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Published ahead of print on March 11, 2005, doi:10.1165/rcmb.2004-0374OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 32, pp. 553-559, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2004-0374OC

Competition between Elastase and Related Proteases from Human Neutrophil for Binding to {alpha}1-Protease Inhibitor

Brice Korkmaz, Pierre Poutrain, Eric Hazouard, Michèle de Monte, Sylvie Attucci and Francis L. Gauthier

INSERM U618 "Protéases et Vectorisation pulmonaires"; and IFR 135 "Imagerie Fonctionnelle," University François Rabelais, Tours, France

Correspondence and requests for reprints should be addressed to Francis Gauthier, INSERM U618 Protéases et Vectorisation pulmonaires, University François Rabelais, 10 Bd Tonnellé, 37032 Tours Cedex, France. E-mail: gauthier{at}univtours.fr


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The protease–antiprotease imbalance that is characteristic of most inflammatory lung disorders depends on the spatial-temporal regulation of active inhibitor and protease concentrations in lung secretions. We have studied the competition between the three main serine proteases from human neutrophil primary granules in their binding to {alpha}1-Pi, the main serine proteases inhibitor in lung secretions. Elastase was the only target of {alpha}1-Pi when identical molar amounts of purified inhibitor and the three proteases were tested together. The other two proteases were only inhibited once elastase was saturated. Elastase remained the preferred target of inhibitors when bronchoalveolar lavage fluids from patients with lung pneumonia and acute respiratory distress syndrome were used as the source of inhibitors, in spite of the presence of additional inhibitors in lung secretions. Since neutrophil proteases are expressed at the neutrophil surface, we also measured residual activities of membrane-bound proteases after purified neutrophils were incubated with bronchoalveolar fluids. Again, elastase was the preferred target of the inhibitors. We conclude that protease 3 and cathepsin G are not controlled as efficiently as elastase in lung secretions, a feature that must be taken into account when developing inhibitor-based anti-inflammatory therapies.

Key Words: {alpha}1-protease inhibitor • {alpha}1-antitrypsin • bronchoalveolar lavage • protease inhibitor • serine protease


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The closely related serine proteases human neutrophil elastase (HNE), protease 3 (Pr3), and cathepsin G (Cat G) are stored at similar concentrations in the primary granules of human neutrophils (1). They are released to break down elastin fibers and other extracellular matrix components when neutrophils are recruited and activated at inflammatory sites, and are therefore major factors in chronic inflammation (2–5). However, the role of each one at inflammatory sites has not been yet clearly established because they have similar physicochemical and enzymatic properties. This has impaired the specific measurement of their activities in bronchoalveolar lavage (BAL) fluids and expectorations, into which they are released simultaneously from their azurophil granules when neutrophils are activated. HNE has long been considered to be the main enzyme causing proteolytic damage during severe lung inflammation. But no experiment has yet investigated the fates of the three serine proteases from primary granules after neutrophil activation and degranulation. Their fates could differ significantly because they are controlled differently by local endogenous inhibitors, and other regulatory compounds such as DNA (6), autoantibodies (7), heparin (8), and bacterial proteases (9). Their bioavailability also depends on their binding to cell membranes (1, 10) and on their affinities for peptide or protein substrates (11). As a result of this spatial-temporal regulation at inflammatory sites, the physiologic function of active proteases may differ more than is predicted by in vitro analyses of their physicochemical and enzymatic properties.

