Interaction between leukocyte elastase and elastin: quantitative and catalytic analyses.

Solubilization of elastin by human leukocyte elastase (HLE) cannot be analyzed by conventional kinetic methods because the biologically relevant substrate is insoluble and the concentration of enzyme-substrate complex has no physical meaning. We now report quantitative measurements of the binding and catalytic interaction between HLE and elastin permitted by analogy to receptor-ligand systems. Our results indicated that a limited and relatively constant number of enzyme binding sites were available on elastin, and that new sites became accessible as catalysis proceeded. The activation energies and solvent deuterium isotope effects were similar for catalysis of elastin and a soluble peptide substrate by HLE, yet the turnover number for HLE digestion of elastin was 200-2000-fold lower than that of HLE acting on soluble peptide substrates. Analysis of the binding of HLE to elastin at 0 degrees C, in the absence of significant catalytic activity, demonstrated two classes of binding sites (Kd=9.3x10(-9) M and 2.5x10(-7) M). The higher affinity sites accounted for only 6% of the total HLE binding capacity, but essentially all of the catalytic activity, and dissociation of HLE from these sites was minimal. Our studies suggest that interaction of HLE with elastin in vivo may be very persistent and permit progressive solubilization of this structurally important extracellular matrix component.

Elastase inhibitors of the respiratory tract.

Lung secretions contain several elastase inhibitors although alpha 1-antitrypsin (alpha 1AT) and antileukoprotease (ALP) are the major ones. Studies of lung alpha 1AT show that the inhibitor is usually partly inactive. In patients with established lung disease this is due to a combination of proteolytic degradation, complex with enzyme and oxidation at the active site. Studies in subjects with normal lungs demonstrate that the alpha 1AT is also partly inactivated although not by any of the recognised mechanisms. Furthermore no difference in function is found between current smokers and nonsmokers. ALP is largely an inhibitor of the major airways although it is still present in the lower airway secretions collected by lavage in healthy subjects. The proportions of alpha 1AT to ALP vary in patients with alpha 1AT deficiency (1:9), established emphysema (1:1) and subjects with healthy lungs (10:1). These differences affect the ability of the lavage fluids to inhibit neutrophil elastase during prolonged incubation with enzyme substrate. The results suggest that the relative concentrations of alpha 1AT and ALP, particularly in close proximity to lung substrates, may determine the degree of connective tissue destruction.

Inhibition of human leukocyte elastase bound to elastin: relative ineffectiveness and two mechanisms of inhibitory activity.

Human leukocyte elastase (HLE) has been demonstrated on lung elastic fibers in areas of pulmonary emphysema. In vitro studies in our laboratory have shown that HLE-elastin complexes may be remarkably stable. We tested the possibility that elastin-bound HLE may retain catalytic activity in the presence of inhibitors that are effective against free HLE and found: (1) alpha-1-proteinase inhibitor (alpha 1PI), antileukoprotease (ALP), and eglin C inhibited free HLE on an approximately 1:1 molar basis, measured with either 3H-elastin or a synthetic peptide substrate; (2) the ability of each inhibitor to control catalytic activity of HLE when complexed with elastin was impaired (e.g., in a 24-h assay, a 70-fold molar excess of alpha 1PI gave only 93% inhibition of HLE); and (3) a chloromethyl ketone inhibitor of HLE gave qualitatively similar results, although at the low enzyme concentrations used it was a less effective inhibitor of free and elastin-bound enzyme than were the polypeptide inhibitors. Further, we found evidence for two distinct mechanisms of inhibition of elastin-bound HLE. alpha 1PI and eglin C prevented elastin solubilization largely by enhancing net dissociation of HLE from the complexes; enzyme remaining bound to the substrate retained essentially full activity. In contrast, ALP and the chloromethyl ketone prevented elastin solubilization by binding to the complexes and inhibiting the enzyme in situ. These results may have implications regarding progressive elastin solubilization in vivo and should stimulate further investigation of enzyme activity in heterogeneous systems in which one or more reactants are insoluble.

