INTRODUCTION

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Tight regulation of local protease activity is critical to the maintenance of the physiologic function of the lung and other tissue sites (15, 40, 48). Serine proteases such as neutrophil elastase are among the proteases found within the human airway (15, 16, 20, 38, 40). Serine protease activity must be tightly regulated in order to protect local tissue from protease-mediated injury. This is accomplished in vivo through the presence of a group of proteins which specifically inhibit serine protease activity (32, 40, 48). Among the key antiproteases in the airway is ₁-protease inhibitor (₁PI) (32, 40, 48). ₁PI is a 51-kDa protein member of the serpin class of serine protease inhibitors (32, 40, 48). ₁PI forms a nearly irreversible enzymatically inactive complex with various serine proteases, including human neutrophil elastase (HNE), porcine pancreatic elastase (PPE), and trypsin (29, 40, 48). The active site of ₁PI provides a putative cleavage site for the target enzymes, and it is the methionine 358 within that location which is key for both the protease specificity and inhibitory activity of ₁PI. Modifications of this methionine markedly decrease the inhibitory activity of ₁PI (29, 40, 48).

A number of laboratories have shown that exposure of ₁PI to various oxidant sources, including activated phagocytes (6, 14, 28, 34, 39, 50) and cigarette smoke (17, 41), results in rapid inactivation of the protein due to oxidation of methionine 358 (31). Since the hereditary decrease in ₁PI activity leads to early-onset emphysema (10), it has been postulated that oxidant-mediated inactivation of ₁PI, resulting in decreased local regulation of serine protease activity, is an important contributor to the pathophysiology of cigarette-associated chronic obstructive lung disease (6, 30).

An imbalance of protease-antiprotease activity is also detected in the airways of patients with cystic fibrosis-associated lung disease (5, 16, 18, 19, 35). Most of this elevated protease activity is due to an increase in HNE activity (23, 43, 45). Studies indicate that the untoward protease imbalance is a result of both elevated amounts of HNE and the presence of functionally inactive ₁PI (1, 2, 5, 35, 38, 44). The reason for the presence of inactive ₁PI is unclear.

The onset of progressive lung disease in cystic fibrosis usually coincides with the development of persistent colonization and infection of the airway with Pseudomonas aeruginosa. P. aeruginosa secretes a number of potentially cytotoxic factors, including the phenazine derivative pyocyanin (21, 24, 25, 51). Under aerobic conditions, exposure of pyocyanin to NADH or other cell-derived reducing equivalents, leads to redox cycling of the compound with resultant formation of superoxide (O₂^·) and hydrogen peroxide (H₂O₂) (4, 11, 22, 24, 25). Given the ability of pyocyanin to induce oxidant production, we hypothesized that this P. aeruginosa-derived product could contribute to the pathogenesis of the protease-mediated component of cystic fibrosis lung disease by serving as an additional source of oxidant-mediated inactivation of ₁PI. The in vitro work reported herein supports the ability of redox cycling of pyocyanin to inactivate ₁PI. However, the mechanism may not directly involve oxidant production.

	MATERIALS AND METHODS

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Reagents. NADH, CuZn superoxide dismutase (SOD), trypsin, porcine pancreatic elastase (PPE), H₂O₂, methionine, histidine, catalase, and dimethylthiourea (DMTU) were obtained from Sigma Chemical Co., St. Louis, Mo. ₁PI was purchased from Calbiochem, La Jolla, Calif. The PPE preparations contain chymotrypsin-trypsin (25 to 100 U of trypsin activity/mg of protein) as an impurity. 5,5 Dimethyl-N-pyrroline 1-oxide (DMPO) was purchased from the Oklahoma Medical Research Foundation, Oklahoma City, Okla.

