Pediatric Pulmonology 11:249-253 (1991)

Antineutrophil Elastase Activity in Cystic Fibrosis Serum Andre M. Cantin, MD, Sylvie Lafrenaye, MD, and Raymond 0. Begin, MD Summary. The antigenic concentrations of alpha-1-antitrypsin (a1AT) were measured in 13 patients with cystic fibrosis (CF) and in 11 healthy subjects. Serum alAT was purified by immunoaffinity chromatography and the antielastase activity of the purified a1AT was determined by measuring the molar ratio necessary to inhibit human neutrophil elastase (HNE). The association rate constant of alAT with HNE was determined in a timed assay. The capacity of CF serum a1AT to form complexes with porcine pancreatic elastase was studied by polyacrylamide gel electrophoresis. Antigenic concentrations of a1AT pmol/L were markedly increased in the serum of all patients with CF (61.9 5 4.3 pmol/L) in comparison to a reference standard (36.7 ? 1.8 pmol/L; P < 0.0001). CF serum a1AT was fully active against HNE, and its association rate constant in the presence of HNE was similar to that of healthy subjects, In addition, CF serum a1AT formed complexes with porcine pancreatic elastase that were electrophoretically indistinguishablefrom those of normal serum a1AT. These results indicate that patients with CF have increased serum a1AT concentrations and that this antiprotease, when purified from serum, functions normally. Pediatr Pulmonol. 1991; 11249-253. Key words: Alpha-1-antitrypsin; immunoaffinity chromatography; porcine pancreatic elastase complexes; polyacrylamide gel electrophoresis

INTRODUCTION

Proteolytic degradation of extracellular structural proteins comprising the lung connective tissue is a cardinal feature of cystic fibrosis (CF)-related lung disease. ' The burden of free active human neutrophil elastase (HNE) within the airway secretions of most patients is very high.2,3 Secretory leukoprotease inhibitor (SLPI), and alpha- I -antitrypsin (a1AT) are two major neutrophil elastase inhibitors.435Although a 1AT is present in the CF lung, it is inactive in most The mechanisms by which a1AT is inactivated probably involve oxidative and proteolytic processes. Direct evidence of a 1AT oxidative inactivation in the CF lung is lacking. However, the presence of massive numbers of neutrophils capable of releasing hydrogen peroxide (H202), as well as myeloperoxidase, a neutrophi1 enzyme that converts chloride to hypochlorite in the presence of H,O,, suggests that a large oxidant burden is present in the airways .' In addition, bronchial secretions from patients with CF can directly inactivate a l A T through elastase-mediated proteolysis.6 However, it is not clear whether proteolytic and/or oxidative processes within the CF lung can affect the function of systemic alAT. Goldstein et al. studied the serum a l A T function in five patients with CF.3 While they concluded that CF serum a 1AT was functional, their results showed that the binding capacity of CF serum a l A T to human neutrophil elastase ranged from 60 to 93%. Alpha-1-antitrypsin 0 1991 Wiley-Liss, Inc.

from normal serum is known to inhibit HNE in a 1:l molar ratio, yet only 77% of the active a l A T can be detected as a complex with HNE.8*9 Whether the CF serum a1AT binding capacities reflect active or partially inactivated serum a 1AT is unknown. Today a l A T is available for intravenous therapy in patients with hereditary a1AT deficiency; therefore the question of whether a 1AT intravenous therapy may be useful in CF is now open." However, a major distinction between these two groups of patients is that the lung a l A T deficiency results from a systemic d A T deficiency in hereditary emphysema, and from a local lung inflammatory response in CF. The effects of the lung's inflammatory response in CF on the functional capacity of systemic a l A T are unknown. The present study was undertaken to compare the antigenic and functional concentrations of a1AT in the From the Unitt de Recherche Pulmonaire, Centre Hospitalier Universitaire de Sherbrooke, Sherbrooke, Quebec, Canada. Received October 8, 1990; (revision) accepted for publication May 28, 1991.

