = group III. Lower panel shows elastase activity in yig per 108 cells. Upper panel shows elastase activity per 25 /ug of lysosomal protein. Bars indicate mean ± SE.
130 r
r= 056 p < 0.01
r = 0.63 p < 0.002
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20
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Fig. 2. Relationships of maximal expiratory flow at 50 per cent of vital capacity (Vmax50), ratio of closing capacity to total lung capacity (CC/TLC), and slope of phase III (AN 2 /L), as dependent variables, to smoking in pack-years and to neutrophil elastase content per 108 neutrophil leukocytes (PMN).
frequently by MEFV curves and the singlebreath N 2 washout test. Only 3 persons had abnormal MMEF, two had abnormal SGaw, eight had abnormal Vmax 50 , using the predicted values of Cherniack and Raber (23); practically identical results of abnormal Vmax 50 values were obtained if the predicted values and lower limits of normal were taken from Knudson and co-workers (36). The single-breath washout revealed an abnormal alveolar slope in 8 subjects, an abnormal closing volume in five, and abnormal plethysmographic CC/TLC in nine. Eight subjects had both abnormal single-breath N 2 test results and abnormal MEFV curves; the remaining two had either an abnormal MEFV curve or an abnormal result of the single-breath N 2 test. The value for DLCO was abnormal in one person. Values for P 6 0 were abnormal in 4 subjects. Of interest is that P 6 0 was less (P < 0.05), whereas upstream resistance was significantly greater (P < 0.0025) in group III as compared to group II (table 2), suggesting that the
946
LAM, ABBOUD, CHAN-YEUNG, AND RUSHTON
abnormalities detected in group III subjects by the MEFV curve and single-breath N 2 test were the results of both loss of elastic recoil and intrinsic airway dysfunction. Neutrophil elastase concentrations are shown in table 1 and figure 1. As a group, elastase concentrations of Pi MZ subjects were not significantly different from those of 26 normal healthy M subjects whom we had studied (30). There was no correlation in Pi MZ subjects between elastase concentration and age, sex, or a family history of chronic airflow obstruction. Unlike control subjects, however, Pi MZ subjects showed a significant correlation of elastase activity per 108 cells with smoking in pack-years (r = 0.38, P < 0.05). Elastase concentration, expressed as micrograms of elastase per 108 neutrophil leukocytes, was significantly higher among Pi MZ subjects in group III as compared to group II (P < 0.002), and was significantly lower in group II as compared to 20 healthy M subjects more than 30 years of age (P < 0.05). When elastase activity was expressed in terms of micrograms of elastase per 25 fig of lysosomal protein, values were larger in group III as compared to group II; however, the difference was not significant (P > 0.05). Similar results were obtained when SLAPN was used as a substrate for measuring esterase activity. The potential relationships between pulmonary function measurements as dependent variables and such possible risk factors as smoking history, neutrophil elastase concentration, per-
ipheral blood neutrophil count, and neutrophil elastase per cubic millimeter of blood (equivalent to elastase per 108 cells X neutrophil count per cubic millimeter), as independent variables, were analyzed statistically in groups II and III combined, using single and multiple-linear regression analyses. In figure 2 are shown the relationships between smoking in pack-years and neutrophil elastase content per 108 cells, as separate independent variables, and Vmax 50 , CC/ TLC, and AN2 per liter expressed as per cent of predicted values. Vmax 50 showed a negative correlation with both smoking and elastase; however, the correlation was significant only for elastase. Both CC/TLC and AN2 per liter showed a significant correlation with smoking and neutrophil elastase. The inverse correlations of smoking and neutrophil elastase with SGaw, FEVX as a per cent of FVC, and static lung recoil are shown in figure 3; these were significant except for the correlation between smoking and FEVX as a per cent of FVC. Indicated in table 3 is that whereas smoking showed a significant correlation with SGaw, CC/TLC, AN2 per liter, and P 6 0 , and no significant correlation with FEVX as a per cent of FVC, MMEF, Vmax 50 , and the ratio of closing volume to vital capacity, there was significant correlation between neutrophil elastase content per 108 cells and all of these lung function tests. Similar results were obtained when SLAPN was used for measuring esterase activity. When neutrophil elastase was expressed as activity per
