INDUCTION OF INFLAMMATORY MEDIATOR RELEASE (12-HYDROXYEICOSATETRAENOIC ACID) FROM HUMAN PLATELETS BY PSEUDOMONAS-AERUGINOSA

The role of platelets in acute and chronic infection has been widely d iscussed in various disease processes. We studied the effects of two l ipolytic enzymes (phospholipase C, lipase) secreted by Pseudomonas aer uginosa strains isolated from cystic fibrosis (CF) patients with regar d to 12-hydroxyeicosatetraenoic acid (12-HETE) generation from human p latelets. Both phospholipase C (PLC) and lipase were secreted into the culture supernatant at the end of the logarithmic growth phase. Indee d, only culture supernatants obtained from the late logarithmic/early stationary phase of CF strains induced the generation of 12-hydroxyeic osatetraenoic acid (12-HETE) (from 15+/-9 to 370+/-98 ng, n = 7). Puri fied P. aeruginosa lipase itself generated only small amounts of 12-HE TE from human platelets with a maximum of 30+/-7 ng/10(8) platelets at the highest concentration tested (20 nkat). A partially purified cult ure supernatant from P. aeruginosa strain PAO1 containing phospholipas e C and lipase, but lacking glycolipid and protease activities, induce d time- and dose-dependently a significant 12-HETE generation from hum an platelets. Maximal 12-HETE generation was observed at the highest e nzyme concentrations tested (PLC: 1.35 nkat, lipase: 3.7 nkat/10(8) pl atelets). To analyze whether lipase exhibits a modulatory role on PLC- induced 12-HETE generation from human platelets we inhibited lipase ac tivity in the P. aeruginosa partially purified culture supernatant by treatment with the lipase-specific inhibitor hexadecylsulfonylfluoride (AMSF) leaving the activity of PLC unaffected (lipase-free culture su pernatant). The capacity of lipase-free culture supernatant to induce 12-HETE generation was diminished by up to 100% depending on the PLC a ctivity. In parallel, when human platelets were stimulated with purifi ed P. aeruginosa lipase in combination with partially purified culture supernatant or with purified phospholipase C from Clostridium perfrin gens the generation of 12-HETE was further increased up to and fold de pendent on PLC as well as lipase concentrations. Our results suggest t hat the simultaneous secretion of lipase and PLC by P. aeruginosa resi ding in a chronically infected host may result in severe pathological effects which cannot be explained by the sole action of the individual virulence factor on inflammatory effector cells.