This research was supported by a grant from the Canadian Cystic Fibrosis Foundation. A.M.C. is a scholar ofthe Fonds de la Recherche en Santk du Qutbec. S.L. is a recipient of a fellowship from the Canadian Cystic Fibrosis Foundation. Address correspondence and reprint requests to Dr. A . M . Cantin, CHUS, 3001, 12e Ave Nord, Sherbrooke, QC J I H 5N4, Canada,

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serum of patients with CF to that of normal subjects, using sensitive and accurate assays of a 1AT antineutrophil elastase activity. Anti-HNE activity was analyzed by: 1) measuring the molar concentration of a l A T necessary to inhibit HNE; 2) assessing the time required for a l A T to inactivate HNE (K association); and 3) observing the formation of complexes between a1AT and porcine pancreatic elastase (PPE).

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Thirteen patients with diagnostic criteria of CF" and 11 normal volunteers were enrolled into this study after informed consent had been obtained. The average age of the patients was 16 k 2 years and that of the controls was 27 -+ 3 years. One subject from each group was a cigarette smoker while all others were lifetime nonsmokers. All patients had evidence of chronic lung infection characterized by purulent sputum production. Sputum cultures from ten patients revealed the presence of Pseudomonus aeruginosa with or without Staphylcoccus uureus, whereas three patients had S . uureus without detectable P . ueruginosu. Serum a1AT Purification

Alpha- 1-antitrypsin was purified from each serum sample according to the methods described by Sugiura et a1.I2 Briefly, an anti-alAT immunoabsorbent column was prepared by covalently binding 7 mL of an antibody specific for human a1AT (Dimension Laboratories Inc., Mississauga, Ont.) to 5 gm cyanogen-bromide activated Sepharose 4B gel (Pharmacie Fine Chemicals, Piscataway, NJ). The column was equilibrated in 0.1 M Tris, 0.5 M NaC1, 0.005 M Na,EDTA, pH 8.3 (buffer A), before loading 2 mL serum. The column was thoroughly washed in buffer A to remove contaminating proteins. a l A T was eluted with 0.1 M Na2C0,, 0.5 M NaCl, and 0.005 M Na,EDTA. The pH of the eluted material containing a 1AT was immediately neutralized by adding an equal volume of buffer A. Subsequently, the samples were concentrated on an Amicon YM-10 membrane.

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CYSTIC

FIBROSIS Fig. 1. Alpha-1-antitrypsin levels in the serum of patients with cystic fibrosis and of healthy subjects. Antigenic levels of a1AT were determined by radial immunodiffusion. Results represent the mean 2 standard error of the mean for 11 healthy subjects and 13 patients with CF.

quently, 200 pL elastase solution was added to 200 pL purified a 1AT, and incubated at 23°C for 2 hours. At the end of the incubation period, 100 FL methoxy-succinylalanyl-alanyl-prolyl-valyl nitroanilide (MeOSAAPVNA, 1 mM)I3 was added to the reaction mixture and residual elastase activity was measured in a spectrophotometer (DU-7, Beckman Instruments Inc., Fullerton, CA) as the change in absorbance at 410 n d m i n . The percentage of a1 AT molecules capable of binding and inactivating human neutrophil elastase was calculated according to the following formula; % active/total a l A T = (A/ B) X 100; where A is elastase concentration in the reaction mixture and B is the minimal a l A T concentration at which no elastase activity is detectable in the reaction mixture. Association Rate Constant of alAT With HNE