The major naturally occurring inhibitor in lung epithelial lining fluid is the polyvalent serpin inhibitor {alpha}1-protease inhibitor ({alpha}1-Pi); it inhibits HNE, Pr3, and Cat G. There are several other inhibitors in lung secretions, and these may also contribute to the overall control of serine protease activity. Some belong to the serpin family: one is monocytes neutrophil elastase inhibitor (MNEI), which inhibits all three serine proteases (12, 13); another is antichymotrypsin (ACT), which is more specific for chymotrypsin-like proteases including Cat G (14). Others are low Mr inhibitors of the chelonianin family, like SLPI (also called mucous protease inhibitor [MPI]) and elafin (15). There may also be small amounts of {alpha}2-macroglobulin in lung secretions (16). The association rate constant for the interaction between purified {alpha}1-Pi and HNE is higher than those of the other two neutral proteases from neutrophils (2, 17–20). But in vivo conditions are very different from those in vitro that use purified material; all three proteases are present in lung secretions and compete to bind to {alpha}1-Pi and other natural inhibitors that act in concert with the serpin to control proteolytic activity. These proteases are also partly bound to neutrophil membranes, which may result in different rates of inhibition by physiologic inhibitors (21).

No competition experiments have been performed to determine the rate at which each protease is inhibited in biological fluids because specific substrates for each protease were not available until very recently. We have developed fluorogenic substrates with intramolecularly quenched fluorescence that enable us to measure subnanomolar concentrations of HNE, Pr3, and Cat G (22–24). These substrates have now been used to measure the activities of equimolar amounts of HNE, Pr3, and Cat G mixed with purified {alpha}1-Pi or with inflammatory BAL fluids supernatants containing a mixture of all the inhibitors in lung secretions. The residual activity of each protease was also measured at the surface of activated neutrophils after their incubation with BAL fluids. The way free and membrane-bound neutrophil proteases are controlled by purified {alpha}1-Pi or BAL fluids will help to know which protease(s) should be targeted by therapeutic inhibitors during the treatment of lung diseases.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Materials
HNE (EC 3.4.21.37), Pr3 (EC 3.4.21.76), {alpha}1-Pi, and ACT were obtained from Athens Research and Technology (Athens, GA). Cat G (EC 3.4.21.20) was from ICN Pharmaceuticals (Orsay, France), trypsin (EC 3.4.21.4), polymorphprep, and lymphoprep from Nycomed Pharma (Oslo, Norway). Polyclonal anti–{alpha}1-Pi antibodies were purchased from Behring (Marburg, Germany). Igepal CA-630 was from Sigma (St Louis, MO). All other reagents were of analytical grade.

Collection of BAL Fluids
All patients were referred to the Department of Pneumology or to the Medical Intensive Care Unit of Bretonneau University Hospital (Tours, France). They were suffering from putative acute bacterial pneumonia and/or fulfilled the diagnostic criteria for acute respiratory distress syndrome (ARDS), and underwent BAL for diagnostic or therapeutic investigations. The decision to undertake BAL and patient care were taken by the clinicians. The protocol was approved by the local institution Ethics Committee.

BAL was performed via a fibroptic bronchoscope (Olympus BF-33; Olympus, Paris, France) using 50-ml aliquots of warmed (30°C) sterile 0.9% NaCl. BAL fluid samples were collected under sterile conditions, and the fifth fraction, which is devoided of contaminating epithelial and bronchial cells, was immediately filtered through a double-layer of sterile cotton gauze moistened with 0.9% NaCl to remove debris and mucus. A fraction of this fifth filtered aliquot was taken for biochemical and enzymatic analysis. BAL fluids were immediately buffered in 50 mM Hepes, pH 7.4, 0.15 M NaCl, 1 mM EDTA, and 1 mM DTT, and centrifuged first at 1,000 x g for 10 min and then at 3,500 x g for 15 min. Cell-free supernatants were aliquoted and frozen at –80°C. Total proteins in BAL fluid supernatants were determined by the Bradford procedure.

Isolation of Blood Polymorphonuclear Leukocytes
Human polymorphonuclear leukocytes (PMNs) were purified from 8-ml samples of peripheral blood collected from healthy volunteers into EDTA-containing tubes (24). Cells were counted and neutrophils quantified as reported earlier (25). Cell viability was checked by trypan blue exclusion. Purified PMNs were kept at room temperature in buffer with gentle shaking and washed with PBS containing 4 mM EGTA just before use.

