Roles of human peripheral blood leukocyte protein kinase C and G proteins in inflammatory mediator release by isogenic Escherichia coli strains

Abstract

The signal transduction pathway (protein kinase C [PKC], calcium influx, and G protein involvement) was studied with isogenic Escherichia coli strains expressing different types of adhesins (MSH+/- MS-Fim+/-, P-MRH+/- P-Fim+/-, and S-MRH+/- S-Fim+/-) or varying only in the expression of E. coli alpha-hemolysin. As target cells, human polymorphonuclear granulocytes (PMN) and a lymphocyte-monocyte-basophil (LMB) cell suspension were used. The alpha-hemolysin-producing (Hly+) strain E. coli K-12(pANN5211) induced calcium influx in a dose-dependent manner in both cell types. No calcium influx was detected after stimulation with the hemolysin-negative (Hly-) E. coli bacteria independent of the type of fimbriae. With Hly+ bacteria, a dose-dependent activation of PKC was observed in both cell types. The Hly- E. coli K-12 induced PKC to a lesser degree, expressing kinetics different from those of E. coli K-12(pANN5211) (Hly+). E. coli MSH+ MS-Fim+ was the most potent activator for PKC. Membrane preparations from leukocytes stimulated with Hly+ E. coli K-12(pANN5211) showed increased binding of [3H]guanylylimidodiphosphate, a nonhydrolyzable GTP analog, and increased GTPase activity compared with leukocytes stimulated with Hly- E. coli K-12. The amounts of GTPase activation and [3H]guanylylimidodiphosphate binding were similar for all Hly- E. coli bacteria in human PMN as well as in human LMB; no activation was obtained for E. coli bacteria without any type of fimbriae. GTP-gamma-S, a nonhydrolyzable GTP analog, inhibited the leukotriene B4 (LTB4) generation from human PMN by Hly- bacteria, unlike E. coli K-12(pANN5211). However, in the presence of NaF, a predominant activator of Gi, LTB4 generation by Hly+ and by Hly- bacteria was significantly enhanced. For LMBs only LTB4 generation by Hly+ bacteria was increased in the presence of GTP-gamma-S. NaF decreased the chemiluminescence induced by all E. coli strains. Our results thus indicate that (i) Hly+ and Hly- bacteria induce the activation of distinct G proteins, e.g., Gi, to different degrees, (ii) LTB4 generation and chemiluminescence response are differently regulated, and (iii) in comparison with PMN, a different signal transduction pathway is activated by E. coli bacteria in LMBs.

Association of RTX toxins with erythrocytes

“…The difference in binding levels between HlyA and LktA or EhxA also argues in favor of a specific interaction for HlyA. Existence of a receptor per se for HlyA is supported by several studies demonstrating cell signalling events triggered by sublytic doses of HlyA on leukocytes (11,12,17). Interestingly, greater specificity in the requirements of HlyA structural domains would be expected than were discernable in these assays if HlyA binds in a specific manner.…”

Pore formation by the Escherichia coli alpha-hemolysin: role for mediator release from human inflammatory cells

“…Thus, the pore formation leading to calcium influx was thought to be the active principle for inflammatory mediator release from human target cells. Previously, we have shown that the E. coli alpha-hemolysin leads to inflammatory mediator release from human leukocytes and platelets via a distinct signal transduction cascade including G proteins, calcium influx, and protein kinase C activation (23,28). Furthermore, it is established that the hydrolysis of the membrane inositol phospholipids represents one major step in the cellular signalling process by the E. coli alpha-hemolysin (18,24,32).…”

Effect of growth factors on Escherichia coli alpha-hemolysin-induced mediator release from human inflammatory cells: involvement of the signal transduction pathway

“…Hemolysin-producing (Hly+) Escherichia coli strains are isolated from patients with extraintestinal infections such as urinary tract infections, bacteremia, and septicemia (15). The pathogenic relevance of the alpha-hemolysin has been proven in several animal as well as in vitro models (3,21,23). After interaction with soluble hemolysin or with hemolysin-producing E. coli bacteria, human neutrophils produce reactive oxygen species, generate the chemotactically active leukotriene B4 (LTB4) and release cytoplasmic and lysosomal enzymes, e.g., P-glucuronidase and lysozyme; human basophils release histamine (23).…”

“…In contrast, E. coli alpha-hemolysin inhibits cytokine (interleukin 6 , tumor necrosis-factor alpha [TNF-cx], and IL-11) release from human monocytes (22). The cellular responses to E. coli alpha-hemolysin are mediated by a complex signal transduction cascade which is dependent on the cell type (21). In this regard, substantial evidence for the fact that protein kinase C and guanine nucleotide-binding proteins (G proteins) play an essential role in the cellular activation process leading to reactive oxygen metabolites and mediators of inflammation such as leukotrienes from human neutrophils was obtained (21,24).…”

“…The cellular responses to E. coli alpha-hemolysin are mediated by a complex signal transduction cascade which is dependent on the cell type (21). In this regard, substantial evidence for the fact that protein kinase C and guanine nucleotide-binding proteins (G proteins) play an essential role in the cellular activation process leading to reactive oxygen metabolites and mediators of inflammation such as leukotrienes from human neutrophils was obtained (21,24). IL-8, a recently described 6to 10-kDa protein known for its neutrophil chemotactic activity, is another potent mediator of host response released during injury and infection (1).…”