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INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA
Institut National de la Santé et de la Recherche Médicale U618 (Protéases et Vectorisation Pulmonaires) (S.A., A.G., B.K., M.F., P.Di., F.G.) and l'Institut Fédératif de Recherches 135 (S.A., P.Di., F.G.), Université François Rabelais, Tours, France; Institut National de la Santé et de la Recherche Médicale U613, Université de Bretagne Occidentale, Brest, France (P.De.); and Debiopharm S.A., Lausanne, Switzerland (F.S.)
Received February 24, 2006; accepted April 19, 2006.
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Abstract |
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-trypsin inhibitor and designed to control the excess proteolytic activity in the sputum of cystic fibrosis patients. We analyzed its resistance to the proteolysis it is likely to encounter at inflammatory sites in vivo. EPI-hNE4 resisted hydrolysis by neutrophil matrix metalloproteases (MMPs) and serine proteases that are released from activated neutrophils in inflammatory lung secretions, including MMP-8 and MMP-9, and the elastase-related protease 3 and cathepsin G. It also resisted degradation by epithelial lung cell MMP-7 but was broken down by the Pseudomonas aeruginosa metalloelastase pseudolysin, when used in a purified system, but not when this protease competed with equimolar amounts of neutrophil elastase. We also investigated the inhibitory properties of EPI-hNE4 at the surface of purified blood neutrophils and in the sputum of cystic fibrosis patients where neutrophil elastase is in both a soluble and a gel phase. The elastase at the neutrophil surface was fully inhibited by EPI-hNE4 and formed soluble complexes. The elastase in cystic fibrosis sputum supernatants was inhibited by stoichiometric amounts of EPI-hNE4, allowing titration of the protease. But the percentage of inhibition in whole sputum homogenates varied from 50 to 100%, depending on the sample tested. EPI-hNE4 was rapidly cleaved by the digestive protease pepsin in vitro. Therefore, EPI-hNE4 seems to be an elastase inhibitor suitable for use in aerosols to treat patients with cystic fibrosis.
). Neutrophils recruited to inflammatory sites release reactive oxygen species and active serine proteases, one of which, neutrophil elastase (HNE), has generated the largest interest until now. Neutrophils also release the related serine proteases, protease 3 (Pr3) and cathepsin G (cat G), so that the overall proteolytic activity overwhelms the extracellular inhibitors present in the CF lung. The uninhibited HNE and related proteases degrade extracellular matrix molecules (Owen and Campbell, 1995). HNE is also involved in a variety of functions that prolong inflammatory responses, making it an attractive target for anti-inflammatory compounds. The HNE burden stimulates IL-8 production and lung permeability by degrading interendothelial and interepithelial junction proteins, thus fostering the continued recruitment of neutrophils and their transmigration (reviewed in Moraes et al., 2003). HNE is also a potent secretagogue, causing airways to become plugged in CF (Fahy et al., 1992). Lastly, it impairs the function of mucocilia (O'Riordan et al., 1997) and reduces the phagocytosis of apoptotic neutrophils by cleaving the phosphatidyl serine receptor on macrophages (Vandivier et al., 2002), thus exacerbating the defective clearance of cells and mucus.
Several attempts have been made to use natural and recombinant protein inhibitors as antiproteases to treat the inflammation of CF lungs (McElvaney et al., 1991, 1992; Griese et al., 2001). Synthetic antielastase molecules such as acyl-enzyme inhibitors and transition state inhibitors have also been tested (Ohbayashi, 2002), but no satisfactory results have yet been obtained, because of the toxicity and side effects of synthetic inhibitors or the unsuitability of the protein molecules for in vivo use (Konstan and Davis, 2002; Chughtai and O'Riordan, 2004). Nevertheless, most of the inhibitors tested have beneficial effects that have encouraged further studies (Chughtai and O'Riordan, 2004).
