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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 14, 2001 as doi:10.1096/fj.01-0572fje. |
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B activation and downstream events 1
Institute of Medical Microbiology, Johannes Gutenberg-University Mainz, D-55101 Mainz, Germany
2Correspondence: Institute of Medical Microbiology and Hygiene, Obere Zahlbacher Strasse 67, D-55101 Mainz, Germany. E-mail: MattHusmann@web.de or walev{at}mail.uni-mainz.de
SPECIFIC AIMS
We questioned whether transient membrane permeabilization by streptolysin O, which creates very large lesions, will trigger transcriptional events in the affected cells.
PRINCIPAL FINDINGS
Human keratinocyte (HaCaT) and endothelial cells (EC) represent natural targets during streptococcal infections. Therefore, we chose these cells for our study. All experiments were conducted with wild-type SLO and with the inactive SLO mutant N402C, which retains the ability to bind cells but cannot oligomerize to form functional channels. Experiments conducted with this mutant yielded negative results, which are not shown.
1. Recovery from toxin attack occurs only at low toxin doses
Cells were exposed to SLO at concentrations ranging from 10 ng to 1000 ng/ml for 15 min, 37°C. One set of cells was immediately stained with trypan blue; a parallel set was postincubated in toxin-free medium. Cellular ATP was determined in the latter after another 4 h and values were expressed as percent of the nontoxin-treated controls. As shown in Fig. 1
, HaCaT cells exhibited a marked capacity to replenish their ATP content.
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Endothelial cells displayed an analogous behavior but had a distinctly smaller repair capacity. Cell recuperation was limited to low SLO doses. When > 90% of the cells were permeabilized (trypan blue positive), recovery of ATP levels no longer occurred.
2. Activation of NF-
B in SLO-treated cells
EMSAs were conducted using extracts of cells that had been stimulated with tumor necrosis factor 
(TNF-) (HaCaT) or PMA (EC) or reversibly permeabilized with SLO, using an NF-B oligonucleotide probe. Supershift experiments were conducted with antibodies against p65 or with an irrelevant control antibody. Binding of activated p65 to the oligonucleotide probe was detected in EC treated with PMA and in those that had been permeabilized with SLO.
Immunofluorescent staining was used to localize the p65 subunit of NF-B. p65 was located in the cytoplasm of control HaCaT cells and translocated to the nucleus after stimulation with TNF-. When SLO was applied at a dose that effected 
80% trypan blue positivity, p65 was also found to be translocated to the nucleus at 4 h (after repair had been completed) in the majority of cells.
3. Evidence for transcriptional activation of interleukin 6 (IL-6) and IL-8 in SLO-treated HaCaT cells
To assay for SLO-dependent enhancement of steady-state mRNA levels, RT-PCR analyses were performed for IL-6 and IL-8, two important proinflammatory cytokines under the regulation of NF-B. Unstimulated cells yielded essentially negative RT-PCR signals for both cytokines whereas TNF- induced mRNA synthesis of both cytokines, as expected. The toxin doses used ranged from 20 ng/ml (10% trypan blue positivity) to 500 ng/ml (95% positivity). When 95% of the cells were trypan blue positive, recovery of ATP levels was incomplete, but mRNA levels were still enhanced, indicating marked activation on a per cell basis.
That SLO can activate the IL-8 promoter was shown in transient transfection assays with EC. An IL-8-LUC-hybrid construct was induced in SLO-treated cells (1.5±0.036-fold, n=4) whereas luciferase activity in EC transfected with the parental vector was slightly suppressed (0.8±0.04-fold, n=4).
4. Transiently permeabilized cells produce IL-6 and IL-8
Evidence was sought that SLO-stimulated cells produced the two candidate cytokines. We also wished to determine whether paracrine effects invoked, for example, by products released by dead or dying cells might induce cytokine production in bystander cells. This question had not been addressed in any study before. HaCaT cells were exposed to SLO for 1 h at 4°C to allow toxin binding in the absence of pore formation. Supernatants containing residual toxin were removed, and the cells were washed three times and incubated in toxin-free medium at 37°C for 20 min. This led to pore formation as evidenced by trypan blue positivity. Cell supernatants (designated ‘I’) were then removed and applied to naive cells, whereas the permeabilized cells received fresh medium. After 6 h, these supernatants (designated ‘II’) were assayed together with supernatants ‘I’ for IL-6 (Fig. 2
). Results of cytokine determinations in respective supernatants I are shown as shaded columns and those in supernatants II as open columns. Supernatants of cells treated with TNF- served as positive controls.
