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 (O2-) metabolites (luminol-enhanced chemiluminescence), and to release beta-glucuronidase and histamine (fluorophotometry from human granulocytes. Therefore, human neutrophilic granulocytes (PMN; 1 x 10(7)) or human peripheral blood mononuclear cells (5 x 10(6)) were treated with purified P. aeruginosa PLC-H (up to 10 units) as well as culture supernatants (cutoff: MW > 50,000) of P. aeruginosa PAOl capable of producing both PLC-H and PLC-N, and PAOl 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 P. perfringens activated human leukocytes for a significant (p < 0.05) increase in inflammatory mediator release. In this regard, purified PLC-H (10 units) from P. aeruginosa activated human PMN for a significant increase in the generation of oxygen metabolites (30 +/- 5.4 x 10(3) cpm) and in leukotriene B4 (6.1 +/- 2.0 ng), in the release of beta-glucuronidase (15.8 +/- 1.1%) and of histamine (25.8 +/- 6.2%) as compared to the corresponding control values (3 +/- 1 x 10(3) 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.


CONCLUSIONS
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.
König
B
B
Ruhr-Universität Bochum, Deutschland.
Vasil
M L
ML
König
W
W
D001409:Bacillus cereus / Q000201:enzymology; D003016:Clostridium perfringens / Q000201:enzymology; D005966:Glucuronidase / Q000378:metabolism; D006098:Granulocytes / Q000187:drug effects* / Q000378:metabolism*; D006461:Hemolysis; D006636:Histamine Release / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D018836:Inflammation Mediators / Q000502:physiology*; D007975:Leukotriene B4 / Q000378:metabolism; D008163:Luminescent Measurements; D008970:Molecular Weight; D011552:Pseudomonas Infections / Q000209:etiology; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000472:pathogenicity*; D017382:Reactive Oxygen Species / Q000378:metabolism; D010738:Type C Phospholipases / Q000302:isolation & purification / Q000633:toxicity*; D014774:Virulence
D018836:Inflammation Mediators; D017382:Reactive Oxygen Species; D007975:Leukotriene B4; D010738:Type C Phospholipases; D005966:Glucuronidase