Enzyme Assays
The specific Abz-peptidyl-EDDnp fluorogenic substrates were Abz-APEEIMRRQ-EDDnp for HNE, Abz-TPFSGQ-EDDnp for Cat G, and Abz-VADCADQ-EDDnp/Abz-VADnVADQ-EDDnp for Pr3 (22, 24). Stock substrate solutions (2–5 mM) were prepared in 30% (vol/vol) N,N-dimethylformamide and diluted to 0.5 mM with 50 mM Hepes buffer pH 7.4. The stock solution of the cys-containing Pr3 substrate contained 10 mM dithiothreitol. Free HNE and Pr3 were titrated with {alpha}1-Pi, the titer of which had been determined using bovine trypsin titrated with p-nitrophenyl-p'-guanidinobenzoate (26). A 1:1 stoichiometry was used for HNE; that for the Pr3/inhibitor complex was 1:1.3 to allow for partial {alpha}1-Pi degradation via the "substrate pathway" (27). Membrane-bound active proteases were quantified by comparing the rates of hydrolysis of their specific substrates to those of titrated proteases working under the same experimental conditions. Trypsin was prepared as a 2 x 10–4 M stock solution in 100 mM Tris/HCl pH 8, 50 mM CaCl2, then used in the same buffer as HNE and Pr3. The hydrolysis of Abz-peptidyl-EDDnp substrates was followed by measuring the fluorescence at {lambda}ex = 320 nm and {lambda}em = 420 nm in a Hitachi F-2000 spectrofluorometer (Hitachi, Les Ulis, France). The system was standardized using Abz-FR-OH prepared by the total tryptic hydrolysis of an Abz-FR-pNA solution, and its concentration was determined from the absorbance at 410 nm, assuming E410 nm = 8,800 M–1 cm–1 for p-nitroanilide. Concentrations of Abz-peptidyl-EDDnp substrate were determined by measuring the absorbance at 365 nm, using E365 nm = 17,300 M–1 cm–1 for EDDnp.

Cat G was titrated with ACT based on a 1:1 stoichiometry and by {alpha}1-Pi based on a 1.2:1 stoichiometry.

Free HNE, Pr3, and Cat G were measured at 37°C in 50 mM Hepes buffer (pH 7.4), 150 mM NaCl, supplemented with 0.05% Igepal CA-630 (vol/vol) to take into account the great propensity of these proteases to adhere to plastic and glass surfaces when in dilute solution. The activities of free proteases were also measured in the detergent-free buffer (10 mM PBS, pH 7.4) used for membrane-bound proteases to allow comparison between free and membrane-bound activities.

Membrane-bound activities were quantified by comparing the rates of hydrolysis by membrane-bound HNE, Pr3, and Cat G to those of the titrated proteases under the same experimental conditions (24). PMNs (1 x 105 to 5 x 105 cells), or purified proteases, were incubated with 20 µM Abz-peptidyl-EDDnp in polypropylene microplate wells (Hard-Shell Thin-Wall Microplates; MJ Research, Waltham, MA) at room temperature in activity buffer (10 mM PBS, pH 7.4). The fluorescence was recorded at {lambda}ex = 320 nm and {lambda}em = 420 nm using a microplate fluorescence reader (Spectra Max Gemini; Molecular Devices, St. Grégoire, France) under continuous stirring.

Measurement of the BAL Fluids' Inhibiting Capacity
BAL fluids were diluted 1:10 to 1:200 in activity buffer and incubated with each protease (2.5 · 10–8 M final) for 20–30 min at 37°C in a volume of 60 µl. Aliquots of 50 µl were then mixed with 250 µl activity buffer, and residual protease activities were measured by adding substrates (5 µM) specific for each protease. The absence of endogeneous proteolytic activity in BAL fluids was checked by mixing concentrated BAL fluids diluted 1:6 in reaction buffer with specific substrates and recording residual activities.