EPI-hNE4 (DX-890) is a low molecular mass (6237 Da) inhibitor of HNE that was discovered and patented by Dyax Corp (Cambridge, MA). It was engineered using phage display (Roberts et al., 1992) and is expressed in Pichia pastoris. It is derived from the second Künitz domain of the light chain (bikunin) of the naturally occurring human protease inhibitor, inter--trypsin inhibitor (residues 285-340; Swiss-Prot accession number PO2760), from which it differs at five positions (Becher et al., 2006). Replacement of the critical P1 Arg297 residue by an Ile residue confers a specificity for neutrophil elastase and makes it a potent HNE inhibitor with a Ki value of 5.45 x 10-12 M and a kon value of 8 x 106 M-1 s-1 (Delacourt et al., 2002). It protects the lungs of rats against HNE and CF sputum soluble fraction instilled into the trachea when given intratracheally or i.v. (Delacourt et al., 2002); it also protects the lungs of rats against repeated injuries (Honore et al., 2004).
We showed previously that EPI-hNE4 retains its inhibitory capacity upon nebulization (Grimbert et al., 2003). We have now assayed its resistance to those proteases it will encounter in vivo when given as an aerosol. These tests used serine proteases related to HNE (Pr3 and cat G) and matrix metalloproteases (MMP-8 and MMP-9) secreted by activated neutrophils or released from dead cells at inflammatory sites, MMP-7, the matrix metalloprotease with collagenolytic activity released from epithelial lung cells, and the bacterial metalloprotease pseudolysin secreted by Pseudomonas aeruginosa. We also tested the ability of EPI-hNE4 to inhibit membrane-bound HNE at the surface of purified neutrophils and the HNE in whole CF sputum because sputum elastase is present in both soluble and heterogenous gel phases. Finally, we determined the effect of pepsin on EPI-hNE4, because aerosolized drugs rapidly reach the digestive tract.
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Materials and Methods |
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1-PI, and antichymotrypsin were obtained from Athens Research and Technology (Athens, GA). Cathepsin G (EC 3.4.21.20
[EC]
) was from ICN Pharmaceuticals (Orsay, France). MMP-7 (EC 3.4.24.33
[EC]
), proMMP-8 (EC 3.4.24.34
[EC]
), proMMP-9 (EC 3.4.24.35
[EC]
), and their respective fluorogenic substrates dinitrophenyl-RPLALWRS-OH, dinitrophenyl-PLAYWAR-OH, and dinitrophenyl-PLGMWSR-OH were from Calbiochem (VWR, Strasbourg, France). Pseudolysin (EC 3.4.24.26
[EC]
) was kindly provided by Professor Jean Wallach (Université Claude Bernard, Lyon, France) or was purchased from Calbiochem (VWR). Porcine pepsin (EC 3.4.23.1
[EC]
), bovine trypsin (EC 3.4.21.4
[EC]
), N-chlorosuccinimide, and peroxidase-coupled goat anti-rabbit-IgG were from Sigma (St. Quentin Fallavier, France). Lymphoprep and PBS without calcium and magnesium were from Invitrogen (Paisley, UK). Patients. Sputum samples were collected after informed consent from 12 adult CF patients chronically infected with P. aeruginosa. Approval was obtained from the ethics committee of our institution.
Processing of Sputum Samples. Samples were homogenized in 10 mM PBS, pH 7.4 (3 ml/g sputum) and centrifuged at 10,000g for 10 min at 4°C. The pellets were suspended in PBS, and supernatants were centrifuged for another 10 min at 20,000g for further analysis.
Isolation of Blood Neutrophils. Human neutrophils from healthy volunteers, who gave their informed consent, were purified essentially as described previously (Korkmaz et al., 2005). The neutrophils recovered at the bottom of the Lymphoprep gradient were suspended in 200 µl of PBS. Erythrocytes were lysed by placing them in 5 ml of sterile distilled water for 20 s; osmolarity was restored by adding 5 ml of 2x concentrated PBS containing 8 mM EGTA. The suspension was then centrifuged at 500g for 10 min at 25°C. The neutrophil pellet was washed twice in PBS containing 4 mM EGTA, and neutrophils were suspended in this buffer at approximately 3 x 103 cells/µl and kept at room temperature with gentle stirring. The final pellet suspension was assessed by direct microscopic observation to consist of >99% neutrophils. It was used within a few hours of purification.