The rate of association of a l A T with HNE is a sensitive indicator of the functional state of alAT. We therefore measured the association rate constant (Ka) of a l A T in the presence of HNE.14 Human neutrophil elastase was titrated against a 1AT freshly isolated from Antigenic and Functional Concentrations of a1 AT normal serum of an individual with the M l M l phenoThe antigenic concentrations of a l A T in serum and in type. All subsequent assays to determine the association the purified samples were determined by radial immuno- rate constants were done with the same lot of HNE. To diffusion (LC-partigen, Hoechst Canada Inc., Montreal, determine the K association of a l A T for HNE, equimoQC).' To determine whether a l A T from CF patients lar concentrations of functional purified a 1AT were could inactivate human neutrophil elastase in a 1:1 molar incubated with 3.4 nM HNE in buffer B, at 23°C. The ratio, the following experiments were performed. Puri- reaction was stopped at various time points ( 1 6 3 0 0 s) by fied a l A T from normal and CF serum was diluted in adding 0.1 M MeOSAAPVNA. Residual elastase activ100 mM HEPES, 0.5 M NaCl, 0.1% Brij-35, pH 7.5 ity was measured immediately after the addition of the (buffer B) to concentrations of 0-16 nM. Human neutro- chromogenic substrate by monitoring the change in phil elastase (Elastin Products Co., Pacific, MO) was absorbance of the solution at 410 nm. The K association diluted in this buffer to a concentration of 4 nM. Subse- was calculated using the following formula: Ka = 1/

Serum al-Antitrypsin in Cystic Fibrosis

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Fig. 2. Characterization of the functional capacity of purified serum-derived or1AT from patients with cystic fibrosis and healthy subjects, to inhibit neutrophil elastase. A: Human neutrophil elastase inhibitory capacity of purified alAT as evaluated in a time-Independentassay. A 100% activehotal a1AT ratlo indicates that alAT can inhibit an equimolar concentration of

NORMAL

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FIBROSIS HNE. B: Rate of inhibition of an equimolar concentrationof HNE by purified serum-derived a1 AT from CF and healthy subjects. Results are expressed as the mean 2 standard error of the mean, of the association rate constant (K assoc.) of d A T for HNE.

(enzyme concentration) X (T1/2), where T1/2 represents all a l A T purified from serum of healthy subjects was the time needed to inhibit 50% of the elastase activity. found to be fully active. In addition, the functional Each sample of purified serum a l A T was measured 3 capacity of a l A T in serum of patients with CF times. (100.2 2 4.1%) was not different from that of purified a1AT of healthy subjects (103.1 2 9.6%; P > 0.2) (Fig. a1 AT-Elastase Complex Formation 2A). However, since patients had a 1.7-fold increase in To examine the capacity of a l A T purified from the antigenic a1AT, the antineutrophil elastase activity of CF serum of CF and healthy subjects to bind PPE, 100 nM serum was increased to the same extent as the increase in a l A T was incubated with 25 nM PPE (Sigma Chemical antigenic concentrations of a 1AT. Co., St. Louis, MO) in buffer B at 23°C for 2 hours. At the end of the incubation period, the samples were K Association of CF Serum a1AT separated by electrophoresis on a sodium dodecyl sulfate Although the number of a l A T molecules required to 7.5% polyacrylamide gel. inhibit 4 nM HNE was similar in CF and healthy subjects, the rate at which inactivation occurred remained Statistical Analysis unknown. To determine whether inactivation of elastase All data are expressed as the arithmetic mean k molecules of CF serum a 1AT might be slower than that standard error of the mean. Comparisons between groups of a l A T from healthy subjects, K association was were considered significant when P d 0.05, using the measured in a time-dependent assay of a1AT anti-HNE two-tailed Student's t-test. activity. The results of these experiments revealed that not only was CF a l A T capable of inhibiting an equimolar concentration of HNE, but the rate at which this inactiRESULTS vation occurred was similar to that observed with a 1AT Antigenic Concentrations of Serum a1 AT from healthy subjects (CF Ka, 0.77 +- 0.16 X lO7M-ls-'; The concentration of serum a 1AT was markedly normal Ka, 0.82 f 0.19 X 107M-'sec-*; P > 0.2) increased in patients with CF (61.9 ? 4.3 pmol/L) when (Fig. 2B). compared to that of healthy subjects (36.7 & 1.8 p,mol/ a1AT and Porcine Pancreatic Elastase L; P < 0.0001) (Fig. 1). Complex Formation Function of Serum alAT Since oxidized a 1AT is unable to form complexes with The functional capacity of serum a l A T was consid- PPE, we compared the electrophoretic patterns of a1AT ered to be 100% if the molar concentration of a l A T from CF and healthy subjects incubated in the presence of needed to inhibit all elastase activity was equal to the PPE. As can be seen in Figure 3, purified a 1AT from the molar concentration of elastase present in the reaction serum of three CF and three healthy subjects formed mixture, using a time-independent assay. As expected, similar complexes with PPE. The electrophoretic patterns

