Role of hemolytic and nonhemolytic phospholipase C from Pseudomonas aeruginosa for inflammatory mediator release from human granulocytes.
Role of hemolytic and nonhemolytic phospholipase C from Pseudomonas aeruginosa for inflammatory mediator release from human granulocytes.
复制标题
铜绿假单胞菌溶血性和非溶血性磷脂酶 C 在人粒细胞释放炎症介质中的作用。
DOI：
10.1159/000237441
复制
发表时间：
1997
影响因子：
2.8
通讯作者：
Wolfgang König
中科院分区：
文献类型：
--
作者：
B. König;M. Vasil;Wolfgang König
背景
铜绿假单胞菌磷脂酶C（PLC）是严重铜绿假单胞菌感染发病机制的关键成分。然而，铜绿假单胞菌可以产生溶血性（PLC-H）以及非溶血性（PLC-N）变体，两者都具有约77 kD的MW。在过去，研究没有区分两种类型的PLC在诱导炎症介质从人类细胞。
方法
我们比较了铜绿假单胞菌PLC-H和PLC-N产生白三烯B4（通过HPLC）和氧（O2-）代谢物（鲁米诺增强的化学发光）以及释放β-葡萄糖醛酸酶和组胺（来自人粒细胞的荧光光度法）的能力。因此，人类嗜中性粒细胞（PMN; 1 x 10（7））或人外周血单核细胞（5 × 10（6））用纯化的铜绿假单胞菌PLC-H处理（最多10个单位）以及培养上清液（截止日期：Mff> 50，000）的能够产生PLC-H和PLC-N两者的铜绿假单胞菌PAO 1，和产生任一种或两种磷脂酶缺陷的PAO 1突变株。对照是来自产气荚膜梭菌的PLC-H和来自蜡状芽孢杆菌的PLC-N。
结果
含有PLC-H的铜绿假单胞菌培养物上清液、纯化的铜绿假单胞菌PLC-H以及来自产气单胞菌的PLC-H激活人白细胞，使炎症介质释放显着增加（p < 0.05）。在这方面，纯化的PLC-H（10单位）从铜绿假单胞菌激活人PMN的氧代谢产物的产生显着增加（30 +/- 5.4 x 10（3）cpm）和白三烯B4（6.1 +/- 2.0 ng），β-葡萄糖醛酸酶释放（15.8 +/- 1.1%）和组胺（25.8 +/- 6.2%），与相应对照值（3 +/- 1 x 10（3）cpm; 0.2 +/- 0.1 ng; 5.1 +/-1.0%，5.1 +/- 1.5%）相比。不含PLC或仅含PLC-N以及来自B的PLC-N的培养物上清液。蜡状，未能激活或仅轻微刺激人粒细胞释放炎性介质。
结论
因此，数据提供了证据，铜绿假单胞菌PLC-H可以是炎症介质释放的有效诱导剂，至少在体外。因此，我们的研究结果有助于了解铜绿假单胞菌PLC的病理生理作用。
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.
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.