TABLE 3 C O R R E L A T I O N COEFFICIENTS BETWEEN V A R I O U S P U L M O N A R Y F U N C T I O N TESTS AS DEPENDENT V A R I A B L E S A N D SMOKING A N D N E U T R O P H I L ELASTASE A C T I V I T Y A G A I N S T T R I T I A T E D ELASTIN AS INDEPENDENT V A R I A B L E S , IN MZ SUBJECTS MORE T H A N 30 YEARS OF AGE
Pulmonary Function Test SGaw
FEV^FVC, % MMEF Vmax S o CV/VC CC/TLC A N 2 per liter P60
Smoking (pack-years) -0.48* - 0 . 3 2 (NS) - 0 . 3 5 (NS) - 0 . 3 9 (NS) 0.41 (NS) 0.59t + 0.64tt -0.55*
Elastase/10 8 Neutrophils
Elastase/ mm of blood
-0.45* -0.55 1 " -Q.58ft -0.56 1 " 0.45* 0.47* 0.63tt -0.50*
- 0 . 4 1 (NS) -0.68** -0.69** -0.66** 0.44* 0.49* 0.70** - 0 . 3 7 (NS)
NS = not significant; P < 0.05. For definitions of other abbreviations, see table 2. *P P **P t1 > f
SUMMARY We studied the relationships among neutrophil elastase, smoking, and pulmonary function in 32 subjects with intermediate alphax-antitrypsin deficiency (Pi MZ phenotype). Nineteen subjects were detected in population studies, and 13 were detected in a family study of patients with chronic airway obstruction. Of the 32 subjects, 10 less than 30 years of age and 12 more than 30 years of age had normal lung function. The remaining 10 subjects, all more than 30 years of age, had abnormal lung function as determined by one or more of the following indices: maximal expiratory flow, specific conductance, closing capacity, slope of phase III of the single-breath N 2 test, lung elastic recoil, or upstream resistance. The neutrophil elastase content of all Pi MZ subjects as a group was not significantly different from that of a control group of 26 subjects of Pi M phenotype. However, in Pi MZ subjects more than 30 years of age, those with abnormal lung function had greater neutrophil elastase content than did those with normal lung function. The 12 MZ subjects more than 30 years of age with normal lung function had less neutrophil elastase content as compared to the 20 M control subjects more than 30 years of age. In Pi MZ subjects more than 30 years of age, neutrophil elastase content correlated significantly with tests of maximal expiratory flow, airway conductance, closing capacity, and slope of phase III, whereas smoking showed a correlation with the latter 4 measurements, but not with tests of maximal expiratory flow. Neutrophil elastase and smoking interacted as significant factors related to abnormal lung function in Pi MZ subjects more than 30 years of age. We conclude that in MZ antitrypsin-deficient subjects neutrophil elastase content is a significant risk factor related to lung function abnormalities and that neutrophil elastase and smoking interact in producing abnormal lung function in Pi MZ subjects.
Introduction T h e association of alphaj-antitrypsin deficiency with emphysema and the induction of emphysema in laboratory animals by leukocyte extracts led to the hypothesis that emphysema may result (Received in original form November in revised form March 13,1979)
14, 1978 and
iFrom the Respiratory Division, Department of Medicine, University of British Columbia, Vancouver General Hospital, Vancouver, B. C. V5Z 1L6, Canada.
from relatively uninhibited digestion of lung tissue by elastolytic enzymes released by leukocytes (1). Accordingly, one would expect that lung damage in antitrypsin deficiency may be related to concentrations of leukocyte elastase. Galdston
2 Supported by Grant No. MA 5523 from the Medical Research Council of Canada, and by the Canadian Lung Association and the British Columbia Lung Association. 3 Presented in part at the annual meeting of the American Thoracic Society, Boston, Mass., May 1978. 4 Canadian Lung Association Fellow.
AMERICAN REVIEW OF RESPIRATORY DISEASE, VOLUME 119, 1979
941
942
LAM, ABBOUD, CHAN-YEUNG, AND RUSHTON
a n d co-workers (2) suggested that persons with i n t e r m e d i a t e alphaj-antitrypsin deficiency m i g h t be less likely to develop emphysema if they had small concentrations of leukocyte proteases; however, Klayton a n d co-workers (3), in a study of elderly subjects with i n t e r m e d i a t e antitrypsin deficiency, found that airway obstruction was related to smoking b u t n o t to leukocyte proteolytic enzymes. Recently, Kidokoro a n d co-workers (4) found that the degree of l u n g function abnormality in p a t i e n t s with severe alphaj-antitrypsin deficiency was related to leukocyte elastase concentration. T h e p u r p o s e of o u r study was to evaluate the relationships of n e u t r o p h i l elastase concentrations to i m p a i r m e n t of p u l m o n a r y function in subjects with i n t e r m e d i a t e a l p h a ^ a n t i t r y p s i n deficiency of the Pi MZ p h e n o t y p e , a n d to determ i n e w h e t h e r smoking a n d n e u t r o p h i l elastase interact as risk factors in Pi MZ subjects.