HNE Assays. Free HNE activity was measured at 37°C in 50 mM HEPES buffer, pH 7.4, 0.75 M NaCl, 0.05% Igepal CA-630 (v/v) using a fluorescence resonance energy transfer peptide substrate Abz-APEEIMRRQ-EDDnp (Korkmaz et al., 2004). HNE was titrated with 1-PI, the titer of which had been determined using bovine trypsin titrated with p-nitrophenyl-p-guanidinobenzoate. All HNE concentrations reported throughout the text refer to that of the active protease in the reactional mixture. The hydrolysis of the Abz-peptidyl-EDDnp substrate was followed by measuring the fluorescence at 
ex = 320 nm and em = 420 nm in a Hitachi F-2000 spectrofluorometer. The system was standardized using Abz-FR-OH prepared from the total tryptic hydrolysis of an Abz-FR-p-nitroanilide solution; its concentration was determined from the absorbance at 410 nm, assuming 
410 nm = 8800 M-1 cm-1 for p-nitroaniline. The concentrations of Abz-peptidyl-EDDnp substrate were determined by measuring the absorbance at 365 nm using 365 nm = 17,300 M-1 cm-1 for EDDnp. Membrane-bound HNE activity was quantified by comparing the rate of hydrolysis of Abz-APEEIMRRQ-EDDnp with that of titrated HNE in a detergent-free buffer (10 mM PBS, pH 7.4) at 28°C.
Titrated HNE was used to titrate a stock solution (10 mg/ml in 10 mM acetate buffer, pH 4.0) of EPI-hNE4. One to 30 µl of a 100-fold diluted EPI-hNE4 stock solution in 50 mM HEPES buffer, pH 7.4, 150 mM NaCl, were incubated with a constant amount (10-8 M final) of HNE in 300 µl of reaction buffer for 10 min at 37°C. The residual HNE activity was measured by adding 5 µl of substrate (15 µM final). The EPI-hNE4 molar concentration was calculated from the value at the equivalence point (I/E molar ratio = 1).
Inhibition of HNE-Related Neutrophil Serine Proteases by EPI-hNE4. Free Pr3 and cat G were titrated with 1-PI and 1-antichymotrypsin, respectively (Attucci et al., 2002; Korkmaz et al., 2004). Activities were measured in 50 mM HEPES buffer, pH 7.4, 0.75 M NaCl, 0.05% (v/v) Igepal CA-630 for Pr3 and in 50 mM HEPES buffer, pH 7.4, 0.1 M NaCl, 0.01% (v/v) Igepal CA-630 for cat G, using Abz-VADCADQ-EDDnp and Abz-TPFSGQ-EDDnp that are specifically cleaved by Pr3 and cat G, respectively (Attucci et al., 2002; Korkmaz et al., 2004).
Pr3 or cat G (10-8 M final) was incubated for 10 min at 37°C with a range of EPI-hNE4 concentrations up to an inhibitor/protease molar ratio of 100. The residual activity of each protease was then measured. The effects of Pr3 and cat G on the inhibition of HNE by EPI-hNE4 were tested at a subsaturating concentration of EPI-hNE4 ([EPI-hNE4]/[HNE] = 0.75) using equimolar concentrations of the proteases (4.7 x 10-8 M final).
Resistance of EPI-hNE4 to Host and Bacterial Metalloproteases. ProMMP-8 and proMMP-9 were activated (Korkmaz et al., 2004), and the activities of MMP-8 and MMP-9 (10-8 M final) were measured with 20 µM (final) dinitrophenyl-PLAYWAR-OH and dinitrophenyl-PLGMWSR-OH fluorogenic substrates, respectively, in the MMP buffer (50 mM Tricine, pH 7.5, 0.2 M NaCl, 10 mM CaCl2, and 50 µM ZnCl2) at 37°C. MMP-7 activity was measured with 20 µM (final) dinitrophenyl-RPLALWRS-OH in the HNE-Pr3 buffer at 37°C. Pseudolysin activity was measured at a 10-8 M (final) concentration with 15 µM (final) fluorogenic substrate Abz-VADCAPQ-EDDnp in the HNE-Pr3 buffer at 37°C.