Effect of alpha-1-proteinase inhibitor on neutrophil chemotaxis.

Factors that modulate neutrophil migration into the lung are poorly understood. However, there is evidence that neutrophil activation by formylmethionylleucylphenylalanine (FMLP) depends upon a surface proteinase with chymotrypsin-like activity. This suggests that chymotrypsin inhibitors such as alpha-1-proteinase inhibitor (alpha 1PI) could modify neutrophil migration in response to FMLP. We have studied neutrophil chemotaxis using the multiple blind well assay system. This article presents evidence that alpha 1PI is an inhibitor of neutrophil migration in response to FMLP. The effect is related to the inhibitory function of the protein. Alpha-1-antichymotrypsin is more potent than alpha 1PI as an inhibitor of this movement, whereas antileukoprotease is less potent. The results suggest that a cell membrane-bound serine proteinase (perhaps cathepsin G) is necessary for the enhancement of cell movement after receptor binding of FMLP. Oxidized alpha 1PI or a 4,000-D peptide cleaved from alpha 1PI by porcine pancreatic elastase or human neutrophil elastase are capable of enhancing cell motility. The results suggest that alpha 1PI may play a role in cell migration into the lung during acute inflammatory process.

Elastase inhibitors in sputum from bronchitic patients with and without alpha 1-proteinase inhibitor deficiency: partial characterization of a hitherto unquantified inhibitor of neutrophil elastase.

Anti-elastase function in sputum sol-phase from patients with alpha 1-proteinase inhibitor (alpha 1PI) deficiency was compared with sol-phase from patients with cigarette smoke-induced bronchitis and emphysema. Both alpha 1PI (2P less than 0.01) and anti-leucoprotease (ALP) (2P less than 0.01) concentrations were lower in sol-phase from the alpha 1PI-deficient group, although alpha 2-macroglobulin (alpha 2M) levels were similar. There was no difference in alpha 1PI function between the two groups, but the inhibitor was only congruent to 30% active. The absolute neutrophil elastase (NE) inhibitory capacity was similar in both groups (median 185 micrograms of NE inhibited/ml of sputum, range 80-480, for the alpha 1PI-deficient group; median 175, range 80-300, for the bronchitic group). A substantial proportion of NE inhibition in secretions could not be accounted for by the amount of alpha 1PI, ALP and alpha 2M present (median 74.8%, range 43.2-97.4, for alpha 1PI-deficient sol-phase; median 50.0%, range 0-80.8, for bronchitic sol-phase). Gel filtration of sol-phase demonstrated the presence of NE inhibition in the low molecular weight fractions which was markedly sensitive to changes in substrate concentration and ionic strength, in contrast to purified alpha 1PI and ALP. Sputum sol-phase from both groups failed to prevent hydrolysis of elastin-fluorescein or succinyltrialanyl-p-nitroanilide by NE completely during prolonged incubation in the presence of an excess of functional inhibitors. This was more apparent in secretions from subjects with alpha 1PI deficiency and may explain why such patients have a more rapidly progressive form of emphysema.

Determination of elastase inhibitory activity of alpha 1-proteinase inhibitor and bronchial antileukoprotease: different results using insoluble elastin or synthetic low molecular weight substrates.

We have determined the effect of two elastase-specific synthetic low molecular weight substrates, L-pyroglutamylprolylvaline-paranitroanilide and succinyltrialanyl-paranitroanilide, together with insoluble elastin-fluorescein, on the determination of the neutrophil elastase (NE) inhibitory capacity of purified alpha 1-proteinase inhibitor (alpha 1-PI) and bronchial antileukoprotease (ALP). In addition, the inhibitory capacities of mixtures of alpha 1-proteinase inhibitor, antileukoprotease and alpha 2-macroglobulin prepared in ratios similar to that in lung secretions were determined. Purified inhibitors, alone or in combinations, inhibited about 1 mole neutrophil elastase per mole inhibitor when assessed using synthetic substrates. However, when elastin-fluorescein was used in the assay system, the purified inhibitors showed an inhibitory capacity that was 40-85% of the value obtained using synthetic substrates. Even less inhibition was observed when mixtures of inhibitors were assessed using elastin-fluorescein (23-44% of the value for synthetic substrates). Our data indicate that results of elastase inhibitor activity measurements depend on the type of substrate which has been used.