Ability of ₁PI to form a complex with serine proteases. ₁PI (10 µg/ml) in H₂O was incubated with either PPE (50 to 200 µg/ml) or trypsin (50 to 200 µg/ml) for 15 to 30 min at 37°C. Aliquots of the reaction mixture were then added to Laemmli solubilizing buffer and subjected to standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results were similar regardless of whether or not the gels were run under reducing conditions. The ₁PI-protease complex is well known to remain associated under these conditions (7, 31, 46). Detection of an ₁PI-protease complex was assessed by either (i) staining the gel with silver and determining the presence or absence of a new band with an apparent molecular weight consistent with a complex of ₁PI and the protease; or (ii) transfer of the SDS-PAGE-separated sample to nitrocellulose. The nitrocellulose was then blocked with 4% bovine serum albumin-Tris-buffered saline for 1 h and then incubated overnight with a 1:100 dilution of rabbit anti-human ₁PI (Sigma). The blots were then washed three times, and immunoreactive protein was determined with antirabbit immunoglobulin G linked to alkaline phosphatase. The ₁PI-protease complex was manifested as the presence of an ₁PI immunoreactive band with an apparent molecular weight higher than that of an ₁PI standard.

₁PI activity. ₁PI activity was measured in terms of ₁PI's ability to inhibit PPE-mediated cleavage of succinyl-L-alanyl-L-alanyl-L-alanyl-p-nitroanilide (Sigma) according to the spectrophotometric assay of Travis and Johnson (47).

Cell culture of human lung epithelial cells. The A549 human lung carcinoma cell line which resembles type II alveolar epithelial cells was maintained in continuous culture with Dulbecco's modification of Eagle's medium (DMEM) obtained through the University of Iowa Cancer Center, Iowa City. The culture medium was routinely supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin-streptomycin, and 1% glutamine. HBE cells were cultivated in a similar manner, except that the primary culture medium was 50% DMEM and 50% HAM (hepatocyte medium F12; University of Iowa Cancer Center). For each set of experiments, cells from the stock culture were seeded (2.5 × 10⁴ to 5 × 10⁴ cells/well) into 24- or 48-well plates. Cells were maintained at 37°C in 5% CO₂ until they were at least 80% confluent (usually 72 h), at which time they were utilized in the desired experiments.

Purification of pyocyanin. Pyocyanin was extracted from culture supernatant of P. aeruginosa PAO1 (ATCC 15692; American Type Culture Collection, Rockville, Md.) by serial chloroform extractions followed by sequential extractions with acid and neutral water as previously detailed (9). After the completion of five separation sequences, the pyocyanin was crystallized and dried under vacuum. It was resuspended in water and stored at 4°C until used.

Effect of pyocyanin redox cycling on ₁PI-protease complex formation. Experiments were performed in which ₁PI (or, in some cases, the target protease) was exposed to either NADH- or cell-mediated redox cycling of pyocyanin. ₁PI (10 µg/ml) was added either to a solution of NADH (6 mM) in H₂O or to an A549 or HBE cell monolayer in Hanks' balanced salt solution after which the desired concentration of pyocyanin was added. The system was then incubated for 30 min at 37°C. At this point, trypsin or PPE (200 µg/ml) was added for an additional 15 min (37°C). The solutions were assessed for the formation of ₁PI-protease complex as described above.

EPR and spin trapping. For spin trapping experiments, the spin trap DMPO (100 mM) and diethylenetriaminepentaacetic acid (DTPA [0.1 mM]) were included in the reaction mixture of interest. After the desired time of incubation, the reaction mixture was transferred to an electron paramagnetic resonance (EPR) quartz flat cell and placed into the cavity of the EPR spectrometer (ES 300; Brüker, Karlsrühe, Germany). EPR spectra were then obtained at 25°C with the following parameters: 4.00 × 10⁹ gain, 335.544-s sweep time, 100-kHz modulation frequency, 0.501-G modulation amplitude, 80-G sweep width, 9.76-GHz frequency, and 20 mW of power. For detection of the pyocyanin radical, 40 µM pyocyanin was added to a 44 µM solution of NADH and 0.1 mM DTPA, which had been sparged with N₂ for 20 min. The pyocyanin and NADH were then allowed to react at 25°C in the continuous presence of N₂ and subjected to EPR spectroscopy with a Varian E-104A EPR spectrometer (Varian Associates, Inc., Palo Alto, Calif.) with a 1-s time constant, 8-min scan time, 2.5 × 10⁴ gain, 100-kHz modulation frequency, and 20 mW of power.

