EFFECTS OF TOXIN-A FROM CLOSTRIDIUM-DIFFICILE ON MAST-CELL ACTIVATIONAND SURVIVAL

Toxins A and B from Clostridium difficile are the main cause of antibi otic-associated diarrhea and pseudomembranous colitis. They cause flui d accumulation, necrosis, and a strong inflammatory response when inoc ulated in intestinal loops. Since mast cells are a rich source of infl ammatory mediators, abundant in the gut, and known to be involved in C . difficile-induced enteritis, we studied the in vitro effect of toxin A on isolated mast cells, Normal rats sensitized by infection with Ni ppostrongilus brasiliensis were used to isolate peritoneal mast cells (PMC). PMC from naive rats were stimulated with calcium ionophore A231 87 as a model of antigen-independent activation, and PMC from sensitiz ed rats were stimulated with N. brasiliensis antigens to study immunog lobulin E-dependent mast cell activation. After 4 h, toxin A did not i nduce release of nitric oxide or histamine in naive PMC. However, 10 n g of toxin per mi caused a significant release of tumor necrosis facto r alpha (TNF-alpha). In contrast, 1 mu g of toxin per mi inhibited ant igen or A23187-induced histamine release by PMC. Toxin A at 1 mu g/ml for 4 h caused disruption of actin which aggregated in the cytoplasm a nd around the nucleus. After 24 h, chromatin condensation, cytoplasmic blebbing, and apoptotic-like vesicles were observed; DNA fragmentatio n was documented also. These results suggest that mast cells may parti cipate in the initial inflammatory response to C. difficile infection by releasing TNF-alpha upon interaction with toxin A. However, longer exposure to toxin A affects the release of inflammatory mediators, per haps because of the alteration of the cytoskeleton and induction of ap optosis. The impaired functions and survival of mast cells by C. diffi cile toxin A could hamper the capacity of these cells to counteract th e infection, thus prolonging the pathogenic effects of C. difficile to xins.