Deoxyribonucleic acid (DNA) polymorphism of the alpha 1-antitrypsin gene in chronic lung disease.

Specific gene probes were used to study restriction fragment length polymorphisms of the human alpha 1-antitrypsin gene. A polymorphism due to loss of a recognition site for the restriction enzyme Taq I was identified in eight of 42 patients with bronchiectasis and nine of 49 patients with pulmonary emphysema, none of whom had alpha 1-antitrypsin deficiency. Among a control group without lung disease the polymorphism was significantly less frequent, being found in only five of 101 apparently healthy blood donors. The deoxyribonucleic acid (DNA) polymorphism was also present in two of 14 unrelated patients with alpha 1-antitrypsin deficiency, indicating a lack of association with any specific alpha 1-antitrypsin protein phenotype. The polymorphism identified in this study may be a new marker for genetic predisposition to chronic lung disease.

Lung lavage fluid from patients with alpha 1-proteinase inhibitor deficiency or chronic obstructive bronchitis: anti-elastase function and cell profile.

The anti-elastase composition of bronchoalveolar lavage (BAL) fluid from alpha 1-proteinase inhibitor (alpha 1PI) deficient and bronchitic patients was determined by immunological and functional assays, together with the cell profile of the BAL fluid. alpha 1PI, anti-leucoprotease and alpha 2-macroglobulin were present in all the samples BAL fluid alpha 1PI concentrations were significantly lower in the group with serum alpha 1PI deficiency. Lavage fluid from alpha 1PI deficient subjects inhibited less porcine pancreatic elastase than bronchitic BAL fluid (P less than 0.005). However, the alpha 1PI was only about 50% active as an inhibitor in both groups. There was no difference in the amount of neutrophil elastase (NE) inhibited per ml of lavage fluid or per mol of the measured inhibitors in the secretions, but both groups inhibited more enzyme than would be expected for these inhibitors (alpha 1PI deficient: median 4.78 mol of NE/mol of known inhibitors, range 0.88-78.80; bronchitic: 1.14, 0.21-4.66), suggesting that an additional inhibitor is present. The total leucocyte and neutrophil counts were elevated (2P less than 0.01) in the lavages of alpha 1PI deficient patients, suggesting a greater potential elastase burden than subjects with normal alpha 1PI.

The effect of assay conditions on the measurement of anti-elastase function in lung secretions.

We have determined the effect of altering assay conditions on the observed neutrophil elastase inhibitory capacity in lung secretions from emphysematous patients with normal serum alpha 1 PI. alpha 1 PI, ALP and alpha 2-macroglobulin were detected in all samples. Measurement at low enzyme concentration (less than 33.6 nmol/l) caused a 43% reduction in observed neutrophil elastase inhibitory capacity of sputum sol-phase, while inhibition by secretions in buffer without added NaCl was 20% greater than in the presence of 0.2 mol/l NaCl. Increasing the concentration of the synthetic substrate Suc-[Ala]3-pNA in the assay from 0.45 mmol/l to 7.2 mmol/l reduced the observed inhibitory capacity by 53% and the use of elastin-fluorescein gave lower results for inhibitory capacity than the Suc-[Ala]3-pNA (median 0.26 mol neutrophil elastase/mol measured inhibitors (range 0-0.72) with elastin; 1.40 mol neutrophil elastase/mol measured inhibitors (0.80-3.21) with Suc-[Ala]3-pNA). Assay conditions therefore greatly influence the results. In addition these findings suggest the presence of an additional inhibitor of neutrophil elastase in these secretions.

Studies of proteinases and their inhibitors in lung secretions.