Inhibition of Membrane-Bound Proteases by BAL Fluids
HNE, Pr3, and Cat G at the surface of PMNs were measured as reported before (24). The number of cells was adjusted so that total proteases concentration was in the nanomolar range. Cells were incubated for 15–60 min under stirring with the volume of BAL fluid needed to inhibit about one-third of the total protease activity and the activity of each protease was measured. The experiment was repeated using double and triple volumes of BAL fluid. Fluorescence was recorded with the Gemini fluorometer at room temperature.

Electrophoresis and Western Blotting
Equimolar amounts of purified HNE, Pr 3, and Cat G (5 x 10–8 M) were incubated with a molar excess of {alpha}1-Pi in PBS, 4 mM EGTA, for 2 min at room temperature and the mixtures analyzed by SDS-PAGE on 12% acrylamide/bisacrylamide gels. The resolved proteins were transferred to nitrocellulose membranes and the complexes detected by incubation with rabbit polyclonal anti–{alpha}1-Pi antibodies (diluted 1:1,000) followed by peroxidase-coupled goat polyclonal anti-rabbit-IgG antibodies (diluted 1:15,000), using the Renaissance Plus kit (ICN). BAL fluids from patients with pneumonia and ARDS (3 or 10 µl depending on their total protein content) were also analyzed by imunoblotting for {alpha}1-Pi distribution using the same procedure.

Statistics
Protease activities from the various combinations were analyzed using an exact nonparametric Kruskal-Wallis (test for k independent samples), followed, when significant, by an exact nonparametric permutation test with general scores (test for two independent samples). Both tests were performed with StaXact software (Cytel Software Corp., Cambridge, MA).


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Endogenous inhibitors are present at inflammatory sites, where they control activities of the HNE, Pr3 and Cat G released into the extracellular space from recruited, activated neutrophils. The major serine protease inhibitor in lung secretions, {alpha}1-Pi, inhibits all three proteases, so that there may be competition between them for binding to {alpha}1-Pi and to any other inhibitors, like SLPI that can inhibit both HNE and Cat G, and elafin that inhibits HNE and Pr3. We first analyzed enzymatically the partition of equimolar amounts of HNE, Pr3 and Cat G on purified {alpha}1-Pi to determine how each protease is controlled in the presence of the others and hence identify their roles in the proteolytic degradation of lung tissue during inflammatory disorders.

Distribution of Equimolar Amounts of HNE, Pr3, and Cat G on Purified {alpha}1-Pi
Aliquots of titrated HNE, Pr3, and Cat G (3 · 10–8 M final) were each incubated with an identical molar amount of titrated {alpha}1-Pi (3 · 10–8 M) for 30 min. This is long enough for complete association of the inhibitor with each of the proteases, according to the association rate constants (2, 17–19). We measured residual activities with their specific fluorogenic substrate (22–24). HNE was fully inactivated under these conditions, Pr3 and Cat G retaining ~ 20–30% of activity due to partial degradation of the inhibitor via the substrate pathway (Figure 1). This was confirmed by Western blotting using anti {alpha}1-Pi antibodies (insert, Figure 1) that showed an additional band of lower Mr after incubation of {alpha}1-Pi with Pr3 and Cat G, but not after incubation with HNE, which binds to {alpha}1-Pi stoichiometrically.



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  		Figure 1. Residual activities of HNE, Pr3, and Cat G (3.10–8 M final) after incubation with a stoichiometric amount of {alpha}1-Pi. The complete inhibition of HNE denotes that all the {alpha}1-Pi has formed inactive complexes with HNE, whereas the partial inhibition of Pr3 and Cat G shows that part of {alpha}1-Pi has been inactivated via the substrate pathway during the course of the interaction. This is confirmed by Western blotting (inset) showing an additional band of lower Mr corresponding to inactivated {alpha}1-Pi, in the lanes for Pr3 and Cat G, but not in the HNE lane.

