Gastroenterology

Gastroenterology

Volume 122, Issue 4, April 2002, Pages 1048-1057
Gastroenterology

Basic Research
Clostridium difficile toxin A triggers human colonocyte IL-8 release via mitochondrial oxygen radical generation,☆☆

https://doi.org/10.1053/gast.2002.32386Get rights and content

Abstract

Background & Aims: Clostridium difficile toxin A causes mitochondrial dysfunction resulting in generation of oxygen radicals and adenosine triphosphate (ATP) depletion. We investigated whether mitochondrial dysfunction is involved in nuclear factor κB (NF-κB) activation and interleukin (IL)-8 release from toxin A–exposed enterocytes. Methods: NF-κB activation and IL-8 release in response to toxin A were correlated with reactive oxygen intermediate (ROI) generation and ATP production in HT-29 monolayers or HT-29 cells exposed to ethidium bromide (EB) to inhibit mitochondrial function. Results: HT-29 cells exposed to EB showed damaged mitochondria and diminished resting levels of ATP. ROI production in EB-treated cells exposed to toxin A for 30 minutes was significantly reduced. Exposure of wild-type HT-29 cells to toxin A resulted in increased oxygen radical generation and IL-8 production (P < 0.01 vs. control) that was inhibited by antioxidant pretreatment. Degradation of IκB was observed within 30 minutes of toxin exposure, before ras homologue (Rho) glucosylation, and was followed by nuclear translocation of NF-κB. Toxin A did not increase IL-8 levels in EB-treated cells, whereas IL-8 release in response to IL-1β was not affected. Conclusions: Our data support an early role for mitochondria-derived ROIs in stimulation of IL-8 release from colonocytes by toxin A. ROI generation is independent of Rho inactivation and involves nuclear translocation of NF-κB before release of IL-8.

GASTROENTEROLOGY 2002;122:1048-1057

Section snippets

Materials and methods

Toxin A was purified to homogeneity from culture supernatants of C. difficile strain 10463 (American Type Culture Collection, Rockville, MD) as previously described.12 Protein concentration was determined by the bicinchoninic acid protein assay reagent method (Pierce Laboratories, Rockford, IL). Monoclonal mouse anti–IκB-α was obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and the anti–NF-κB (p65 subunit) antibody was from Boehringer Mannheim (Mannheim, Germany). The anti–IL-8

Toxin A stimulates IL-8 release from HT-29 colonocytes

Cultured HT-29 colonocytes exposed to 10 nmol/L toxin A released increased amounts of IL-8 compared with untreated monolayers, which was statistically different from control at 3, 8, and 24 hours of toxin exposure (Figure 1A).

. Effect of BHA on toxin A–induced IL-8 production and ROI generation from HT-29 cells. (A) Subconfluent HT-29 monolayers were incubated at 37°C with either medium alone or medium containing toxin A (10 nmol/L) for the indicated time points. In some experiments, cells were

Discussion

We show here that IL-8 release from enterocytes after exposure to toxin A is dependent on an oxidative burst originating from mitochondria and transduced via the IκB–NF-κB pathway. These observations extend our earlier report9 that toxin A localizes to mitochondria of target cells within 5–10 minutes of binding to its plasma membrane receptor and that such binding is followed by a sharp decrease in cellular ATP and a concomitant increase in ROIs. Although most of the cellular effects of C.

Acknowledgements

The authors thank Dr. T. C. O'Brien from the Department of Surgery at Beth Israel Deaconess Medical Center for providing colonic tissues for these experiments.

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  • Cited by (0)

    Address requests for reprints to: J. Thomas Lamont, M.D., Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, Massachusetts 02215. e-mail: [email protected]; fax: (617) 667-2767.

    ☆☆

    Supported by National Institutes of Health grants DK 34583 (to J.T.L.) and DK 47343 (to C.P.).

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