	RESULTS

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Pyocyanin-NADH blocks ₁PI-protease complex formation. Several laboratories, including our own, have shown that addition of NADH to pyocyanin leads to the reduction of pyocyanin to the pyocyanin radical (4, 11, 22, 24-26). Under aerobic conditions, the pyocyanin radical will transfer an electron to O₂, leading to the formation of O₂^· (4, 11, 22, 24-26). Consistent with these earlier data (4, 11, 22, 24-26), when NADH and pyocyanin were incubated under conditions in which O₂ was previously depleted from the system by N₂ bubbling, an EPR spectrum indicative of the pyocyanin radical was detected (data not shown). When the same reaction was performed under aerobic conditions, the pyocyanin radical was no longer seen, but O₂. production was readily detected by DMPO spin trapping (data not shown).

View larger version (88K): [in this window] [in a new window]   FIG. 1.   Immunoblot with antisera to 1PI in which gel samples were comprised of 10 µg of 1PI alone (lane 1); 1PI which had been incubated with trypsin for 30 min at 37°C (lane 2); or 1PI and 200 µg of trypsin along with 6 mM NADH and pyocyanin at a concentration of 12.5 µM (lane 3), 50 µM (lane 4), and 100 µM (lane 5). The arrow designates the location of the complex formed by 1PI and trypsin. The results are representative of 10 experiments.

View larger version (81K): [in this window] [in a new window]   FIG. 2.   Immunoblot with antisera to 1PI. The gel sample in lane 1 consisted of 1PI which had been incubated for 30 min at 37°C with 200 µg of PPE. Lane 2 shows results obtained under the same conditions as lane 1, except that 6 mM NADH and 100 µM pyocyanin were added prior to the addition of PPE to the reaction mixture. The results are representative of five separate experiments.

View larger version (39K): [in this window] [in a new window]   FIG. 3.   Immunoblot with antisera to 1PI in which gel samples were comprised of 10 µg of 1PI plus 200 µg of trypsin which had been incubated for 30 min at 37°C. Lanes 1 to 3 show the results obtained when 1PI was previously exposed to 6 mM NADH and 100 µM pyocyanin alone (lane 1), or with SOD (300 U/ml; lane 2) or catalase (5,000 U/ml; lane 3) added, for 20 min prior to the addition of trypsin to the reaction mixture. Samples in lanes 4 to 6 were obtained under the same conditions as those of lanes 1 to 3, except that the trypsin rather than the 1PI was previously exposed to 6 mM NADH and 100 µM pyocyanin alone (lane 4), or with SOD (300 U/ml; lane 5) or catalase (5,000 U/ml; lane 6) added, for 30 min prior to its interaction with 1PI. The results are representative of three experiments.

Biochemical evidence of NADH-pyocyanin inactivation of ₁PI. As confirmation that exposure to NADH-pyocyanin leads to loss of ₁PI functional activity, the ability of NADH-pyocyanin-treated ₁PI to inhibit PPE enzymatic activity was assessed. ₁PI exposed to NADH-pyocyanin exhibited an impaired ability to inhibit PPE cleavage of succinyl-L-alanyl-L-alanyl-L-alanyl-p-nitroanilide. In the absence of ₁PI, cleavage of succinyl-L-alanyl-L-alanyl-L-alanyl-p-nitroanilide by PPE yielded a change in A₄₁₀ of 0.132 ± 0.012 U/min (mean ± standard error [SE]; n = 10). Addition of ₁PI decreased this to 0.040 ± 0.005 U/min (mean ± SE; n = 10). When ₁PI was first exposed to NADH-pyocyanin for 10 min, its ability to inhibit PPE activity was significantly decreased (P < 0.001 by analysis of variance) because A₄₁₀ was 0.076 ± 0.009 U/min (mean ± SE; n = 10). NADH-pyocyanin had no effect on PPE activity as assessed in this assay (data not shown).