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Fig. 3. Electrophoretic pattern of a1AT in the absence (odd rows) and presence(even rows) of porcine pancreatic elastase. Alpha-1-antitrypsin was purifiedfrom the serum of patients with CF (rows 1-6) and healthy subjects (rows 7-12). Subsequently, 100 nM a l A T was incubated with 25 nM PPE in 0.1 M Tris, 0.5 M NaCI, 0.1% Brij-35, pH 7.5, for 2 hours at 23°C. At the end of the incubation, the samples were reduced, heated to 100°C, and analyzed by electrophoresis in a sodium dodecyl sulfate 7.5% polyacrylamide gel.

of the other CF and healthy subjects were similar to those presented in Figure 3 .

DISCUSSION The lung disease associated with CF is characterized by a large influx of polymorphonuclear leukocytes into the airways. Neutrophil degranulation results in the accumulation of large amounts of elastase. In spite of the presence of a l A T , most of the lung elastase is free and active.’ The major mechanism by which a l A T is inactivated in the CF lung seems to be through proteolytic degradation by excess neutrophil elastasc6 The present study demonstrates that free a1AT molecules from CF serum inactivate HNE in a 1:1 molar ratio as do a l A T molecules from healthy subjects. Furthermore, the rate at which the inactivation occurs is similar to the rate of association of normal alAT.I4 Since the rate of association is a sensitive index of a l A T functional capacity, these results suggest that the severe inflammatory lung disease associated with CF does not adversely affect systemic a 1AT anti-HNE function. Finally, the rate of association of oxidized a 1AT with porcine pancreatic elastase is OI4; however, CF serum a l A T was found to form complexes with PPE that were electrophoretically indistinguishable from those formed by normal a 1AT. This further supports the conclusion that CF serum a1AT is not oxidized. Active a l A T is capable of inactivating an equimolar amount of HNE; however, due to rapid degradation of a1AT-HNE complexes without recovery of elastase activity, only 77% of the a 1AT can be detected as bound to HNE.‘ Goldstein ct al. observed that incubation of five

CF serums with a molar concentration of elastase equal to that of the serum a l A T resulted in 77% (mean, 76.6 k 11.9%; range, 60-93%) of the CF a l A T binding to HNE.3 This is exactly the amount of complex formation to be expected if CF serum a l A T is fully active, as suggested in the present study. Several investigators have reported the presence of a1AT-HNE complexes in the serum of patients with CF.33’6If some of the systemic a l A T is complexed to HNE, then it is no longer available to react further with elastase and should, therefore, be detected as inactive a1 AT. In contrast, we found no evidence of inactive CF serum a l A T . The likely explanation for this apparent contradiction is that the ratio of complexed a 1AT to total a1AT in CF serum is too small to be detectable by assays of a1AT function. Consistent with this concept are reports indicating that the amount of a l A T complexed to HNE in CF serum is less than 2 pgimL, or 0.04% of the total serum ( x ~ A T . ~ . ~ ~ As previously reported ,3.17.I* we have found that the antigenic amounts of a 1AT are increased twofold in patients with CF. Since CF serum a l A T is fully active, the CF serum antineutrophil elastase activity is necessarily increased. In spite of this increased systemic antiHNE protection, the CF lung is constantly exposed to free active HNE.2,3.6.19-2 1 The presence of free elastase in the CF lung may have many deleterious effects, such as the proteolytic destruction of lung connective tissue, the potent stimulation of mucus secretion, and the cleavage of both CR1 on neutrophils and C3bi on opsonized Pseudomonas,which creates severe deficiencies in complement-dependent phagocytosis and killing of Pseudornonas bacteria by neutrophils. 1,22-24 These data strongly suggest that attempts to lessen the lung elastase burden are warranted. One approach that has been used successfully in patients with a l A T deficiency and emphysema is to supplement the systemic circulation with human plasmaderived a 1AT in order to reestablish the lung proteaseantiprotease balance. l o However, since the lung elastase burden persists despite increased systemic anti-HNE activity, it is likely that systemic a 1AT supplementation in CF would require frequent and massive doses of a 1AT to maintain a lung environment free of elastase activity. Consistent with this concept are the results of a recent study in which active elastase was detectable at the CF respiratory epithelial surface only 12 hours after intravenous infusion of 90 mg/kg C X ~ A TIn . ~contrast, ’ protective levels of active a 1AT are maintained for 1 week after an infusion of 60 mg/kg a l A T in patients with a l A T deficiency and emphysema. l o In the absence of a detectable systemic a 1AT dysfunction, it would seem rational to direct therapeutic strategies to the local correction of the lung elastase burden in CF. One such approach may be through the use of aerosolized a l A T , which has