 
The three enzymes (3 · 10–8 M each) were then mixed in a buffered solution containing {alpha}1-Pi (3 · 10–8 M) and the mixture incubated for 30 min at 37°C. The residual activity of each protease was then measured using appropriate substrates (Figure 2). When the three proteases competed for binding to {alpha}1-Pi, the hydrolysis of the HNE-specific substrate Abz-APEEIMRRQ-EDDnp was almost totally inhibited, whereas that of substrates of Pr3 (Abz-VADCADQ-EDDnp and Cat G (Abz-TPFSGQ-EDDnp) remained unchanged (Figure 2B). Thus, only HNE interacted with the inhibitor. Repeating the experiment with a twice the concentration of {alpha}1-Pi resulted in additional inhibition of Pr3 but not of Cat G (Figure 2C). When the total amount of {alpha}1-Pi was identical to the combined concentrations of the three enzymes, only a little Cat G activity remained, due to partial inactivation of the inhibitor via the substrate pathway during its interaction with Pr3 and Cat G (Figure 2D).



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  		Figure 2. Residual activities of a mixture of equimolar amounts (3.10–8 M final) HNE, Pr3, and Cat G (protease mix.) before (A) and after incubation with 3.10–8 M final {alpha}1-Pi (B), 6.10–8 M final {alpha}1-Pi (C), and 9.10–8 M final {alpha}1-Pi (D). The incomplete inhibition in D confirms the existence of the substrate pathway. Results are the medians and extremes of at least three experiments.

 
These results confirm that {alpha}1-Pi binds to and inhibits the three serine proteases of human neutrophil primary granules and that it binds much better to HNE, as this enzyme was fully inactivated before the other two were significantly inhibited.

Inhibition of Free HNE, Pr3, and Cat G by Inflammatory BAL Fluids
As {alpha}1-Pi is the major inhibitor in lung lining fluid, HNE should be better controlled than the other two when all are present as active proteases in inflammatory BAL fluids or lung expectorates. However, there are other inhibitors in lung lining fluid that may compete with {alpha}1-Pi for the control of overall serine protease activity. We therefore used BAL supernatants as a source of inhibitors to mimic pathophysiologic conditions. BAL fluids were obtained from patients with pneumonia and ARDS. They were selected so that their cell and protein contents differed greatly, but none had any HNE, Pr3, or cat G activity in its supernatant after centrifugation. Thus inhibitors were in excess in the seven BAL fluids used (Table 1). We checked by Western blotting that most of the {alpha}1-Pi in all BAL fluids was active and could form irreversible, SDS-resistant complexes (Figure 3). Each BAL fluid supernatant was first titrated with purified HNE to determine its total HNE-inhibiting capacity. The total HNE-inhibiting capacity of each BAL fluid supernatant was measured as the amount of HNE inhibited by ml of BAL fluid. It varied from 6.5 nM to 1.71 µM, depending on the BAL fluid used (Table 1). These values were proportional to total {alpha}1-Pi concentrations, when those were high enough to be quantified by a routine immunonephelemetry procedure (not shown). The volume of each BAL fluid was adjusted so that they all inhibited almost totally (90–100%) the activity of a 25 nM (final concentration) solution of HNE. The same BAL fluid volumes were then incubated with Pr3 and with Cat G (25 nM final), and the residual activities of each protease measured. The experiment was repeated using the same BAL fluid volumes but mixing proteases by pairs (HNE-Pr3; HNE-Cat G; Pr3-Cat G) and then all together, so that the total protease molar content in the last experiment was about three times that of the HNE inhibitors in BAL fluids.


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  		TABLE 1. Patient status and bronchoalveolar lavage fluid characteristics

 


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  		Figure 3. Western blotting of seven BAL fluid samples obtained from patients with pneumonia and ARDS, as revealed with anti {alpha}1-Pi antibodies. The major band in all samples corresponds to native {alpha}1-Pi. Upon incubation of BAL fluids with HNE, native {alpha}1-Pi is converted into an ~ 80-kD band corresponding to the irreversible complex. Inset shows a representative result of this conversion (S7).