Epithelial cell-mediated redox cycling of pyocyanin inactivates ₁PI. In vivo redox cycling of pyocyanin in the P. aeruginosa-infected airway would likely occur via epithelial cell-mediated reduction of the compound rather via its interaction with extracellular NADH or NADPH. We have previously shown that incubation of monolayers of A549 (3, 12, 37) or HBE cells (12) with pyocyanin results in the formation of O₂^·, as detected by spin trapping. Since initial reduction of pyocyanin would likely occur intracellularly, it was unclear whether cell-mediated redox cycling of pyocyanin could inactivate extracellular ₁PI. In order to assess this, A549 (or HBE) monolayers were incubated with ₁PI (10 µg/ml) ± pyocyanin (10 to 200 µM) for 30 min. PPE or trypsin was then added, and after 15 min of additional incubation, formation of ₁PI-protease complex in the extracellular milieu was assessed by immunoblot analysis. As shown in Fig. 4 and 5, the presence of pyocyanin resulted in a marked decrease in the subsequent ability of ₁PI to form a complex with either trypsin or PPE. This was dependent on the concentration of pyocyanin present. As with NADH-pyocyanin, supernatants from pyocyanin-treated, but not control, epithelial cells often exhibited evidence of ₁PI proteolysis (Fig. 4).

View larger version (57K): [in this window] [in a new window]   FIG. 4.   Immunoblot with antisera to 1PI in which gel samples were comprised of supernatants removed from monolayers of HBE cells 30 min following the addition of 1PI (lane 1), 1PI plus 50 µM pyocyanin (lane 2), and 1PI plus 200 µM pyocyanin (lane 3), which were then mixed with trypsin for 30 min at 37°C. Each sample was subjected to SDS-PAGE and immunoblot analysis as described in Materials and Methods. The results are representative of three experiments.

View larger version (52K): [in this window] [in a new window]   FIG. 5.   Immunoblot with antisera to 1PI in which gel samples were comprised of supernatants removed from monolayers of A549 cells 30 min following the addition of 1PI (lane 2) or 1PI plus 100 µM pyocyanin (lane 3), which were then mixed with trypsin for 30 min at 37°C. The samples were then subjected to SDS-PAGE and immunoblot analysis as described in Materials and Methods. Lane 1 contains only 1PI, which was not incubated with trypsin and is included as a reference. The results are representative of three experiments.

Role of reactive oxygen intermediates in pyocyanin-associated inactivation of ₁PI. The ability of neutrophils to inactivate ₁PI is linked to oxidation of the protein by reactive oxygen species, particularly those arising from the reaction of H₂O₂ and myeloperoxidase (MPO) (6, 14, 34, 52). Accordingly, we postulated that oxidant species resulting from pyocyanin-induced O₂^· formation were responsible for NADH-pyocyanin-mediated inhibition of ₁PI activity. In order to test this, we determined if the presence of SOD, catalase, or the H₂O₂-hydroxyl radical scavenger DMTU blocked NADH-pyocyanin-mediated inactivation of ₁PI. Surprisingly, none of these agents alone nor the combination of SOD and catalase exhibited any ability to block the effect of NADH-pyocyanin (Fig. 3). SOD and catalase are reasonably large proteins, raising the possibility that they were unable to adequately reach the site of ₁PI which required protection. However, we found that methionine, which blocks MPO-mediated inactivation of ₁PI (6) via its ability to spare methionine 358 within the active site of ₁PI (6, 29, 40, 48), did not protect ₁PI from the effects of NADH-pyocyanin (data not shown). In addition, since O₂^· is not generated as a consequence of pyocyanin reduction under anaerobic conditions, we assessed the ability of O₂ depletion on the process by bubbling the experimental system with N₂ prior to the addition of pyocyanin. NADH-pyocyanin retained its ability to inactivate ₁PI under O₂-depleted conditions (Fig. 6). That bubbling of N₂ effectively depleted the system of O₂ was confirmed with the anaerobic indicator resazurin (33).

