Formation of transmembrane pores by staphylococcal alpha-toxin can provoke a spectrum of events depending on target cell species and toxin dose, and in certain cases, repair of the lesions has been observed. Here, we report that transcriptional processes are activated as a response of cells to low toxin doses. Exposure of monocytic (THP-1) or epithelial (ECV304) cells to 40 to 160 ng/ml alpha-toxin provoked a drop in cellular ATP level that was followed by secretion of substantial amounts of interleukin-8 (IL-8). Cells transfected with constructs comprising the proximal IL-8 promoter fused to luciferase or to green fluorescent protein cDNA exhibited enhanced reporter gene expression following toxin treatment. Electrophoretic mobility shift and immunofluorescence assays demonstrated that IL-8 secretion was preceded by activation of NF-B. Transfection experiments conducted with p65/p50 double-deficient cells showed that activation of the IL-8 promoter/reporter by toxin was absolutely dependent on NF-B. In contrast, this transcription factor was not required for lesion repair. Attack of cells by low doses of a pore-forming toxin can lead to transcriptional gene activation, which is followed by production of mediators that may contribute to the initiation and propagation of inflammatory lesions.More than 300 publications dealing with pore-forming bacterial toxins have been published during the past decade, the majority focusing on structural aspects and mechanisms of pore formation (3, 6, 7). Biological consequences other than cell lysis have been described, e.g., secretory responses (4, 5), production of lipid mediators (11), interleukin-1␤ (IL-1␤) maturation (27), and programmed cell death (12). Killing of human keratinocytes by alpha-toxin is due to enhanced permeability for monovalent ions (27). Certain cells can repair a limited number of lesions (24). These findings all indicate that cells attacked by pore-forming toxins are not inevitably and instantly paralyzed and that they retain the capacity to mount active responses to membrane damage. In this investigation, we questioned whether cell damage by alpha-toxin might result in the activation and exploitation of transcriptional mechanisms. NF-B transcription factors are involved in the response to many types of stress. Proteins encoded by NF-B-regulated genes are devoted to intercellular signaling, cell adhesion, and other defense-related functions (9, 10). NF-B has also been implicated in the regulation of apoptosis (1,8). Biochemical features of NF-B include constitutive expression in the cytosol, rapid activation, and translocation into the nucleus.We report that alpha-toxin causes rapid activation of NF-B, which leads to the expression of IL-8 in monocytic THP-1 cells and in ECV304 cells. Transcriptional activation was also shown in 3T3 fibroblasts, where it was found that recovery to sublethal toxin attack was not dependent on activation of this transcription factor. MATERIALS AND METHODSWild type alpha-toxin and the nonlytic toxin mutant H35R were prepar...

There is evidence that low-density lipoprotein (LDL) is modified by hydrolytic enzymes, and that the product (E-LDL) induces selective production of interleukin 8 (IL-8) in endothelial cells. Since nuclear factor-kappaB (NF-kappaB) is a major regulator of IL-8 transcription, we studied its activation in endothelial cells treated with E-LDL. Unexpectedly, the modified lipoprotein not only failed to activate NF-kappaB, but completely blocked its activation by tumour necrosis factor-alpha (TNF-alpha) in EA.hy926-cells, as assessed by electrophoretic mobility shift assays and immunofluorescence. Inhibition occurred upstream of NF-kappaB translocation, as inhibitor of NF-kappaB- (IkappaB)-phosphorylation was suppressed by E-LDL. In contrast to NF-kappaB, transcription factor activator protein-1 (AP-1) proved to be activated. Removal of free fatty acids present in E-LDL obliterated both activation of AP-1 and inhibition of NF-kappaB. Chromatin immunoprecipitation revealed that phosphorylated c-jun, but not NF-kappaBp65 bound to the natural IL-8 promoter. Production of endothelial IL-8 and simultaneous modulation of NF-kappaB in response to hydrolyzed LDL might serve to protect the vessel wall and promote silent removal of the insudated lipoprotein.

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