Extracellular metabolites of Pseudomonas aeruginosa produce bronchoconstriction by different mechanisms

Bacterial supernatants (BS) obtained from broth cultures of Pseudomonas aeruginosa cause bronchoconstriction in sheep, suggesting that BS contain proinflammatory metabolites. In this study we investigated the mechanism(s) responsible for this bronchial effect. BS were obtained from 48 h cultures and sterilized by filtration. Sheep (n = 6) were intubated and swallowed an esophageal balloon for the measurement of specific lung resistance (SRL). Aerosols of BS (3 ml total) immediately increased SRL (541%). Neither aerosolized broth (control) nor inhaled endotoxin in excess of that contained in the BS had an effect. BS challenges were repeated on separate occasions except that the sheep were treated 30 min before challenge with the anticholinergic agent atropine (0.2 mg/kg, intravenously); the anti-allergic agent nedocromil (1 mg/kg, aerosol); the histamine H₁ antagonist chlorpheniramine (2 mg/kg); or the bradykinin (BK) B₂ receptor antagonists NPC-567 (5 mg/ml, aerosol) or NPC-17761 (1 mg/ml aerosol). The results showed that greater than 90% protection (p < 0.05) was achieved when the animals were pretreated with atropine, nedocromil sodium, or either of the two BK antagonists, but only 27 ± 21% protection was seen with chlorpheniramine pretreatment. These findings are characteristic of a BK-mediated response. Analysis of bronchoalveolar lavage fluid obtained before and after BS challenge confirmed that i-kinins, but not histamine, increased (p < 0.05) from 61 ± 7 to 304 ± 55 pg/ml. Control (broth) challenges produced no such change. To identify the metabolites involved, we tested the effects of aerosolizing two suspected components of BS, 1-hydroxyphenazine (1-HP) and pyocyanine (PYO) in five sheep. Aerosols of both substances (1 mg/3 ml buffer) increased SRL (PYO, 183 ± 36%; 1-HP, 228 ± 20%; both p < 0.05). NPC-567 completely inhibited the increase in SRL induced by 1-HP (p < 0.05) but not that produced by PYO. Pretreatment with aerosolized catalase (38 mg/3 ml water), the enzyme that degrades hydrogen peroxide, had the opposite effect on the PYO- and 1-HP-induced bronchoconstriction. Catalase failed to block BK-induced bronchoconstriction. Ultraviolet spectra confirmed the presence of 1-HP in the BS. These data suggest that aerosols of phenazine derivatives contained in BS from P. aeruginosa (as well as BS itself) cause bronchoconstriction, but the mechanism of this bronchial response depends on the predominate phenazine metabolite. Our data suggest that the BS used in this study contained 1-HP, which caused bronchoconstriction through BK B₂ receptor stimulation.