View larger version (48K): [in this window] [in a new window]   FIG. 6.   Immunoblot with antisera to 1PI in which gel samples were comprised of 10 µg of 1PI alone (lane 1) or 10 µg of 1PI alone plus 200 µg of trypsin (lane 2), which had been incubated for 30 min at 37°C. Lanes 3 and 4 show results obtained under the same conditions as lane 2, except that 6 mM NADH and 100 µM pyocyanin were added prior to the addition of trypsin to the reaction mixture. The incubation in lane 3 was performed under standard aerobic conditions, whereas lane 4 reflects results obtained under O2-depleted conditions in which the reaction mixture was bubbled with N2 for 20 min prior to the addition of pyocyanin. The results are representative of three separate experiments.

·

View larger version (77K): [in this window] [in a new window]   FIG. 7.   Immunoblot with antisera to 1PI in which gel samples were comprised of 10 µg of 1PI alone (lane 1) or 10 µg of 1PI alone plus 200 µg of trypsin (lane 2), which had been incubated for 30 min at 37°C. The reaction in lane 3 was performed under the same conditions as those of lane 2, except that the 1PI had been exposed to 14 mM -mercaptoethanol prior to the addition of the trypsin. Although not shown in this figure, addition of -mercaptoethanol after the completion of the incubation of trypsin and 1PI has no effect on complex formation.

	DISCUSSION

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An elevated ratio of protease to antiprotease activity has been detected in the P. aeruginosa-infected airways of cystic fibrosis patients, and this is felt to contribute to the pathogenesis of cystic fibrosis lung disease (23, 43, 45). This imbalance is likely due to the presence of markedly elevated levels of the serine protease HNE as well as a higher than expected frequency of inactive ₁PI (1, 2, 5, 35, 38, 44). Given the known susceptibility of ₁PI to oxidant-mediated inactivation (6, 14, 17, 34, 41, 52), the ability of the redox-active P. aeruginosa secretory product pyocyanin to inactivate ₁PI was assessed. Such a process could contribute to protease-mediated lung injury in cystic fibrosis, as well as potentially necrotizing acute nosocomial P. aeruginosa. Nearly all strains of P. aeruginosa are capable of producing pyocyanin.

In support of this hypothesis, we found that the reaction of NADH with concentrations of pyocyanin reported to be present in sputum sol of P. aeruginosa-infected cystic fibrosis patients (51), in excess of 100 µM, was able to alter ₁PI such that it was no longer able to bind to and inhibit the enzymatic activity of the serine proteases PPE and trypsin (binding studies only). The concentration of pyocyanin present in the lung in the setting of P. aeruginosa pneumonia in the non-cystic fibrosis-hospitalized patient is not known. Our studies were performed with concentrations of ₁PI and serine proteases very close to the mean values of 12 and 88 µg of ₁PI and human neutrophil elastase, respectively, per ml reported to be present in the sputum of P. aeruginosa-infected cystic fibrosis patients (38). Although we would expect a similar effect of pyocyanin exposure on ₁PI binding to HNE, this was not assessed due to the cost-prohibitive requirement of HNE for such studies. ₁PI binding to all serine proteases occurs via the same basic process (32, 40, 48).

Similar to the results with NADH-pyocyanin, in studies in which human lung epithelial cell monolayers were exposed to pyocyanin, ₁PI was inactivated when the protein was present in the extracellular milieu. Thus, in spite of the intracellular site at which pyocyanin reduction likely occurs, there was sufficient interaction between reduced pyocyanin and/or its oxidant products to inactivate ₁PI. Previous spin trapping studies with endothelial cells support the concept that these pyocyanin-derived products do have access to the extracellular space (4).

Regardless of whether NADH or epithelial cells were employed as the means of reducing pyocyanin, incubation of pyocyanin-treated ₁PI with either trypsin or PPE resulted in the formation of proteolytic degradation products of ₁PI. This suggests that redox cycling of pyocyanin leads to modifications of ₁PI which allow it to be cleaved by the protease rather than resulting in the formation of an irreversible complex between ₁PI and the protease. Oxidant-mediated inactivation of another airway protease inhibitor, secretory leukocyte protease inhibitor (SLPI), also renders it more susceptible to proteolysis (49).