Materials and Methods Subjects. Thirty-two subjects with intermediate alphaj-antitrypsin deficiency (Pi MZ phenotype) were studied. They represent 32 of 35 Pi MZ subjects detected from 2 sources: 19 were detected in an epidemiologic health survey of 734 subjects in the Greater Vancouver area, and 13 were from a study of 211 immediate relatives of patients with chronic airflow obstruction who attended the University of British Columbia Respiratory Clinic at the Vancouver General Hospital. T h e concentration of alpha^antitrypsin was determined by radial immunodiffusion using commercially available plates and standards (Behring Diagnostics), and phenotyping was done using acid starch gel electrophoresis with subsequent antigen-antibody crossed electrophoresis (5) or immunofixation (6). All subjects completed the British Medical Research Council Respiratory Questionnaire (7); all regarded themselves as healthy, had not sought medical advice for cardiopulmonary complaints, and were not taking beta-adrenergic agents or corticosteroids. Pulmonary function studies. Forced expiratory volume in one second (FEV^, maximal mid-expiratory flow (MMEF), and forced vital capacity (FVC) were measured with a 13.5-liter Collins spirometer. Functional residual capacity, airway resistance, and thoracic gas volume were measured in a constantvolume body plethysmograph (W. E. Collins, Inc.), according to the techniques of DuBois and associates (8, 9), using an Electronics for Medicine DR8 recorder. Airway resistance was converted to its reciprocal, conductance, which was divided by thoracic gas volume to obtain specific airway conductance (SGaw). Total lung capacity (TLC) was calculated by adding functional residual capacity and inspira-
tory capacity. Residual volume was obtained by subtracting FVC from TLC. Carbon monoxide diffusing capacity (DLCO) was determined by the steady-state method (10). Closing volume was determined by a modification of the technique of Anthonisen and co-workers (11) as described previously (12). T h e slope of the alveolar plateau expressed as per cent of N 2 (AN2) per liter of volume was measured as described by Buist and Ross (13). Closing capacity (CC) was obtained by adding closing volume and plethysmographic residual volume and was expressed as a percentage of TLC (CC/TLC). Maximal expiratory flow volume (MEFV) curves were obtained using a wedge spirometer (Med Science Model 570) and a rapid-response XY recorder (Hewlett Packard Model 7046 A) with the subject breathing air and were then repeated after the subjects had taken 3 vital capacity breaths of a mixture of 80 per cent helium and 20 per cent oxygen, followed by 3-min breathing of the mixture (14). T h e difference in flow between air and helium at 50 per cent of vital capacity (AVmax 50 ) and the volume at which the flow on air and helium were equal, the volume of isoflow (Viso^), were determined as described by Dosman and co-workers (15) and Hutcheon and associates (16). Static deflation pressure volume curves were determined using an esophageal balloon according to the technique of Milic-£mili and co-workers (17) and were expressed as static elastic recoil pressures at various percentages of observed TLC (18). Upstream resistance (19) was calculated at 60 per cent of TLC by dividing elastic recoil at that volume by the corresponding maximal expiratory flow from the flow-volume curve. Predicted values for pulmonary function tests were obtained from the following sources: YEV1 and MMEF from Morris and co-workers (20), lung volumes from Goldman and Becklake (21), AN 2 per liter and closing volume from Buist and Ross (13, 22), Vmax 5Q from Cherniack and Raber (23), Viso^ from Gelb and co-workers (24), SGaw from Pelzer and Thomson (25), steady-state diffusing capacity from Bates and co-workers (26), transpulmonary pressure in males from Turner and co-workers (18), and in females from Gibson and Pride (27). A value was considered abnormal if it was different from the mean predicted value by more than 1.645 times the SD, the value within which 95 per cent of the normal population would fall (28). Values for DLCO and elastic recoil at 60 per cent of TLC (P 60 ) were arbitrarily taken as abnormal if they were less than 70 per cent of predicted. Neutrophil lysosomal elastase determination. T h e method followed the procedure of Janoff and Scherer (29) with slight modifications and has been described in detail in a separate publication (30). Neutrophils were isolated from 30 ml of peripheral venous blood by Hypaque-Ficoll centrifugation followed by Dextran sedimentation according to the method of Boyum (31); the resulting suspension