The study of proteinases and their inhibitors in lung secretions may provide an understanding of the pathogenesis of many chronic lung diseases. However problems arise in the collection and assessment of the samples that leads to variability both within and between subjects. The purpose of the paper is to highlight these problems as well as the methods used to overcome them in secretions obtained from patients with chronic obstructive bronchitis. The results show that between subject variability is high; and may be up to 150% within an individual. Conventional means of reducing variability (standardisation for albumin) may not be appropriate for some proteins, leading to increased variability. The function of inhibitors is probably best expressed as enzyme inhibited/unit of inhibitor though even this shows daily variability within the same individual. An awareness of such variability is necessary for the interpretation of results obtained from patients.

Short term response of patients with bronchiectasis to treatment with amoxycillin given in standard or high doses orally or by inhalation.

The effect of three amoxycillin treatment regimens on purulent secretions of patients with bronchiectasis has been studied. On the basis of information recorded on a diary card the patients were divided into three groups, according to the usual nature of their secretions: seven who produced mucoid sputum, which occasionally became purulent; seven whose secretions were usually mucopurulent but occasionally purulent; and 19 whose secretions were persistently purulent. Treatment with capsules of amoxycillin in a dosage of 250 mg three times a day resulted in clearance of purulent secretions in patients of the mucoid group when they were treated for a clinical exacerbation. The sputum remained clear in these patients for long periods before a further exacerbation (median 6 1/2, range 1-11 months). The mucopurulent-purulent group also responded to this dosage but sputum purulence returned more rapidly (median 9, range 4-31 days). Only three of the 19 (17%) patients with persistently purulent secretions showed a macroscopic response to this dosage, whereas seven (60%) of 12 patients who received the higher dosage (3 g sachets twice a day) responded. Among the failures, some responded to nebulised amoxycillin, suggesting that higher levels of amoxycillin in secretions are required in these patients. Macroscopic clearance of purulent secretions was finally achieved in most of the patients studied. The response was not always predictable from the results of sputum culture. Clearance of secretions by antibiotics was also identified by the patients, using a diary card score. Improvements in well being and in symptoms were noticed even in the group who usually produced mucopurulent and purulent secretions even though they appeared to be clinically stable before treatment.

Elastase inhibitors of sputum sol phase: variability, relationship to neutrophil elastase inhibition, and effect of corticosteroid treatment.

The concentrations of three known elastase inhibitors (alpha 1 proteinase inhibitor, antileucoprotease, and alpha 2 macroglobulin) have been determined in the sputum of six patients with obstructive bronchitis over five consecutive days. Antileucoprotease was the major inhibitor measured and potentially could provide more than 80% of the elastase inhibition, whereas the contribution of alpha 2 macroglobulin was less than 0.2%. Comparison with the inhibitory capacity of the secretions active against human neutrophil elastase showed that the inhibitors could account for only about half of the inhibition measured. This suggests the presence of a substantial amount of unrecognised inhibitor. Corticosteroid treatment in 10 patients reduced the mean alpha 1 proteinase inhibitor concentration (p less than 0.025) from 18.6 micrograms/ml (SD 22.5) to 9.8 (6.6). Antileucoprotease, however, increased (p less than 0.05) from 20.5 micrograms/ml (24.3) to 39.3 (23.4). These changes were associated with an increase in elastase inhibition (p less than 0.025) from 180 (160) micrograms elastase/ml secretion to 310 (130), suggesting a beneficial effect of steroid treatment on the antielastases in lung secretions.

Comparison of concentrations of two proteinase inhibitors, porcine pancreatic elastase inhibitory capacity, and cell profiles in sequential bronchoalveolar lavage samples.