 
HNE was the preferred target of all the BAL fluids tested when all three proteases competed for BAL fluid inhibitors; the median value was ~ 15% of the initial activity (Figure 4B). Pr3 remained more than 90% active. But about half of the Cat G activity was inhibited (Figure 4B), in contrast to the results obtained using purified {alpha}1-Pi (Figure 4A). Thus Pr3 is the least well controlled by the inhibitors in BAL fluids. We noted that the cumulative inhibition of the three proteases was more than that expected from the HNE-inhibiting capacity determined for each BAL fluid. This reflects the contributions of other BAL fluid inhibitors that do not inhibit HNE. One important one may be ACT, which inhibits Cat G but not the other two.



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  		Figure 4. Inhibition of a mixture of equimolar concentrations of HNE, Pr3, and Cat G by BAL fluids: Competition between the three proteases for binding to purified {alpha}1-Pi (A) and to BAL fluid inhibitors (B); competition between pairs of proteases (C), and individual proteases (D), for binding to BAL fluid inhibitors. The results are the median ± the first quartile of seven BAL samples.

 
HNE remained, as expected, the preferred target for inhibitors when the proteases were added in pairs to BAL fluids (Figure 4C); less than 10% of its activity remained after incubation with equimolar amounts of either Pr3 or Cat G (Figure 4C). This value was not statistically different from that obtained when HNE was added alone (P > 0.05) (Figure 4D). In the absence of HNE, Pr3 was ~ 50% inhibited when added alone (P = 0.001) (Figure 4D) or with Cat G (P = 0.0009) (Figure 4C), which further demonstrates that it competes with HNE for binding to {alpha}1-Pi and possibly to elafin. The result also shows that there is no competition between Pr3 and Cat G (P > 0.05) for binding to BAL fluid inhibitors, confirming the previous observation that Cat G is inhibited by a specific inhibitor and not by {alpha}1-Pi. The inhibition of Cat G by BAL fluids remained the same whether it was added alone or in combination with HNE or Pr3. The apparently lesser Cat G inhibition in the presence of HNE was not statistically significant according to the Kruskal-Wallis test (P = 0.09).

Inhibition of Neutrophil-Bound Proteases by BAL Fluid Supernatants
HNE, Pr3, and Cat G are present at the surface of quiescent and activated neutrophils, where they retain enzymatic activity (21, 24). The membrane-bound proteases in lung inflammatory fluids could be responsible for the proteolytic activity that remains in biological fluids even in the presence of inhibitors (28). Because we recently showed that both free HNE and membrane-bound HNE was inhibited by {alpha}1-Pi (Korkmaz and coworkers, unpublished observations), we measured the extent to which BAL fluids controlled membrane-bound activities using purified blood PMNs.

We first measured the activity of each protease at the surface of freshly prepared blood PMNs using its specific substrate. Membrane-bound proteases were quantified by comparing the rate of hydrolysis of each substrate to that obtained with titrated enzymes (24). The concentrations of the three proteases were in the nanomolar range when using 106 cells/ml. We adjusted the number of cells so that the mHNE concentration was 1 nM, which means that concentration of the other two proteases varied slightly around this value. Working at such low membrane-bound protease concentrations is a prerequisite for accurate measurements of kinetics on whole cells. The BAL fluid volume was adjusted so that it inhibited 10–9 M free HNE, and it was added to the cell suspension and the mixture incubated for 60 min at 25°C. Residual protease activities were then measured (Figure 5). Again, HNE was almost totally inhibited, whereas Cat G was ~ 20% inhibited and Pr3 remained fully active (Figure 5B). With a double volume of BAL fluid, Cat G was ~ 40% inhibited and Pr3 20% inhibited (Figure 5C), and this percentage increased only slightly when three volumes of BAL fluid were used. This was most probably due to experimental conditions that require use of a limited number of cells to measure cell surface activities. This results in too low a rate of association between Pr3 or Cat G and {alpha}1-Pi, for the second order reaction to be complete even after incubation for 60 min, as checked with free Pr3 and Cat G at the same concentration. Alternatively, it could be that Pr3 and Cat G, unlike HNE, are inhibited more slowly by {alpha}1-Pi when they are bound to the neutrophil membrane.



