NEUTROPHIL RECRUITMENT IN CLOSTRIDIUM-DIFFICILE TOXIN-A ENTERITIS IN THE RABBIT

Neutrophil infiltration is a prominent feature of Clostridium difficile-associated enteritis and colitis. The aim of this study was to examine the importance of neutrophil recruitment and neutrophil-mediated tissue damage in C. difficile toxin A-induced enteritis. Competitive binding experiments using purified H-3-toxin A demonstrated the presence of a single class of medium affinity receptors on rabbit neutrophils (K-d 7 X 10(-8) M). Pertussis toxin and the nonhydrolyzable GTP analog GTPgamma S both inhibited H-3-toxin A binding (by 56 and 65%, respectively), indicating that the rabbit neutrophil toxin A receptor is G protein linked. Toxin A elicited a dose-dependent (25-200 mu g/ml) stimulation of neutrophil migration in vitro, and this functional effect was also pertussis toxin sensitive (69% inhibition). Treatment of neutrophils with R15.7, a blocking monoclonal antibody to the leukocyte adhesion molecule CD18, inhibited toxin A-stimulated neutrophil migration by 85% in vitro. Pretreatment of rabbits with R15.7 also prevented neutrophil infiltration of toxin A-exposed ileal loops in vivo as determined by histologic examination and by ileal tissue myeloperoxidase levels. Furthermore, R15.7 effected a substantial inhibition of fluid secretion (by 65%), mannitol permeability (by 66%), and histologic damage in toxin A-exposed ileal loops. Anti-CD18 (R15.7) had no inhibitory effect on cholera toxin enterotoxicity. These data demonstrate that C. difficile toxin A is a proinflammatory toxin whose enterotoxic effects are substantially dependent upon neutrophil recruitment.