MMP-7, MMP-8, MMP-9, and pseudolysin (10-10-10-8 M final) were incubated with 2 x 10-6 M EPI-hNE4, in their respective activity buffers, for 30 min at 37°C. The mixtures were adjusted so that the final concentration of EPI-hNE4 was 2 x 10-8 M and assayed against an equimolar amount of HNE. The residual activity of HNE was then measured.
The possible influence of metalloproteases on EPI-hNE4 binding to HNE was checked by mixing equimolar amounts (10-8 M) of each metalloprotease with HNE and incubating the mix with 10-8 M EPI-hNE4. The residual HNE activity was measured and compared with that of mixtures without metalloprotease.
The sensitivity of the HNE-EPI-hNE4 complex to degradation by each metalloprotease was assayed as follows. Equimolar amounts (2 x 10-6 M) of HNE and EPI-hNE4 were incubated for 10 min at 37°C and then with 10-7 M (final) metalloprotease for 30 min at 37°C. The residual HNE activity was measured.
EPI-hNE4 Treatment with N-Chlorosuccinimide. EPI-hNE4 and 1-PI (control) were treated with N-chlorosuccinimide (Korkmaz et al., 2005). The inhibitory capacities of the treated and native forms of the inhibitors were assayed after incubation with equimolar amounts (10-8 M) of HNE for 10 min at 37°C.
Inhibition of HNE at the Surface of Purified Blood Neutrophils by EPI-hNE4. The inhibition of membrane-bound HNE (mHNE) by EPI-hNE4 was measured by incubating purified blood neutrophils whose number was adjusted so that the concentration of active HNE was 2 x 10-9 M (1-5 x 105 cells), with an equimolar amount of EPI-hNE4. Purified neutrophils or purified proteases (controls) were incubated with 15 µM Abz-APEEIMRRQ-EDDnp in polypropylene microplate wells selected for their low binding properties (Hard-Shell Thin Wall Microplates; MJ Research, Waltham, MA). Fluorescence was recorded as before using a microplate fluorescence reader (Spectra Max Gemini; Molecular Devices, Sunnyvale, CA) under continuous stirring. Inhibition time curves were recorded using equimolar amounts of EPI-hNE4 and membrane-bound HNE (2 x 10-9 M final). Inhibitor and substrate were added simultaneously to the neutrophil suspension.
The fate of mHNE-EPI-hNE4 complexes was analyzed by incubating a 50-fold excess of neutrophils (10-7 M mHNE) with a stoichiometric amount of EPI-hNE4 (Korkmaz et al., 2005) for 5 min at room temperature. The suspension was centrifuged, and the supernatant was analyzed by SDS-polyacrylamide gel electrophoresis under nonreducing conditions with 0.02% (w/v) SDS in the sample buffer. Proteins were then blotted onto nitrocellulose and detected with rabbit polyclonal anti-EPI-hNE4 antibodies (1:1000) and peroxidase-coupled goat anti-rabbit-IgG (1:15,000). Detection was performed with an ECL kit (Amersham Pharmacia Biotech, Orsay, France).
Inhibition of HNE in Sputum Samples by EPI-hNE4. The volumes of whole sputum homogenates and sputum supernatants were adjusted so that the active HNE concentration in the reaction medium was 10-8 M. Samples were incubated with a range of EPI-hNE4 concentrations in PBS for 10 min at 28°C, and the residual HNE activity was assayed.
Interaction of EPI-hNE4 with Pepsin. EPI-hNE4 (1.35 x 10-5 M final) was incubated with 5.6 x 10-6 M porcine pepsin in 100 mM citrate buffer, pH 4.1, at 37°C; aliquots were assayed at various times for their capacity to inhibit HNE: [elastase (E)] = [inhibitor (I)] = 3.6 x 10-8 M. Breakdown products were analyzed by reverse-phase HPLC (Korkmaz et al., 2004).
