CLOSTRIDIUM-DIFFICILE TOXIN-A INDUCES THE RELEASE OF NEUTROPHIL CHEMOTACTIC FACTORS FROM RAT PERITONEAL-MACROPHAGES - ROLE OF INTERLEUKIN-1-BETA, TUMOR-NECROSIS-FACTOR-ALPHA, AND LEUKOTRIENES

Clostridium difficile produces a potent enterotoxin and cytotoxin, tox ins A and B, respectively, which appear to be responsible for pseudome nbranous colitis and antibiotic-associated diarrhea. In ire present st udy we explored the neutrophil migration evoked bg toxin A in the peri toneal cavities and subcutaneous air pouches of rats and examined the role of macrophages and their inflammatory mediators in this process. Toxin 4 causes a significant dose-dependent neutrophil influx into the peritoneal cavity, with a maximal response at 0.1 mu g/ml and at 4 h. The depletion of macrophages by peritoneal washing prevents the tor;i n A-induced neutrophil migration into the peritoneal cavity. In contra st, an increase in macrophages induced, bg peritoneal injection of thi oglycolate amplifies this toxin effect on neutrophil migration, Furthe rmore, the injection of supernatants from toxin A-stimulated macrophag es into tile rat peritoneal cavity causes significant neutrophil migra tion. Pretreatment of rats with BWA4C, nordihydroguaiaretic acid, mepa crine, or dexamethasone inhibits the neutrophil migration evoked by to xin A in the peritoneal cavities. However, pretreatment with the cyclo oxygenase inhibitor indomethacin or the platelet-activating factor ant agonist BN52021 fails to alter toxin A-induced neutrophil migration. T oxin A was also injected into air pouches of normal rats or rats pretr eated with anti-interleukin-1 beta (anti-IL-1 beta) or anti-tumor necr osis factor alpha (anti-TNF-alpha) antibodies. Anti-TNF-alpha or anti- IL-1 beta antibodies significantly reduce the neutrophil migration ind uced by toxin A. These data suggest that neutrophil migration evoked b y toxin A is in part dependent on macrophage-derived cytokines, such a s TNF-alpha and IL-1 beta, and leukotrienes. These mediators may help to explain the intense inflammatory colitis caused by C. difficile tox in A in an experimental animal model of this disease.