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  		Figure 5. Residual activity of membrane-bound HNE, Pr3, and Cat G before (A) and after incubation with a representative BAL fluid sample (n°7). PMNs (~ 4 x 105 cells in 150 µl reaction mixture) were reacted with a BAL fluid volume necessary to inhibit 10–9 M free HNE (B), then with twice (C) and three times (D) the BAL fluid volume. Results are the median and extremes of three experiments.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
The pathogenesis of inflammatory lung diseases is mainly due to the imbalance between serine proteases from neutrophil primary granules and their natural inhibitors in the lung. Serine proteases from neutrophils are either secreted or remain bound in an active form to the membranes of recruited, activated PMN (1). The concentration of proteases released from activated neutrophils may greatly exceed that of local inhibitors, as found in cystic fibrosis sputum, leading to an obligate proteolysis by uncontrolled proteases. Proteolysis may also occur in milder inflammatory lung diseases, in the immediate environment of activated PMNs even in the presence of active inhibitors (29, 30). This extracellular proteolysis in the presence of inhibitors is favored by adhesion of neutrophils to vascular endothelium or to the ECM and/or to the tight binding of secreted serine proteases to their protein substrates (31). Membrane attachment of serine proteases is also thought to make them more resistant to inhibitors (1, 21). However, this is not supported by our recent data (B. Korkmaz and colleagues, unpublished observations).

Measuring the activity of each protease in biological fluids and at the cell surface has not been possible until recently because of the lack of specific and sensitive substrates. We used the substrates with intramolecularly quenched fluorescence we raised for HNE, Pr3, and Cat G to carry out competition experiments and thus follow their fate when they are present simultaneously in biological fluids containing polyvalent inhibitors that can control their activity. Among these inhibitors, {alpha}1-Pi is quantitatively the most important in BAL fluids of healthy subjects (32–34). Unlike SLPI and elafin, it is also a polyvalent inhibitor that irreversibly binds HNE, Pr3, and Cat G. Though high levels of SLPI have been reported in BAL fluids of patients with ARDS (35), only one-third of total SLPI in respiratory epithelial lining fluid would be functional (36). We have therefore focused our study on the inhibition of the three proteases of PMN primary granules when they compete for binding to {alpha}1-Pi as it occurs in physiopathologic conditions.

Incubating {alpha}1-Pi with a mixture of equimolar concentrations of the three proteases showed that HNE is by far the preferred target of the inhibitor, as it was totally inhibited before the activities of Pr3 and Cat G were significantly altered. Once HNE was saturated, {alpha}1-Pi bound Pr3 faster than Cat G, in agreement with the values of the association rate constants reported for each purified protease interacting with {alpha}1-Pi. Thus, {alpha}1-Pi binds HNE about ten times faster than Pr3 (2, 20) and more than 100 times faster than Cat G (17, 19, 37). Based on these values, however, there should be some Pr3 inhibition when this protease competes with equimolar amounts of HNE. But Pr3, unlike HNE, binds via a two-step mechanism to {alpha}1-Pi that probably favors the binding of HNE to the inhibitor when these proteases compete for inhibitor binding (20).

Because Pr3 and Cat G are almost not inhibited by {alpha}1-Pi as long as active HNE is present in the solution, HNE is likely to be better controlled in lung secretions than the other two proteases, unless the other inhibitors in lung inflammatory fluids efficiently control their activities. We attempted to answer this question using BAL fluids as a source of inhibitors. They were obtained from patients with pneumonia and ARDS, so that their fluid phase contained no residual proteolytic activity but an excess of protease inhibitors. Analysis of the {alpha}1-Pi content of these BAL fluids showed that most of the inhibitor was present in an active form and that a minor part had formed complexes with or had been degraded by proteases released from neutrophils and other inflammatory cells in lung lining fluid.