NEUTROPHIL ELASTASE AND LUNG FUNCTION IN PiMZ
averaged 98 per cent neutrophils. Contaminating erythrocytes were disrupted by hypotonic lysis (29), and neutrophils were disrupted by sonification (32); lysosomes were then isolated by differential centrifugation in 0.34 M sucrose and were suspended in phosphate-buffered saline (pH, 7.0). Lysosomal membranes were disrupted by freeze-thawing and were sedimented by high-speed centrifugation; the resultant supernatant was used as lysosomal extract and stored at —60° C until assayed. Enzyme assays were done using bovine ligamentum nuchea elastin (Sigma) that was tritiated, and elastase assays were determined as described by Takahashi and associates (33). With each assay, a calibration curve was made using a standard solution of porcine pancreatic elastase (chromatographically purified, NBC); elastase activity of the extract was expressed in equivalent micrograms of porcine pancreatic elastase. Protein concentration of lysosomal extract was determined by the Lowry method (34). Elastase activity was also determined by using a synthetic substrate, succinyl-trialanyl-/?-nitroanilide (SLAPN), according to the method of Bieth and co-workers (35); activity was expressed as a change in optical density at a wavelength of 410 nm. Enzyme activity was expressed per 25 /xg of lysosomal protein and per 108 neutrophils isolated and used to prepare the lysosomal extract. The coefficient of variation of duplicate extractions and elastase assays in the same subject on different days was 19.5 per cent (SO). Statistical analyses of the difference between means of different groups were done using the non-paired t test; simple linear and multiple-regression analyses were used to test the correlation between variables and the different variables of lung function (28). A P value of less than 0.05 was considered significant.
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Results A total of 10 persons, five of 19 survey subjects a n d five of 13 i m m e d i a t e relatives of patients, h a d o n e or m o r e a b n o r m a l results of l u n g function tests. Because all persons with a b n o r m a l l u n g function were m o r e t h a n 30 years of age, all Pi MZ subjects studied were divided into 3 groups: g r o u p I, persons less t h a n 30 years of age, all of w h o m h a d n o r m a l l u n g function (10 subjects); g r o u p II, 12 subjects m o r e t h a n 30 years of age with n o r m a l l u n g function; g r o u p III, 10 subjects older t h a n 30 years with abnorm a l l u n g function. T h e clinical a n d leukocyte d a t a are summarized in table 1. T h e m e a n age ( ± SD) of subjects in g r o u p II, 47.2 ± 10.7 years, was n o t significantly different (P > 0.05) from that for g r o u p III, 52.7 ± 1 1 . 1 years. Subjects in g r o u p II were either ex- or nonsmokers, whereas most of the subjects in g r o u p III were c u r r e n t
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89 61 74 65 45 27 46 72 36 54 57 ± 15
106 ± 18
101±24
MMEF*
0.175 0.175 0.209 0.157 7 ±0.056 ±0.056
0.147 0.147 0.073 0.164 0.167 0.167 0.229 0.666 0.666 0.186 0.186
0.218 ± 0 . 0 6 0
0.216±0.044
SGaw {per sec per cm H20)
115 115 136 82 107 107 104 200 200 140 140 132 132 100 100 96 121 ±33
91 ± 17
91±20
RV*
58 58 51 45 64 64 44 28 28 48 48 59 59 31 31 50 48 ± ±112
89 ± 18
92 ± 19
Vmax50
49 ± 14
55 ± 11
154 52 151 13 85 44 183 35 133 40 100 29 114 27 88 47 127 37 134 22 127 ±31 35 ±12
97 ± 30
88 ± 16
;
Visoy* A V m a x 5 0 ( % )
