This paper is only available as a PDF. To read, Please Download here.
Background. We have found that Clostridium difficile toxins can evoke hepatocyte acute-phase protein synthesis, and that this effect is dependent on a functioning interleukin-1 (IL-1) receptor. The present study was undertaken to determine if C. difficile toxicity, as determined by actin rearrangement and lactate dehydrogenase (LDH) release, also requires a functioning IL-1 receptor.
Methods. Primary hepatocyte cultures were prepared from normal mice, knockout mice deficient in the IL-1-converting enzyme (ICE), and knockout mice deficient in the IL-1 p80 receptor. Hepatocytes were treated for 24 h with C. difficile culture extract, purified C. difficile toxin A, or purified C. difficile toxin B. The actin cytoskeleton was examined using confocal microscopy, and LDH release was measured by spectrophotometric analysis.
Results. C. difficile culture extract, toxin A, and toxin B induced collapse of the actin cytoskeleton in hepatocytes from normal mice. Hepatocytes from both the ICE-deficient mice and the IL-1 p80 receptor-deficient mice demonstrated similar responses to both toxins. These toxins also induced significant LDH release in a concentration-dependent fashion in the normal hepatocytes and the ICE-deficient hepatocytes. However, no significant increase in LDH release was observed in hepatocytes from IL-1 p80 receptor-deficient mice.
Conclusions. C. difficile toxins induce actin cytoskeletal collapse independent of IL-1 or the IL-1 receptor. In contrast, toxin-stimulated LDH release was dependent on the presence of the IL-1 receptor. Thus, separate pathways appear to mediate toxic effects as manifested by actin rearrangement and LDH release.
To read this article in full you will need to make a payment
Purchase one-time access:Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
One-time access price info
- For academic or personal research use, select 'Academic and Personal'
- For corporate R&D use, select 'Corporate R&D Professionals'
Subscribe:Subscribe to Journal of Surgical Research
Already a print subscriber? Claim online access
Already an online subscriber? Sign in
Register: Create an account
Institutional Access: Sign in to ScienceDirect
- Antibiotic-associated pseudomembranous colitis due to toxin-producing clostridia.N. Engl. J. Med. 1978; 298: 531
- Comparison of two toxins produced by Clostridium difficile.Infect. Immun. 1981; 34: 1036
- Biological activities of toxins A and B of Clostridium difficile.Infect. Immun. 1982; 35: 1147
- Purification and properties of toxins A and B of Clostridium difficile.FEMS Microbiol. Lett. 1986; 33: 31
- Protection against experimental pseudomembranous colitis in gnotobiotic mice by use of monoclonal antibodies against Clostridium toxin A.Infect. Immun. 1991; 59: 1192
- Epidermal growth factor attenuates Clostridium difficile toxin A- and B-induced damage of human colonic mucosa.Am. J. Physiol. 1997; 273: G1014
- Nontoxigenic strains of Clostridium difficile lack the genes for both toxin A and toxin B.J. Clin. Microbiol. 1991; 29: 2666
- Molecular characterization of the Clostridium difficile toxin A gene.Infect. Immun. 1990; 58: 480
- Sensitivity in culture of epithelial cells from rhesus monkey kidney and human colon carcinoma to toxins A and B from Clostridium difficile.Toxicon. 1992; 30: 419
- Effects of Clostridium difficile toxins A and B in rabbit small and large intestine in vivo and on cultured cells in vitro.Infect. Immun. 1988; 56: 582
- Effects of Clostridium difficile toxins given intragastrically to animals.Infect. Immun. 1985; 47: 349
- Toxins A and B from Clostridium difficile differ with respect to enzymatic potencies, cellular substrate specificities, and surface binding to cultured cells.J. Clin. Invest. 1997; 100: 1734
- Clostridium difficile toxin B is more potent than toxin A in damaging human colonic epithelium in vitro.J. Clin. Invest. 1995; 95: 2004
- Biochemical studies of the effect of Clostridium difficile toxin B on actin in vivo and in vitro.Infect. Immun. 1987; 55: 1610
- Mechanism of action of Clostridium difficile toxin B: Role of external medium and cytoskeletal organization in intoxicated cells.Eur. J. Cell Biol. 1989; 48: 191