Serum ul-Antitrypsin in Cystic Fibrosis

recently been shown to restore effectively the lung epithelial lining fluid antineutrophil elastase capacity in CF patients.26 In conclusion, the present study indicates that serum a 1AT from patients with CF is capable of inactivating an equimolar amount of HNE. In addition, the rate at which CF serum a1 AT inactivates HNE and its capacity to form complexes with PPE are similar to that of aIAT from normal serum. The increase in CF serum a1 AT concentration therefore results in an increase in CF serum anti-HNE activity. ACKNOWLEDGMENTS

We thank Ginette Bilodeau and Marc Martel for excellent technical assistance. REFERENCES I . Bruce MC, Poncz L, Klinger JD, Stern RC, Tomashefski JF, Dearborn DG. Biochemical and pathological cvidence for proteolytic destruction of lung connective tissue in cystic fibrosis. Am Rev Respir Dis. 1985; 132529-535. 2 . Suter S , Schaad UB, Tegner H , Ohlsson K, Desgrandchamps D, Walvogel FA. Levels of free granulocyte elastase in bronchial secretions from patients with cystic fibrosis: effects of antimicrobial treatment against Pseudomonas aeruginosu. J Infect Dis. 1986; 153:902-909. 3. Goldstein W, Doring G . Lysosomal enzymes from polymorphonuclear leukocytes and protease inhibitors in patients with cystic fibrosis. Am Rev Respir Dis. 1986; 134:49-56. 4. Vogelmeier C, Hubbard RC, Fells GA, Schnebli HP, Thompson RC, Fritz H, Crystal RG. Anti-neutrophil elastase defense of the normal human respiratory epithelial surface provided by the secretory leukoprotease inhibitor. J Clin Invest. 1991; 87:482488, 5. Pannell R, Johnson D, Travis J . Isolation and properties of human a1 -proteinase inhibitor. Biochemistry. 1974; 13:543%5445. 6. Cantin A, Bilodeau G, Begin R. Granulocyte elastase-mediated proteolysis of alpha- I -antitrypin in cystic fibrosis bronchopulmonary secretions. Pediatr Pulmonol. 1989; 7: 12-17. 7. Mohammed JR, Mohammed BS, Pawluk LJ, Bucci DM, Baker NR, Davis WB. Purification and cytotoxic potential of myeloperoxidase in cystic fibrosis sputum. J Lab Clin Med. 1988; 112:711-720. 8. Travis J, Salvesen GS. Human plasma proteinase inhibitors. Annu Rev Biochem 1983; 526.5-709. 9 . Baumstark JS, Ting Lee C, Luby RJ. Rapid inactivation of a I-protease inhibitor (a1 -antitrypsin) by elastase. Biochim Biophys Acta. 1977:482:400411. 10. Wewers MD, Casolaro AM, Sellers SE, Swayze SC, McPhaul KM, Wittes JT, Crystal RG. Replacement therapy for alphaI-antitrypsin deficiency associated with emphysema. N Engl J Med. 1987; 316:1055-1062.

