Role of Hemolytic and Nonhemolytic Phospholipase C from Pseudomonas aeruginosa for Inflammatory Mediator Release from Human Granulocytes

Background: Pseudomonas aeruginosa phospholipase C (PLC) is a critical component in the pathogenesis of severe P. aeruginosa infections. However, P. aeruginosa can produce a hemolytic (PLC-H) as well as a nonhemolytic (PLC-N) variant, both having a MW of about 77 kD. In the past, studies did not distinguish between both types of PLC with regard to the induction of inflammatory mediators from human cells. Methods: We compared the ability of P. aeruginosa PLC-H and PLC-N to generate leukotriene B4 (by HPLC) and oxygen (O––2) metabolites (luminol-enhanced chemiluminescence), and to release β-glucuronidase and histamine (fluorophotometry from human granulocytes. Therefore, human neutrophilic granulocytes (PMN; 1 × 107) or human peripheral blood mononuclear cells (5 × 106) were treated with purified P. aeruginosa PLC-H (up to 10 units) as well as culture supernatants (cutoff: MW > 50,000) of P. aeruginosa PAO1 capable of producing both PLC-H and PLC-N, and PAO1 mutant strains deficient in the production of either or both phospholipases. Controls were PLC-H from Clostridium perfringens and PLC-N from Bacillus cereus. Results: PLC-H-containing P. aeruginosa culture supernatant, purified P. aeruginosa PLC-H as well as PLC-H from C. perfringens activated human leukocytes for a significant (p P. aeruginosa activated human PMN for a significant increase in the generation of oxygen metabolites (30 ± 5.4 × 103 cpm) and in leukotriene B4 (6.1 ± 2.0 ng), in the release of β-glucuronidase (15.8 ± 1.1%) and of histamine (25.8 ± 6.2%) as compared to the corresponding control values (3 ± 1 × 103 cpm; 0.2 ± 0.1 ng; 5.1 ± 1.0%, 5.1 ± 1.5%). Culture supernatants containing no PLC or only PLC-N, as well as PLC-N from B. cereus, failed to activate or only slightly stimulated human granulocytes for inflammatory mediator release. Conclusion: The data thus provide evidence that P. aeruginosa PLC-H can be a potent inducer of inflammatory mediator release, at least in vitro. Our results therefore contribute to the understanding of the pathophysiological role of P. aeruginosa PLCs.