The experiments with BAL fluids instead of purified {alpha}1-PI showed that HNE remained the preferential target of inhibitors when it was incubated with a mixture of equimolar amounts of HNE, Pr3, and Cat G. This was true for all BAL fluids tested, despite their very different cell counts, total protein, and HNE inhibitor contents. This confirms that {alpha}1-Pi is the main inhibitor in lung secretions and is present mainly in an active form. However, there was some inhibition of Pr3 and of Cat G before HNE was totally inhibited, suggesting a significant contribution from other inhibitors. Our competition experiments show that Cat G alone or in combination with HNE and/or Pr3 is inhibited in the same manner by BAL fluids, which means that it does not compete for binding to HNE or Pr3 inhibitors. ACT does not inhibit HNE or Pr3 but rapidly inhibits Cat G (17, 19, 37), and therefore appears to be a good candidate. However, ACT retains only 15% of its inhibitory function when purified from lung lavages (38). This could be due to a conformation change from an active to an inactive state that occurs within the lung and is important in the pathogenesis of chronic lung bronchitis and emphysema (39). Unlike Cat G, Pr3 competes with HNE for irreversible binding to {alpha}1-Pi both in a purified system and when BAL fluids are the source of inhibitors. Pr3 is significantly inhibited only in the absence of HNE, which means that it could be less rapidly inhibited than HNE when it is released into the lung, and thus cause more proteolytic damage than HNE, unless its activity is controlled by other, so far unidentified, mechanism(s). Unlike HNE, however, Pr3 remains mainly membrane-bound after neutrophil degranulation (10, 40) and both proteases could be differently regulated when bound to membranes. We therefore tested the inhibition of membrane-bound proteases by the inhibitors in BAL fluids from patients with acute lung inflammatory diseases. Again, HNE was the preferred target of BAL fluid inhibitors at the surface of purified neutrophils, even though the experimental conditions, which limited the number of cells analyzed kinetically, did not allow precise investigation of the kinetics of inhibition of Pr3 and Cat G by BAL fluid inhibitors. Nevertheless, we found no inhibition of membrane-bound Pr3 under conditions where free Pr3 was partially inhibited by {alpha}1-Pi.

Thus, HNE is always the preferred target of {alpha}1-Pi the major inhibitor in respiratory epithelial lining fluid, whatever the conditions. It might therefore be the best controlled serine protease from neutrophil primary granules involved in proteolytic damage during lung inflammatory disorders. Lung proteolytic degradation depends on the rate at which these proteases are inhibited once they are released from their intracellular stores. The protease inhibitors at inflammatory sites are the main agents controlling active proteases, but the speed at which they act depends on their concentrations and those of the proteases. This is why protease inhibitors fail to protect substrates in the immediate pericellular zone where the local protease concentration is optimal (29, 41). The local enzyme-inhibitor ratio is the dominant determinant of the size and duration of such events (41). Because Pr3 and Cat G are less rapidly inhibited by the inhibitors in BAL fluids than is HNE, they could play a major role in the pathogenesis of lung tissue injury.


   Acknowledgments
 
The authors thank Catherine Girardin for technical assistance, Joseph Bieth for helpful advice, and Owen Parkes for editing the English text.


   Footnotes
 
This work was supported in France by Vaincre la Mucoviscidose. BK holds a grant from the Fondation pour la Recherche Médicale (FRM).

Conflict of Interest Statement: B.K. has no declared conflicts of interest; P.P. has no declared conflicts of interest; E.H. has no declared conflicts of interest; M.d.M. has no declared conflicts of interest; S.A. has no declared conflicts of interest; and F.G. has no declared conflicts of interest.

Received in original form November 24, 2004

Received in final form March 10, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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