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I I . Wood RE, Boat TF, Doershuk CF. Cystic fibrosis. Am Rev Respir Dis. 1976; 113:833-878. 12. Sugiura M, Hayakawa S, Adachi T, Ito Y , Hirano K, Sowaki S. A simple one step purification of human al-proteinase inhibitor by immunoabsorbant column chromatography. J Biochem Biophys Methods. 1981; 5243-249. 13. Castillo MJ, Nakajima K, Zimmerman M, Powers JC. Sensitive substrates for human leukocyte and porcine pancreatic elastase: a study of the merits of various chromophoric and fluorogenic leaving groups in assays for serine proteases. Anal Biochem 1979; 99153-64. 14. Beatty K, Bieth J, Travis J. Kinetics of association of serine proteinases with native and oxidized a-I-antitrypsin and a-1antichymotrypsin. J Biol Chem 1980; 255:393 1-3934. 15. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680-685. 16. Hollsing AE, Lantz B, Bergstrom K, Malmborg AS, Strandvik B. Granulocyte elastase-a 1-antiproteinase complex in cystic fibrosis: sensitive plasma assay for monitoring pulmonary infections. J Pediatr. 1987; 111:20&211. 17. Schidlow DV, Kueppers F. Trypsin binding activity of alpha,macroglobulin in cystic fibrosis and other lung diseases. Am Rev Respir Dis. 1970; 121:31-37. 18. Tomasova H, Vavrova V, Dvorak P, Rybak M, Srajer J. Activity of trypsin-like proteinases and their inhibitors in serum of children with cystic fibrosis. In: Adam G , Valassi-Adam H, eds. Proceedings of the 12th Annual Meeting of the European Working Group on Cystic Fibrosis. Athens: S . Lennis, 1983:119-122. 19. Davis WB, Fells GA, Chernick MS, Di Saint’Agnese P, Crystal RG. A role for neutrophils in the derangements of the bronchial wall characteristic of cystic fibrosis. Am Rev Respir Dis. 1983; 127:207. 20. Jackson AH, Hill SL, Afford SC, Stockley RA. Sputum sol-phase proteins and elastase activity in patients with cystic fibrosis. Eur J Respir Dis. 1984; 65:114-124. 21. Suter S, Schaad UB, Roux L, Nydegger UE, Walvogel FA. Granulocyte neutral proteases and Pseudomonas elastase as possible causes of airway damage in patients with cystic fibrosis. J Infect Dis. 1984; 149523-531. 22. Sommerhoff CP, Nadel JA, Basbaum CB, Caughey GH. Neutrophi1 elastase and cathepsin G stimulate secretion from cultured bovine airway gland serous cells. J Clin Invest, 1990; 85:682689. 23. Berger M, Sorensen RV, Tosi MF, Dearborn DG, Doring G . Complement receptor expression on neutrophils at an inflammatory site, the Pseudornonas-infected lung in cystic fibrosis. J Clin Invest. 1989; 84:1302-1313. 24. Tosi MF, Zakem H, Berger M. Neutrophil elastase cleaves C3bi on opsonized pseudomonds as well as CR 1 on neutrophils to create a functionally important opsonin receptor mismatch. J Clin Invest. 1990; 86:30&308. 25. McElvaney N, Hubbard R, Fells G, Healy J , Chernick M, Crystal RG. Intravenous a 1-antitrypsin therapy to reestablish anti-neutrophi1 elastase defenses of the pulmonary epithelial surface in cystic fibrosis. Am Rev Respir Dis. 1990; 141:A83. 26. McElvaney NG, Hubbard RC, Birrer P, Chernick MS, Caplan DB, Frank MM, Crystal RG. Aerosol al-antitrypsin treatment for cystic fibrosis. Lancet 1991; 337:392-394.

Antineutrophil elastase activity in cystic fibrosis serum.

The antigenic concentrations of alpha-1-antitrypsin (alpha 1AT) were measured in 13 patients with cystic fibrosis (CF) and in 11 healthy subjects. Ser...
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