Bronchoalveolar lavage is used to obtain cells and proteins from the lower respiratory tract for diagnosis and research. Uncertainity exists about which site in the lung is sampled by the lavage fluid and what effect different lavage volumes have on recovery of the constituents of lavage fluid. Dilution of alveolar lining fluid by lavage fluid is variable and results are usually expressed as protein ratios to surmount this problem. We have compared cell profiles and the concentrations of two proteinase inhibitors--the low molecular weight bronchial protease inhibitor antileucoprotease and alpha 1 proteinase inhibitor, together with alpha 1 proteinase inhibitor function and its relationship to the cell profile in sequential bronchoalveolar lavage fluid samples from patients undergoing bronchoscopy. There was no difference in total or differential cell counts or albumin or alpha 1 proteinase inhibitor concentrations between the first and second halves of the lavage. Both the concentration of antileucoprotease and the ratio of antileucoprotease to albumin were, however, lower in the second half of the lavage (2p less than 0.01 and 2p less than 0.05 respectively). There was no difference in the function of alpha 1 proteinase inhibitor (assessed by inhibition of porcine pancreatic elastase--PPE) between aliquots (0.28 mole PPE inhibited/mol alpha 1 proteinase inhibitor; range 0-1.19 for the first half and 0.37 mol PPE inhibited/mol alpha 1 proteinase inhibitor; range 0.10-0.80 for the second half). About 60-70% of alpha 1 proteinase inhibitor in each half of the lavage fluid was inactive as an inhibitor. The function of alpha 1 proteinase inhibitor did not differ between bronchitic smokers and ex-smokers. Alpha 1 proteinase inhibitor function was not related to the number of total white cells, macrophages, or neutrophils in the lavage fluid. Contamination of lavage by red blood cells was found to alter the concentration of alpha 1 proteinase inhibitor but not its function when aliquots with and without erythrocytes were compared. These results show that the only difference between the two halves of these lavage samples is in the amount of antileucoprotease present, suggesting that more proximal secretions are being harvested early in the lavage procedure. Much of the alpha 1 proteinase inhibitor present in the samples is functionally inactive, but this is not clearly related to any particular cell type or to smoking habits, and does not differ between different stages of the lavage procedure. Much of the alpha1 proteinase inhibitor present in the samples is functionally inactive, but this is not clearly related to any particular cell type or to smoking habits, and does not differ between different stages of the lavage procedure. Finally, the presence of erythrocytes probably does affect alpha(1) proteinase inhibitor concentration and such samples should be excluded from analysis.

The effect of catalase and methionine-S-oxide reductase on oxidised alpha 1-proteinase inhibitor.

Oxidative damage to alpha 1-proteinase inhibitor (alpha 1-PI) may be important in the pathogenesis of emphysema. We have studied the ability of 2 enzymes (catalase and methionine-S-oxide reductase) to prevent and reverse oxidation of alpha 1-PI by hydrogen peroxide. Pre-incubation of catalase with H2O2 protected alpha 1-PI from oxidation, but the enzyme could not reverse prior oxidation of alpha 1-PI. In contrast, methionine-S-oxide reductase fully restored activity to H2O2-oxidised alpha 1-PI. Sputum sol-phase from smokers and non-smokers contained alpha 1-PI that was only about 30% active. Functional activity increased in both smokers (p less than 0.025) and non-smokers (p less than 0.05) approximately 2-fold following incubation with the reductase. Western blotting of the samples showed that about 20% of the alpha 1-PI was present as an enzyme-inhibitor complex and 20% was proteolytically cleaved. These observations suggest proteolysis, complexing with enzyme and oxidation are mechanisms of inactivation of alpha 1-PI in lung secretions.

The effect of reducing agents on proteolytic enzymes and oxidation of alpha 1-proteinase inhibitor.

