Basic ResearchClostridium difficile toxin A triggers human colonocyte IL-8 release via mitochondrial oxygen radical generation☆,☆☆
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).
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.
References (33)
- et al.
Early functional effects of Clostridium difficile toxin A on human colonocytes
Gastroenterology
(1997) - et al.
Clostridium difficile toxin A causes early damage to mitochondria in cultured cells
Gastroenterology
(2000) - et al.
Ethidium bromide-induced inhibition of mitochondrial gene transcription suppresses glucose-stimulated insulin release in the mouse pancreatic beta-cell line betaHC9
J Biol Chem
(1998) - et al.
Helicobacter pylori infection activates NF-kappa B in gastric epithelial cells
Gastroenterology
(1997) - et al.
The enterotoxin from Clostridium difficile (ToxA) monoglucosylates the Rho proteins
J Biol Chem
(1995) - et al.
Role of mitochondrial Ca2+ in the oxidative stress-induced dissipation of the mitochondrial membrane potential. Studies in isolated proximal tubular cells using the nephrotoxin 1,2-dichlorovinyl-L-cysteine
J Biol Chem
(1994) - et al.
Release of cytochrome c from heart mitochondria is induced by high Ca2+ and peroxynitrite and is responsible for Ca(2+)-induced inhibition of substrate oxidation
Biochim Biophys Acta
(1999) - et al.
Microbes and microbial toxins: paradigms for microbial-mucosal interactions II. The integrated response of the intestine to Clostridium difficile toxins
Am J Physiol
(2001) - et al.
Clostridium difficile colitis
N Engl J Med
(1994) - et al.
IL-8 release and neutrophil activation by Clostridium difficile toxin-exposed human monocytes
Am J Physiol
(1997)
Roles of intracellular calcium and NF-kappa B in the Clostridium difficile toxin A-induced up-regulation and secretion of IL-8 from human monocytes
J Immunol
p38 MAP kinase activation by Clostridium difficile toxin A mediates monocyte necrosis, IL-8 production, and enteritis
J Clin Invest
Effect of Clostridium difficile toxin A on human intestinal epithelial cells: induction of interleukin 8 production and apoptosis after cell detachment
Gut
Clostridium difficile toxin A stimulates intracellular calcium release and chemotactic response in human granulocytes
J Clin Invest
Bacterial toxins that target Rho proteins
J Clin Invest
Characterization of rabbit ileal receptors for Clostridium difficile toxin A. Evidence for a receptor-coupled G protein
J Clin Invest
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.).