Consistent with our previous work (3, 4, 37) and that of others (4, 8, 11, 22, 24-26, 42), we found by using EPR techniques that the addition of NADH to pyocyanin leads to the initial formation of pyocyanin radical, which in the presence of O₂ transfers that electron to O₂ to form O₂^·. O₂^· production was also observed upon the addition of pyocyanin to human airway epithelial cells. Although not specifically measured, production of O₂^· would lead in turn to the formation of H₂O₂ via the dismutation reaction of O₂^· with itself (36). In view of previous observations that oxidation of methionine 358 at the ₁PI active site is the mechanism whereby MPO-H₂O₂ inactivates ₁PI (29, 40, 48), we suspected a similar process was involved in the inactivation of ₁PI by pyocyanin. However, several pieces of data argue against such a mechanism. Free methionine failed to protect ₁PI from the effect of pyocyanin, in contrast to its reported ability to protect the protein from inactivation by the MPO-H₂O₂ system (6). Neither SOD, catalase, DMTU, nor depletion of O₂ from the system altered the ability of NADH-pyocyanin to inactivate ₁PI. These data argue strongly against a role for pyocyanin-derived O₂^· and/or H₂O₂ in the ability of this P. aeruginosa-derived compound to inactivate ₁PI. Thus, pyocyanin-mediated inactivation of ₁PI inhibition does not likely occur via oxidation of methionine at the ₁PI active site.

These data raise the possibility that a direct interaction between ₁PI and the pyocyanin radical itself, rather than reactive oxidant species generated from the interaction of reduced pyocyanin and O₂, modifies ₁PI such that it loses its ability to bind serine proteases. Under this hypothesis, the pyocyanin radical would directly modify ₁PI by the transfer of an electron (reduction) to an amino acid constituent of the protein resulting in either direct modification of the active site or alternatively a conformational change in the molecule that decreases its ability to irreversibly bind serine proteases. This is consistent with the fact that the pyocyanin radical is predominantly a reducing radical. Supporting this hypothesis, exposure to the reducing agent -mercaptoethanol also resulted in a loss of the ability of ₁PI to bind trypsin. We are unaware of studies investigating the potential for reduction rather than oxidation of ₁PI to decrease its ability to bind serine proteases. What amino acid(s) would be the target of such reduction is difficult to predict. This hypothesis will need to be further examined with previously generated site-specific mutations of ₁PI as well as by molecular analysis of the pyocyanin-modified protein.

Although the exact mechanism remains to be definitively defined, our data are consistent with the potential for pyocyanin present in the P. aeruginosa-infected airway to cause a local inhibition of ₁PI activity, thereby contributing to protease-mediated tissue injury. It is likely that the pathophysiology responsible for the protease-antiprotease imbalance which marks the airway of cystic fibrosis patients is multifactorial. There is an increased burden of HNE due to the large neutrophilic infiltrate that marks this disease state. Oxidants produced by the same neutrophils, particularly those derived from MPO-H₂O₂, also likely serve to inactivate ₁PI. Interestingly, recent data suggest that pyocyanin, through its ability to induce interleukin 8 release from airway epithelial cells, may contribute to this neutrophilic infiltration of the airway (13). Besides ₁PI, the airway contains other antiproteases, such as (SLPI), capable of inhibiting HNE (49). SLPI is also susceptible to oxidant-mediated inactivation (27, 49) due to the presence of a methionine residue at its active site. Whether SLPI can also be inactivated by pyocyanin is unknown and is worthy of investigation.

In summary, the present work demonstrates that, once reduced, pyocyanin can in turn interact with ₁PI in such a way that there is a loss of the protein's ability to form an irreversible complex with and inhibit the enzymatic activity of serine proteases. Although the exact mechanism requires further delineation, occurrence of these events in vivo could contribute to the pathogenesis of serine protease-mediated injury in P. aeruginosa-infected lungs of patients with cystic fibrosis chronic lung disease and/or acute nosocomial P. aeruginosa pneumonia.