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; Witko-Sarsat et al., 1999). Because HNE, Pr3, and cat G can compete for substrate and/or inhibitor binding physiologically, we first measured the inhibition of each of these purified proteases by EPI-hNE4 at different inhibitor/protease molar ratios. HNE (10-8 M) was fully inhibited by an equimolar amount of EPI-HNE4, in agreement with the low Ki for this interaction (Delacourt et al., 2002), but Pr3 and cat G were not significantly inhibited in these conditions, even by a 100-fold molar excess of inhibitor (Fig. 1).
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). The neutrophil metalloproteases MMP-8 and MMP-9, the epithelial MMP-7, and the bacterial metalloprotease pseudolysin from P. aeruginosa are among the main proteases that may break down inhibitors in CF sputum. We incubated EPI-hNE4 (2 x 10-6 M) with 10-10 to 10-8 M concentrations of each of these metalloproteases for 30 min at 37°C and measured the residual activity of HNE incubated with equimolar amounts (2 x 10-8 M final) of the treated inhibitor. EPI-hNE4 was totally resistant to all of the proteases except pseudolysin, which degraded approximately 50% of the inhibitor when used at 10-8 M. Hypothesizing that this concentration of pseudolysin can be present in lung secretions, we incubated equimolar amounts of HNE and pseudolysin (10-8 M) before adding the inhibitor. EPI-hNE4 remained fully active under these conditions as a result of its rapid binding to HNE and the resistance of the complex to degradation by the metalloprotease. The same result was obtained using MMP-7, MMP-8, and MMP-9 as competing proteases.
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; Heinzel-Wieland et al., 1991; Schuster et al., 1996), EPI-hNE4 has no Met residue that could alter its inhibiting properties. Nevertheless, we checked its resistance to inactivation by oxidants by comparing its inhibitory properties with those of 1-PI after incubation with N-chlorosuccinimide. EPI-hNE4 remained fully active, whereas 1-PI had lost all its inhibitory capacity. The oxidative environment of lung inflammatory secretions is therefore unlikely to influence EPI-hNE4 activity.
Inhibition of the Membrane-Bound HNE of Blood Neutrophils by EPI-hNE4. Part of the serine proteases released by activated neutrophils at inflammatory sites remains bound to the surface of activated or dying cells (Owen et al., 1995; Campbell et al., 2000). Any anti-inflammatory therapy that uses inhibitors must ensure that the inhibitor reaches and inhibits all active proteases that might degrade components of the extracellular matrix or other target proteins. We measured the inhibition of mHNE by EPI-hNE4 using purified blood neutrophils. Free HNE and mHNE (both at 2 x 10-9 M final) were inhibited at the same rate by stoichiometric amounts of EPI-hNE4 (Fig. 3A). Thus, mHNE is fully accessible to the inhibitor, and there is no other target protease for EPI-hNE4 at the neutrophil surface. We also showed that mHNE did not remain at the cell surface after cells had been incubated with EPI-hNE4. Western blots of cell supernatants obtained after centrifugation of the inhibitor-containing neutrophil suspension (Fig. 3B) show that EPI-hNE4 formed soluble complexes, suggesting that EPI-hNE4 removes the bound protease from the cell surface, as does 1-PI (Korkmaz et al., 2005).
Inhibition of HNE in CF Sputum by EPI-hNE4. The HNE activity in whole sputum homogenates of 12 patients chronically colonized with P. aeruginosa in sputum supernatants and in suspended pellets was measured using Abz-APEEIMRRQ-EDDnp as substrate (Korkmaz et al., 2004). Concentrations were 0.4 to 4.9 x 10-7 M (median 2.3 x 10-7 M), 0.2 to 3.6 x 10-7 M (median 1.3 x 10-7 M), and 0.4 to 4.6 x 10-7 M (median 2.9 x 10-7 M), respectively. The total HNE activity in fractionated sputum was always higher than that in crude sputum, suggesting that sequestered HNE molecules were released from the pellet when it was suspended in buffer. The HNE in the sputum supernatants of all samples was fully inhibited by stoichiometric amounts of EPI-hNE4, demonstrating that the inhibitor has no other target molecule in the sputum soluble fraction (Fig. 4A). However, inhibition in whole sputum varied from 50 to 100%, depending on the sample used (Fig. 4B). This suggests that either EPI-hNE4 was partially inactivated by unidentified component(s) in some samples or that the partitioning of HNE in sputum samples varied, with a part being trapped and resistant to inhibition. This partitioning agrees with the finding that the total HNE activity in the supernatant plus pellet fractions is higher than that of crude homogenate. We also showed that EPI-hNE4 was stable in crude sputum homogenates: excess inhibitor was incubated with a homogenate whose HNE activity had been stoichiometrically inhibited by EPI-hNE4. Any excess EPI-hNE4 was then titrated with purified HNE. EPI-hNE4 remained fully active under these conditions (not shown), indicating that nothing in sputum alters its inhibitory properties and that it probably remains fully active in vivo.