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Cells stimulated with supernatants of cells permeabilized with SLO displayed essentially no cytokine responses. This was reproduced in two additional experiments with HaCaT and endothelial cells. In contrast, production of both cytokines was observed in supernatants II from the SLO-treated cells. Peak production of IL-6 exceeded the TNF- control manifold. Analogous findings were made when IL-8 was measured (not shown). These results virtually excluded the possibility that cytokine production represented a paracrine phenomenon.
CONCLUSIONS AND SIGNIFICANCE
Nucleated cells can repair a small number of transmembrane pores. This has been shown for complement and perforin lesions, for the relatively small (1–2 nm) -toxin pores, and for the large (
30 nm) SLO pore. The underlying mechanisms are likely as diverse as the pore sizes: -toxin heptamers may be closed by a constriction mechanism; complement pores appear to be shed or endocytosed, whereas SLO pore repair follows yet another Ca2+-dependent pathway that has not been delineated. As shown here, repair occurs spontaneously in the presence of physiological media. That resealed cells truly remain viable has been demonstrated by their ability to endocytose, exocytose, and proliferate. The wide heterogeneity of pore sizes and diversity of repair mechanisms are reflected by significant differences in responses of cells toward the permeabilizing agents.
Previously we observed that NF-B activation occurred in cells after attack by low doses of -toxin. It was of interest to determine whether this response would follow after production and repair of very large lesions such as those created by streptolysin O. The present investigation was of further interest because Kayal et al. recently showed that listeriolysin O production is the key factor responsible for NF-B activation in cells invaded by L. monocytogenes. The listeria model is a paradigm for intracellular infections, and one might accordingly assume that listeriolysin O attacks an intracellular target. No information is available on the status of the plasma membrane permeability barrier during listeria infections.
Our results now indicate that permeabilization of the plasma membrane from the extracellular compartment by streptolysin O can trigger transcriptional activation. One potentially significant deviation from the previous work on listeriolysin was that both IL-6 and IL-8 production occurred whereas an IL-8 response was absent in the invasion experiments.
That NF-B activation and cytokine production might represent a paracrine phenomenon was a possibility that had not been examined in any previous study. It appeared quite conceivable that products released by dead or dying cells might induce transcriptional events in nonpermeabilized neighboring cells. The use of SLO afforded advantages that were exploited in the present study to address this issue. SLO binds at low temperature whereas oligomerization and pore formation are markedly retarded until the temperature is raised. It was thus possible to wash away unbound toxin and then permeabilize the cells by raising the temperature. The effect of supernatants from these permeabilized cells on naive cells could thereby be tested without interference by SLO. The results clearly excluded major paracrine effects, and we consequently conclude that the permeabilized and resealed cells themselves produced the cytokines.
Transcriptional activation, followed by long-term effects thereof, represents an important consequence of transient membrane permeabilization in nucleated cells. Previous calculations indicated that repair of large lesions such as those produced by SLO can occur within a limited range of low toxin concentrations; the repair capacity is in the order of a few lesions per cells. At higher numbers of ‘hits’, the repair capacity will be overrun and cells will die rapidly so that transcriptional activation cannot occur. In the biological situation, the low dose range may gain relevance when adherent bacteria begin to ‘inject’ their toxic products into cells; indeed, viable streptococci were recently shown to provoke IL-6 and IL-8 production in keratinocytes depending on their capacity to produce SLO. Alternatively, critical toxin concentrations may be reached in vivo when toxins diffuse from the sites of production to reach target cells at a distance (Fig. 3
). Future investigations should eventually unravel the complex spectrum of events initiated in cells recovering from transient breakdown of the membrane permeability barrier. These processes are likely of relevance in the context of infection, immunity, and tissue regeneration.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0572fje; to cite this article, use FASEB J. (December 14, 2001) 10.1096/fj.01-0572fje 
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