140 95 71 124 158 162 165 112 155 151 133 ±32
107 ± 2 1
100±32
CV/VC*
125 129 91 123 135 146 126 123 119 117 123 ± 14
107 ± 7
109 ± 13
CC/TLC*
187 187 126 104 166 166 175 210 210 171 171 168 168 167 167 164 164 ±30
82 ± 1 8
101±23
AN2 (per liter)*
217 217 59 124 ± 50
96 96 119 113 107 107 102 196 196 105 105
145 ± 4 2 (n = 8)
116±23 (n = 5)
DLco*
COMPARED T O MEAN D A T A OF MZ SUBJECTS W I T H N O R M A L LUNG F U N C T I O N
77 104 144 65 63 118 67 48 93 111 89 ±30
116 ± 2 1 (n = 9)
93 ± 25 (n = 8)
P 6 0*
1.50 5.20 4.39 2.04 3.22 22.40 2.10 5.02 7.29 7.17 4.21±2.16f
2.06 ± 0.82 (n = 9)
1.51±0.31 (n = 8)
Rus {cm H20 per liter per sec)
Definitions of abbreviations :FEV i = forced expiratory volume in one sec; MMEF = maximal mid-expiratory flow; SGaw = specific airway conductance; R V = residual volume; Vmax 5 o = maximal expiratory flow at 50% of vital capacity; Visoy= volume of isoflow; AVmax 5 o = P e r c e n t increase in Vmax 5 Q with helium 0 2 ; C V / V C = ratio of closing volume to vital capacity; CC/TLC = ratio of closing capacity to total lung capacity; A N 2 per liter = slope of alveolar plateau from single-breath N 2 washout; D L c o = CO diffusing capacity; P 6 0 = static recoil pressure at 60% of T L C ; Rus>= upstream resistance at 60% of T L C . Per cent of predicted values. * Value for Subject 6 was excluded from mean ± S D .
1 2 3 4 5 6 7 8 9 10 Mean±SD
117 93 106 97 86 70 106 100 74 91 94 ± 15
105 ± 1 4
Group 11 (mean+SD)
Group III
103±11
FEV/
Group I (mean+SD)
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Subject
TABLE 2 P U L M O N A R Y F U N C T I O N TEST RESULTS IN 10 Pi MZ SUBJECTS W I T H A B N O R M A L LUNG F U N C T I O N
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945
NEUTROPHIL ELASTASE AND LUNG FUNCTION IN PiMZ
smokers; as expected, the average amount smoked in pack-years was significantly less in group II as compared to group III. Serum alpha^antitrypsin concentrations were very similar in groups II and III. When all Pi MZ subjects were considered, there was a negative correlation between the absolute neutrophil count and age (correlation coefficient r = —0.75, P < 0.001); however, there was no correlation between the absolute neutrophil count and smoking in pack-years or current smoking status. The absolute neutrophil count was significantly greater in group III as compared to group II, despite a slightly greater mean age (P = 0.005). Shown in table 2 are individual pulmonary function results for the 10 subjects in group III, together with mean values for groups I and II; results are expressed as per cent of predicted, except for A^max^, which is expressed as per cent of Vmax 50 , and SGaw and upstream resistance, which are expressed in units of measurement. There was no significant difference in all lung function results between groups I and II; however, as expected, all lung function values except FEVX and DLCO were significantly different in group III as compared to group II. In group III, there was no significant difference in lung function results in subjects who were immediate relatives of patients (Subjects 6 through 10), as compared to survey subjects (subjects 1 through 5). Lung function abnormality was detected most
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11 M^30yrs MZ > 30yrs Normals Normal Abnormal
Fig. 1. Comparison of the neutrophil elastase activity in the different groups. All MZ = groups I, II, and III combined. MZ ^ 30 years Normal = group II. MZ > 30 years Abnormal = group III. Lower panel shows elastase activity in yig per 108 cells. Upper panel shows elastase activity per 25 /ug of lysosomal protein. Bars indicate mean ± SE.
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r= 056 p < 0.01
r = 0.63 p < 0.002
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Fig. 2. Relationships of maximal expiratory flow at 50 per cent of vital capacity (Vmax50), ratio of closing capacity to total lung capacity (CC/TLC), and slope of phase III (AN 2 /L), as dependent variables, to smoking in pack-years and to neutrophil elastase content per 108 neutrophil leukocytes (PMN).
