The Pseudomonas aeruginosa secretory product pyocyanin inactivates alpha1 protease inhibitor: implications for the pathogenesis of cystic fibrosis lung disease

Abstract

Alpha1 Protease inhibitor (alpha1PI) modulates serine protease activity in the lung. Reactive oxygen species inactivate alpha1PI, and this process has been implicated in the pathogenesis of a variety of forms of lung injury. An imbalance of protease-antiprotease activity is also detected in the airways of patients with cystic fibrosis-associated lung disease who are infected with Pseudomonas aeruginosa. P. aeruginosa secretes pyocyanin, which, through its ability to redox cycle, induces cells to generate reactive oxygen species. We tested the hypothesis that redox cycling of pyocyanin could lead to inactivation of alpha1PI. When alpha1PI was exposed to NADH and pyocyanin, a combination that results in superoxide production, alpha1PI lost its ability to form an inhibitory complex with both porcine pancreatic elastase (PPE) and trypsin. Similarly, addition of pyocyanin to cultures of human airway epithelial cells to which alpha1PI was also added resulted in a loss of the ability of alpha1PI to form a complex with PPE or trypsin. Neither superoxide dismutase, catalase, nor dimethylthiourea nor depletion of the media of O2 to prevent formation of reactive oxygen species blocked pyocyanin-mediated inactivation of alpha1PI. These data raise the possibility that a direct interaction between reduced pyocyanin and alpha1PI is involved in the process. Consistent with this possibility, pretreatment of alpha1PI with the reducing agent beta-mercaptoethanol also inhibited binding of trypsin to alpha1PI. These data suggest that pyocyanin could contribute to lung injury in the P. aeruginosa-infected airway of cystic fibrosis patients by decreasing the ability of alpha1PI to control the local activity of serine proteases.

FIG. 1

Immunoblot with antisera to α₁PI in which gel samples were comprised of 10 μg of α₁PI alone (lane 1); α₁PI which had been incubated with trypsin for 30 min at 37°C (lane 2); or α₁PI 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 α₁PI and trypsin. The results are representative of 10 experiments.

FIG. 2

Immunoblot with antisera to α₁PI. The gel sample in lane 1 consisted of α₁PI 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.

FIG. 3

Immunoblot with antisera to α₁PI in which gel samples were comprised of 10 μg of α₁PI plus 200 μg of trypsin which had been incubated for 30 min at 37°C. Lanes 1 to 3 show the results obtained when α₁PI 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 α₁PI 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 α₁PI. The results are representative of three experiments.

FIG. 4

Immunoblot with antisera to α₁PI in which gel samples were comprised of supernatants removed from monolayers of HBE cells 30 min following the addition of α₁PI (lane 1), α₁PI plus 50 μM pyocyanin (lane 2), and α₁PI 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.

FIG. 5

Immunoblot with antisera to α₁PI in which gel samples were comprised of supernatants removed from monolayers of A549 cells 30 min following the addition of α₁PI (lane 2) or α₁PI 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 α₁PI, which was not incubated with trypsin and is included as a reference. The results are representative of three experiments.

FIG. 6

Immunoblot with antisera to α₁PI in which gel samples were comprised of 10 μg of α₁PI alone (lane 1) or 10 μg of α₁PI 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.

FIG. 7

Immunoblot with antisera to α₁PI in which gel samples were comprised of 10 μg of α₁PI alone (lane 1) or 10 μg of α₁PI 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 α₁PI had been exposed to 14 mM β-mercaptoethanol prior to the addition of the trypsin. Although not shown in this figure, addition of β-mercaptoethanol after the completion of the incubation of trypsin and α₁PI has no effect on complex formation.