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), its early control can be a key element in CF therapy. However, classic anti-inflammatory molecules, including oral corticosteroids and high-doses of ibuprofen, have adverse effects that limit their use (Konstan and Davis, 2002). Restoration of the protease-antiprotease balance with proteases inhibitors (McElvaney et al., 1991, 1992; Griese et al., 2001) is a promising alternative because the massive influx of activated polymorphonuclear leukocytes into the CF lung dramatically increases the concentrations of extracellular proteases, especially that of HNE (Birrer et al., 1994). These escape control by their natural inhibitors, 1-PI, SLPI and elafin, as the inhibitors are inactivated by proteolysis and oxidation (Suter and Chevallier, 1991; Birrer et al., 1994; Schuster et al., 1996). Natural and recombinant protein inhibitors have been tried in CF patients (McElvaney et al., 1991, 1992; Griese et al., 2001), but none of them fulfills the requirements for routine use in CF therapy. Previous attempts to investigate the potential of EPI-hNE4 as a therapeutic inhibitor have shown that intratracheal or i.v. administration protects the lungs of rats (Delacourt et al., 2002; Honore et al., 2004). EPI-hNE4 also resists the physical constraints of nebulization (Grimbert et al., 2003).
The present study shows that EPI-hNE4 is not degraded by the HNE-related proteases Pr3 and cat G that are present and active in CF sputum. Likewise, MMP-7, MMP-8, and MMP-9, which are all present in significantly above-normal amounts in CF (Dunsmore et al., 1998; Ratjen et al., 2002), do not cleave EPI-hNE4 under the experimental conditions used. However, the bacterial metalloprotease and major antigen of P. aeruginosa, pseudolysin, does degrade EPI-hNE4 when incubated alone with the inhibitor, but not when it competes with an equimolar concentration of HNE. This is probably due to the rapid formation of HNE-inhibitor complexes that are resistant to further degradation by the metalloprotease. In keeping with previous results (Venaille et al., 1998), we measured no significant pseudolysin activity in the sputum of patients with chronic CF, suggesting that the bacterial protease will not interfere with EPI-hNE4 activity in CF sputum. But degraded forms of natural antiproteases are present in CF sputum (Suter and Chevallier, 1991; Birrer et al., 1994). The proteases that are responsible for this degradation have not yet been identified, although in vitro experiments have shown that host and bacterial proteases can cleave 1-PI and SLPI, altering their inhibitory function (Sponer et al., 1991; Desrochers et al., 1992; Sires et al., 1994; Rapala-Kozik et al., 1999).
Drugs that are to be administered as aerosols must resist oxidation at inflammatory sites. Oxidation of the Met-358 at position P1 of the reactive inhibitory loop of 1-PI (Rosenberg et al., 1984) and of the Met-73 at P1' of SLPI (Heinzel-Wieland et al., 1991) and the Met 25 at P1' of elafin (Zani et al., 2004) dramatically impairs their antielastase activities, and oxidized forms of 1-PI and SLPI are more sensitive to proteolysis than are the native inhibitors (Rapala-Kozik et al., 1999). Thus, oxidation and proteolysis may work together to promote local inflammation. EPI-hNE4 remains fully active under oxidative conditions that completely abolish 1-PI activity because it has no Met residues in its primary structure.
