König B, Ludwig A, Goebel W, König W
AG Infektabwehr, Ruhr-Universität Bochum, Germany.
Infect Immun. 1994 Oct;62(10):4611-7. doi: 10.1128/iai.62.10.4611-4617.1994.
The Escherichia coli alpha-hemolysin represents a potent stimulus for inflammatory mediator release (O2-, beta-glucuronidase release, and leukotriene generation) from human polymorphonuclear granulocytes, for histamine release from a suspension of human lymphocyte/monocyte basophil cells (LMB), and for serotonin release and 12-hydroxyeicosatetraenoic acid generation from human platelets. In contrast, the E. coli alpha-hemolysin leads to a downregulation of cytokine release (interleukin-1 beta [IL-1 beta], IL-6, and tumor necrosis factor alpha) from human LMB. Recently, it became apparent that the E. coli alpha-hemolysin is composed of several functional structures. We analyzed the role of pore formation, pore stability, and calcium-dependent membrane binding for inflammatory mediator release by using washed bacteria as well as culture supernatants of isogenic recombinant E. coli strains expressing no hemolysin (Hly-), the wild-type hemolysin (Hly+), or hemolysin molecules deficient or modulated in defined functions (pore formation, calcium-dependent membrane binding, or pore stability). In human granulocytes and platelets, mutant hemolysin with enhanced pore stability did not lead to a further increase in induction; mutant hemolysin deficient in pore-forming activity or calcium-dependent membrane binding no longer induced leukotriene B4 generation or beta-glucuronidase release compared with the wild-type hemolysin. Similar results were obtained with regard to histamine release from human LMB. The induction of cytokine release from human LMB differed depending on the type of mutant E. coli alpha-hemolysin. The wild-type hemolysin, the mutant hemolysin with enhanced pore-forming activity, and, to a lesser degree, the mutant hemolysin deficient in pore-forming activity decreased cytokine release (IL-1 beta, IL-6, IL-8, and tumor necrosis factor) compared with untreated cells. In contrast, the mutant hemolysin deficient in calcium-dependent membrane binding led to an increase of up to 50% in cytokine release compared with that by unstimulated cells. Our results indicate that simultaneous expression of the pore-forming and calcium-dependent membrane-binding activities of the hemolysin molecule was necessary to obtain the full cellular inflammatory response pattern observed with the wild-type hemolysin.
大肠杆菌α-溶血素是一种强有力的刺激物，可促使人类多形核粒细胞释放炎症介质（超氧阴离子、β-葡萄糖醛酸酶释放以及白三烯生成），促使人类淋巴细胞/单核细胞嗜碱性粒细胞（LMB）悬液释放组胺，促使人类血小板释放5-羟色胺并生成12-羟基二十碳四烯酸。相比之下，大肠杆菌α-溶血素会导致人类LMB释放的细胞因子（白细胞介素-1β[IL-1β]、IL-6和肿瘤坏死因子α）下调。最近，很明显大肠杆菌α-溶血素由几种功能结构组成。我们通过使用洗涤过的细菌以及不表达溶血素（Hly-）、野生型溶血素（Hly+）或在特定功能（孔形成、钙依赖性膜结合或孔稳定性）方面存在缺陷或受到调节的溶血素分子的同基因重组大肠杆菌菌株的培养上清液，分析了孔形成、孔稳定性和钙依赖性膜结合对炎症介质释放的作用。在人类粒细胞和血小板中，孔稳定性增强的突变型溶血素不会导致诱导作用进一步增加；与野生型溶血素相比，缺乏孔形成活性或钙依赖性膜结合的突变型溶血素不再诱导白三烯B4生成或β-葡萄糖醛酸酶释放。关于从人类LMB释放组胺也获得了类似结果。从人类LMB释放细胞因子的诱导情况因突变型大肠杆菌α-溶血素的类型而异。与未处理的细胞相比，野生型溶血素、孔形成活性增强的突变型溶血素以及在较小程度上缺乏孔形成活性的突变型溶血素会降低细胞因子（IL-1β、IL-6、IL-8和肿瘤坏死因子）的释放。相比之下，与未刺激的细胞相比，缺乏钙依赖性膜结合的突变型溶血素会导致细胞因子释放增加高达50%。我们的结果表明，溶血素分子的孔形成和钙依赖性膜结合活性的同时表达对于获得野生型溶血素所观察到的完整细胞炎症反应模式是必要的。