frequently by MEFV curves and the singlebreath N 2 washout test. Only 3 persons had abnormal MMEF, two had abnormal SGaw, eight had abnormal Vmax 50 , using the predicted values of Cherniack and Raber (23); practically identical results of abnormal Vmax 50 values were obtained if the predicted values and lower limits of normal were taken from Knudson and co-workers (36). The single-breath washout revealed an abnormal alveolar slope in 8 subjects, an abnormal closing volume in five, and abnormal plethysmographic CC/TLC in nine. Eight subjects had both abnormal single-breath N 2 test results and abnormal MEFV curves; the remaining two had either an abnormal MEFV curve or an abnormal result of the single-breath N 2 test. The value for DLCO was abnormal in one person. Values for P 6 0 were abnormal in 4 subjects. Of interest is that P 6 0 was less (P < 0.05), whereas upstream resistance was significantly greater (P < 0.0025) in group III as compared to group II (table 2), suggesting that the
946
LAM, ABBOUD, CHAN-YEUNG, AND RUSHTON
abnormalities detected in group III subjects by the MEFV curve and single-breath N 2 test were the results of both loss of elastic recoil and intrinsic airway dysfunction. Neutrophil elastase concentrations are shown in table 1 and figure 1. As a group, elastase concentrations of Pi MZ subjects were not significantly different from those of 26 normal healthy M subjects whom we had studied (30). There was no correlation in Pi MZ subjects between elastase concentration and age, sex, or a family history of chronic airflow obstruction. Unlike control subjects, however, Pi MZ subjects showed a significant correlation of elastase activity per 108 cells with smoking in pack-years (r = 0.38, P < 0.05). Elastase concentration, expressed as micrograms of elastase per 108 neutrophil leukocytes, was significantly higher among Pi MZ subjects in group III as compared to group II (P < 0.002), and was significantly lower in group II as compared to 20 healthy M subjects more than 30 years of age (P < 0.05). When elastase activity was expressed in terms of micrograms of elastase per 25 fig of lysosomal protein, values were larger in group III as compared to group II; however, the difference was not significant (P > 0.05). Similar results were obtained when SLAPN was used as a substrate for measuring esterase activity. The potential relationships between pulmonary function measurements as dependent variables and such possible risk factors as smoking history, neutrophil elastase concentration, per-
ipheral blood neutrophil count, and neutrophil elastase per cubic millimeter of blood (equivalent to elastase per 108 cells X neutrophil count per cubic millimeter), as independent variables, were analyzed statistically in groups II and III combined, using single and multiple-linear regression analyses. In figure 2 are shown the relationships between smoking in pack-years and neutrophil elastase content per 108 cells, as separate independent variables, and Vmax 50 , CC/ TLC, and AN2 per liter expressed as per cent of predicted values. Vmax 50 showed a negative correlation with both smoking and elastase; however, the correlation was significant only for elastase. Both CC/TLC and AN2 per liter showed a significant correlation with smoking and neutrophil elastase. The inverse correlations of smoking and neutrophil elastase with SGaw, FEVX as a per cent of FVC, and static lung recoil are shown in figure 3; these were significant except for the correlation between smoking and FEVX as a per cent of FVC. Indicated in table 3 is that whereas smoking showed a significant correlation with SGaw, CC/TLC, AN2 per liter, and P 6 0 , and no significant correlation with FEVX as a per cent of FVC, MMEF, Vmax 50 , and the ratio of closing volume to vital capacity, there was significant correlation between neutrophil elastase content per 108 cells and all of these lung function tests. Similar results were obtained when SLAPN was used for measuring esterase activity. When neutrophil elastase was expressed as activity per
TABLE 3 C O R R E L A T I O N COEFFICIENTS BETWEEN V A R I O U S P U L M O N A R Y F U N C T I O N TESTS AS DEPENDENT V A R I A B L E S A N D SMOKING A N D N E U T R O P H I L ELASTASE A C T I V I T Y A G A I N S T T R I T I A T E D ELASTIN AS INDEPENDENT V A R I A B L E S , IN MZ SUBJECTS MORE T H A N 30 YEARS OF AGE
Pulmonary Function Test SGaw
FEV^FVC, % MMEF Vmax S o CV/VC CC/TLC A N 2 per liter P60
Smoking (pack-years) -0.48* - 0 . 3 2 (NS) - 0 . 3 5 (NS) - 0 . 3 9 (NS) 0.41 (NS) 0.59t + 0.64tt -0.55*
Elastase/10 8 Neutrophils
Elastase/ mm of blood
-0.45* -0.55 1 " -Q.58ft -0.56 1 " 0.45* 0.47* 0.63tt -0.50*
- 0 . 4 1 (NS) -0.68** -0.69** -0.66** 0.44* 0.49* 0.70** - 0 . 3 7 (NS)
NS = not significant; P < 0.05. For definitions of other abbreviations, see table 2. *P P **P t1 > f