The critical feature of any inhibitors to be used in therapy is whether the inhaled inhibitors can inhibit both gel phase-entrapped and membrane-bound proteases, as the proteases in lung secretions are present in the soluble and gel phases. Chan et al. (2003) have shown that HNE in the soluble phase of mucous bronchial secretions of patients with idiopathic bronchiectasis is not present as the free enzyme but as a proteolytically active member of a supramolecular complex that includes heparan sulfates, syndecan-1, and its physiological regulators 1-PI and SLPI. HNE may also be entrapped in neutrophil extracellular traps (Brinkmann et al., 2004) or in complexes with macrophage-derived lipids (Fujita et al., 1999). We found that EPI-hNE4 stoichiometrically inhibits HNE bound to the membranes of purified blood neutrophils, forming soluble stable EPI-hNE4-HNE complexes. This stoichiometric inhibition occurred in some but not all of the crude sputum homogenates, suggesting that other, unidentified, factors are involved in the regulation of proteolytic activity in the CF sputum of some individuals.
Nevertheless, low molecular mass protein inhibitors seem to be better than high Mr inhibitors at reaching proteases in lung secretions. This is emphasized by the observation that SLPI inhibits the HNE in the supramolecular complexes found in the soluble fraction of sputum, whereas 1-PI does not (Chan et al., 2003). Although aerosolized 1-PI, however, significantly reduces the active HNE concentration in the epithelium lining fluid (McElvaney et al., 1991) and overall proteolysis in the bronchoalveolar lavage fluid (Griese et al., 2001) of CF patients, its effect on the progression of lung disease in CF is not yet known. Oxidation-resistant variants [Met-358 
Val of 1-PI (Rosenberg et al., 1984) and of rSLPI (Met-73 Leu and Met-73, -82, -94, -96 Leu) (Heinzel-Wieland et al., 1991)] have also been constructed to counteract the deleterious effect of oxidation, but they have not yet been tried in humans. Synthetic chemical inhibitors could be attractive alternatives for controlling unwanted proteolysis, but most clinical trials have been ended because of their toxicity and side effects (Chughtai and O'Riordan, 2004). Small protein inhibitors should therefore be preferred for CF therapy.
Protease inhibitors for therapeutic use are administered i.v. or as aerosols. Aerosolized inhibitors may be more efficient at reaching pulmonary tissues, provided they resist the physicochemical conditions of nebulization, are well deposited on the epithelium surface of airways, and do not aggravate any pancreatic insufficiency of CF patients when the excess reaches the digestive tract. Recombinant EPI-hNE4 therefore appears to be a promising molecule: it resists proteolysis, oxidation, and the physical constraints imposed by administration as an aerosol. It also inhibits most of the active HNE in whole CF sputum and is rapidly broken down by pepsin, which prevents its accumulation after the repeated administrations required to treat CF.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: CF, cystic fibrosis; HNE, human neutrophil elastase; Pr3, protease 3; cat G, cathepsin G; IL, interleukin; MMP, matrix metalloprotease; 1-PI, 1-protease inhibitor; PBS, phosphate-buffered saline; Abz, ortho-aminobenzoic acid; EDDnp, N-(2,4-dinitrophenyl)ethylenediamine; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; mHNE, membrane-bound HNE; HPLC, high-performance liquid chromatography; SLPI, secretory leukocyte protease inhibitor; EPI-hNE4, depelstat.
Address correspondence to: Dr. Francis Gauthier, INSERM U618 (Protéases et Vectorisation Pulmonaires), Université François Rabelais, 10 Bd Tonnellé, 37032 Tours Cedex, France. E-mail: gauthier{at}univ-tours.fr
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), Pr3 (
), and cat G (
) (each at 10-8 M) were incubated with various amounts of EPI-hNE4 for 10 min at 37°C. Proteases were assayed with 15 µM concentrations of their specific fluorogenic substrate (Abz-APEEIMRRQ-EDDnp, Abz-VADCADQ-EDDnp, and Abz-TPFSGQ-EDDnp, respectively). Data are means ± S.D. of three individual experiments, each performed in duplicate.
