Clostridium difficile toxin A induces the release of neutrophil chemotactic factors from rat peritoneal macrophages: role of interleukin-1beta, tumor necrosis factor alpha, and leukotrienes.

Clostridium difficile produces a potent enterotoxin and cytotoxin, toxins A and B, respectively, which appear to be responsible for pseudomenbranous colitis and antibiotic-associated diarrhea. In the present study we explored the neutrophil migration evoked by toxin A in the peritoneal cavities and subcutaneous air pouches of rats and examined the role of macrophages and their inflammatory mediators in this process. Toxin A causes a significant dose-dependent neutrophil influx into the peritoneal cavity, with a maximal response at 0.1 microg/ml and at 4 h. The depletion of macrophages by peritoneal washing prevents the toxin A-induced neutrophil migration into the peritoneal cavity. In contrast, an increase in macrophages induced by peritoneal injection of thioglycolate amplifies this toxin effect on neutrophil migration. Furthermore, the injection of supernatants from toxin A-stimulated macrophages into the rat peritoneal cavity causes significant neutrophil migration. Pretreatment of rats with BWA4C, nordihydroguaiaretic acid, mepacrine, or dexamethasone inhibits the neutrophil migration evoked by toxin A in the peritoneal cavities. However, pretreatment with the cyclooxygenase inhibitor indomethacin or the platelet-activating factor antagonist BN52021 fails to alter toxin A-induced neutrophil migration. Toxin A was also injected into air pouches of normal rats or rats pretreated with anti-interleukin-1beta (anti-IL-1beta) or anti-tumor necrosis factor alpha (anti-TNF-alpha) antibodies. Anti-TNF-alpha or anti-IL-1beta antibodies significantly reduce the neutrophil migration induced by toxin A. These data suggest that neutrophil migration evoked by toxin A is in part dependent on macrophage-derived cytokines, such as TNF-alpha and IL-1beta, and leukotrienes. These mediators may help to explain the intense inflammatory colitis caused by C. dificile toxin A in an experimental animal model of this disease.