- Microfilament-disrupting Clostridium difficile toxin B causes multinucleation of transformed cells but does not block capping of membrane Ig.Eur. J. Cell Biol. 1990; 53: 357
- Clostridium difficile toxin A perturbs cytoskeletal structure and tight junction permeability of cultured human intestinal epithelial monolayers.J. Clin. Invest. 1988; 82: 1516
- Clostridium difficile toxin B activates calcium influx required for actin disassembly during cytotoxicity.Am. J. Physiol. 1995; 268: G487
- Role of actin polymerization and adhesion to extracellular matrix in Rac- and Rho-induced cytoskeletal reorganization.J. Cell Biol. 1992; 138: 913
- The enterotoxin from Clostridium difficile (ToxA) monoglycosylates the Rho proteins.J. Biol. Chem. 1995; 270: 13932
- Glucosylation of Rho proteins by Clostridium difficile toxin B.Nature. 1995; 375: 500
- Clostridium difficile toxins A and B are cation-dependent UDP-glucose hydrolases with differing catalytic activities.J. Biol. Chem. 1998; 273: 16021
- UDP-glucose deficiency in a mutant cell line protects against glucosyltransferase toxins from Clostridium difficile and Clostridium sordellii.J. Biol. Chem. 1995; 271: 6925
- Clostridium difficile toxin B acts on the GTP-binding protein Rho.J. Biol. Chem. 1994; 269: 10706
- Involvement of Ras-related Rho proteins in the mechanisms of action of Clostridium difficile toxin A and toxin B.Infect. Immun. 1995; 63: 1421
- In vitro effects of Clostridium difficile toxins on hepatocytes.J. Surg. Res. 1998; 79: 170
- Effects of cytokine antagonists on the hepatic acute-phase response.J. Surg. Res. 1997; 68: 161
- Purification, cloning, expression and biological characterization of an interleukin-1 receptor antagonist protein.Nature. 1990; 344: 633
- Interleukin-1 (IL-1) receptor antagonist binds to the 80 kDa IL-1 receptor but does not initiate IL-1 signal transduction.J. Biol. Chem. 1991; 266: 10331
- An enzyme-release assay for natural cytotoxicity.J. Immunol. Methods. 1983; 64: 313
- A quick and simple method for the quantification of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity.J. Immunol. Methods. 1988; 15: 61
- Mice deficient in IL-1β-converting enzyme are defective in production of mature IL-1β and resistant to endotoxic shock.Cell. 1995; 80: 401
- Altered cytokine export and apoptosis in mice deficient in interleukin-1β converting enzyme.Science. 1995; 267: 2000
- Active interleukin-1 receptor required for maximal progression of acute pancreatitis.Ann. Surg. 1996; 223: 163
- Preparation of isolated rat liver cells.Methods Cell Biol. 1976; 13: 29
- Direct effects of endotoxin on hepatocytes.Arch. Surg. 1988; 123: 340
- Phallotoxin and actin binding assay by fluorescence enhancement.Anal. Biochem. 1992; 200: 199
- Fluorescent phallotoxin, a tool for the visualization of cellular actin.Proc. Natl. Acad. Sci. USA. 1979; 76: 4498
- Nosocomial acquisition of Clostridium difficile infection.N. Engl. J. Med. 1989; 320: 204
- Clostridium difficile colitis in the critically Ill.Dis. Colon Rectum. 1996; 39: 619
- Intestinal secretory factor released by macrophages stimulated with Clostridium difficile toxin A: Role of interleukin 1β.Infect. Immun. 1998; 66: 4910
- Clostridium difficile toxin A induces release of neutrophil chemotactic factors from rat peritoneal macrophages: Role of interleukin-1β, tumor necrosis factor, and leukotrienes.Infect. Immun. 1997; 65: 2740
- Signal transduction pathways and cellular intoxication with Clostridium difficile toxins.J. Cell. Biochem. 1993; 52: 107
- Activation of cellular phospholipase A2 by Clostridium difficile toxin B.J. Cell. Biochem. 1993; 52: 116
- Fecal lactoferrin, interleukin-1β, and interleukin-8 are elevated in patients with severe Clostridium difficile colitis.Clin. Diagn. Lab. Immun. 1997; 4: 719
- Rabbit sucrase–isomaltase contains a functional intestinal receptor for Clostridium difficile toxin A.J. Clin. Invest. 1996; 98: 641
☆Presented at the 23rd Annual Symposium of the Association of Veterans Administration Surgeons, St. Louis, Missouri, May 2–4, 1999.
© 2000 Academic Press. Published by Elsevier Inc. All rights reserved.