No abstract

Toxin A of Clostridium difficile causes severe inflammatory enterocolitis in man and animals that appears to be mediated in part by acute inflammatory cells that migrate into the toxin A-exposed mucosa. To determine the direct effects of toxin A on intestinal epithelial permeability and structure in the absence of other modulating factors, we used cultured monolayers of a human intestinal epithelial cell line (T84). A toxin A concentration of 7 X 10-1 ,ug/ml (3 X i0-' M) nearly abolished monolayer transepithelial resistance within 6-8 h. This marked permeability defect occurred while the monolayers were still confluent. Dual sodium-mannitol flux studies localized the permeability defect to the intercellular tight junction. Cytotoxicity assays and morphological evaluation using Nomarski optics and electron microscopy failed to demonstrate any evidence of cell damage at the time the maximum resistance response was observed. Fluorescent staining for F actin, however, revealed a marked decrease in fluorescent intensity in toxin-treated monolayers versus controls. These data show that toxin A can directly affect the barrier function of this model intestinal epithelium and initially does so by selectively enhancing tight junction permeability. Furthermore, cytoskeletal structure is markedly altered over the same time course, although the integrity of individual cells is maintained. Because the cytoskeleton of intestinal epithelial cells is known to be capable of regulating tight junction permeability, we speculate that the above effects of toxin A on epithelial barrier function result from alterations of the cytoskeleton.

Toxin A from Clostridium diffcile mediates acute inflammatory enterocolitis in experimental animals, while cholera toxin causes noninammatory secretory diarrhea. The purpose of this study was to investigate whether an antagonist to the peptide substance P, a constituent of primary sensory neurons known to participate in inflammatory responses, would inhibit toxin A-mediated enteritis in the rat ileum. Pretreatment of rats with CP-96,345 (2.5 mg per kg of body weight), a substance P antagonist, dramatically inhibited fluid secretion (P < 0.01) and mannitol permeability (P < 0.01) in ileal loops exposed to toxin A. The protective effects, which were dose dependent, caused a significant reduction of inflammation in the lamina propria, reduction of the necrosis of intestinal epithelial cells, and complete inhibition of toxin A-mediated release of rat mast cell protease II, a specific product of rat mucosal mast cells. An inactive enantiomer of the substance P antagonist, CP-96,344, had no effect. In contrast, pretreatment with CP-96,345 had no inhibitory effect on the intestinal effects caused by administration of cholera toxin into the ileal loops. From these data, we conclude that the peptide substance P is involved in the secretory and inflammatory effects of toxin A but not of cholera toxin.Toxigenic strains of Clostridium difficile cause the diarrhea and colitis frequently associated with antibiotic use in children and adults (1). Many studies have shown that these effects appear to be mediated primarily by toxin A (2-4), a 308-kDa protein exotoxin (5) that possesses potent enterotoxic and cytotoxic properties (6). In vivo studies using ileal loops indicate that a major effect oftoxin A is to elicit an acute inflammatory response in the lamina propria. Exposure of rabbit and rat intestinal loops to highly purified toxin A resulted in disruption of the villous architecture, congestion and edema of the mucosa, and an intense infiltration with neutrophils (3,4,7,8). These were accompanied by increased secretion of fluid and increased permeability to mannitol and release of the inflammatory mediators leukotrienes B4 and C4, prostaglandin E2, platelet-activating factor (8, 9), histamine (10), and rat mast cell protease II (RMCPII) (8), a specific mucosa mast cell protease (11). The importance of mast cells and neutrophils is underscored by studies in which pretreatment of experimental animals with ketotifen, a drug that inhibits release of mediators from mast cells and neutrophils, or with an antibody directed against the neutrophil adhesion protein CD18 significantly reduced the inflammation and secretion caused by toxin A (8, 12).The mechanism by which toxin A causes its intestinal effects appears to result from direct effects on intestinal epithelial cells (13) Because of data suggesting possible involvement of SP in inflammatory responses, we postulated that SP might be involved in toxin A-mediated diarrhea. The recent development of potent, highly specific nonpeptide SP antagonists provides unique tool...