We have studied the effect of the mucolytic agent N-acetylcysteine and dithiothreitol on the oxidation of alpha 1-PI by hydrogen peroxide, and their effect on porcine pancreatic elastase and leukocyte elastase. In addition, the effect of S-(carboxymethyl)cysteine (= carbocisteine, a mucolytic agent which does not have reducing properties) was studied in vitro and in patients with chronic obstructive bronchitis. Following addition of 59.6mM N-acetylcysteine, the amidolytic activity of leukocyte elastase was decreased by 55.3% and that of porcine pancreatic elastase by 57.0%. Dithiothreitol (5.7 mM) caused the loss of 97.4% and 67.6% of amidolytic activity of leukocyte elastase and porcine pancreatic elastase respectively whereas S-(carboxymethyl)cysteine had no effect. Similar results were found for the effect on elastolytic activity. Oxidation of alpha 1-PI by 8.6mM H2O2 resulted in partial loss of inhibitory function (mean 68.7% activity of native alpha 1-PI). N-Acetylcysteine and dithiothreitol prevented oxidation of alpha 1-PI when pre-incubated with H2O2 or incubated with alpha 1-PI and H2O2 simultaneously (94.5% and 94.4% activity of native alpha 1-PI for N-acetylcysteine; 78.3% and 87.6% activity for dithiothreitol - p less than 0.025). S-(Carboxymethyl)cysteine, when pre-incubated with H2O2 or incubated concurrently with alpha 1-PI and H2O2, caused a further decrease in the porcine pancreatic elastase inhibitory capacity of alpha 1-PI (53.1% and 63.0% respectively - p less than 0.025). None of the agents reversed oxidative inactivation once it had occurred. S-(Carboxymethyl)cysteine had no effect on alpha 1-PI function in sputum at the dose used.

Inhibitory capacity of alpha 1 antitrypsin in lung secretions: variability and the effect of drugs.

The inhibitory function of alpha 1 antitrypsin (alpha 1AT) has been studied in the lung secretions of 31 patients with chronic obstructive bronchitis. The inhibitory capacity for a single sample showed a wide range (median 0.13 micrograms porcine pancreatic elastase (PPE) inhibited per microgram alpha 1 antitrypsin; range 0-0.55 micrograms), and all but five of 86 samples studied were capable of inhibiting some porcine pancreatic elastase. No sample showed free elastase activity, however. The inhibitory capacity, studied in six patients over five consecutive days, varied daily within the same individual (coefficient of variation 9.0-108.9%). Corticosteroid treatment (40 mg prednisone daily) increased the inhibitory capacity of sputum alpha 1 antitrypsin in 10 patients (2p less than 0.05) from a median value of 0.13 micrograms PPE inhibited per microgram alpha 1AT (range 0.06-0.36) before treatment to 0.22 micrograms PPE inhibited per microgram alpha 1AT (range 0.09-0.65) after treatment. The inhibitory capacity of sputum was higher than in the corresponding bronchoalveolar lavage sample from the same patient (2p less than 0.05; n = 10). The median value for sputum was 0.22 micrograms PPE inhibited per microgram alpha 1AT (range 0-0.55) and the value for lavage fluid was 0.07 micrograms PPE inhibited per microgram alpha 1AT (range 0-0.27).

Effect of antibiotic treatment on sputum elastase in bronchiectatic outpatients in a stable clinical state.

Broad spectrum antibiotic treatment was given on 21 occasions to 15 patients with bronchiectasis who regularly produced purulent, elastase positive secretions. Although the results showed that sputum clearing--that is, changing from purulent to mucoid--largely depended on the pathogenic organism isolated, this was not exclusively the case and in some cases sputum growing sensitive organisms failed to clear whereas clearing occurred in other samples containing resistant organisms or no obvious pathogens. Clearing of sputum was achieved eventually in 12 of the patients and this was associated with the disappearance of elastase activity, although it returned in 10 patients within one week of stopping treatment. There was no change in sputum elastase where the sputum failed to clear. The clearance of elastase activity was associated with a decrease in protein transudation into the lung secretions. The sputum:serum albumin concentration ratio fell (p less than 0.005) from a mean (SD) of 2.32 (1.56) X 10(-2) in these 12 patients before treatment to 1.09 (0.40) X 10(-2) within the first week of treatment, but rose again to 2.07 (1.29) X 10(-2) within one week of stopping treatment. The results suggest that antibiotic treatment when patients are in a stable state may have a beneficial effect on the pathogenic nature of lung secretions and inflammation within the lung.