Role of tumor necrosis factor and nitric oxide in the cytotoxic effects of Clostridium difficile toxin A and toxin B on macrophages.

Clostridium difficile, the bacterium involved in antibiotic-associated colitis, produces two exotoxins, toxin A (TxA) and toxin B (TxB). Although these toxins are well recognized as being cytotoxic to several mammalian cell types, the mechanisms involved are not fully understood. The aim of the present investigation was to examine the cytotoxicity of TxA and TxB to peritoneal macrophages in culture and to investigate whether tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) are involved in the process. As a control, the effect of E. coli LPS was also investigated. TxA, TxB and LPS were dose-dependently cytotoxic to macrophage monolayers, with TxB being the most potent. All of the toxins stimulated the release of TNF-alpha from macrophages. TxB was again the most active in inducing this response. The TNF-alpha released appears to be involved in the action of LPS and TxA, but not of TxB, since a mAb against TNF-alpha inhibited the cytotoxicity of the former two but had no effect on the latter. NO is not involved in the effects of TxA and TxB since these toxins did not induce the production of this mediator in macrophages, even in the presence of IFN-gamma. In addition, L-imino-ethyl-L-ornithine (L-NIO), a NO synthase inhibitor, did not modify the macrophage death caused by TxA or TxB. Although LPS was able to induce the production of high amounts of NO, NO did not mediate the LPS cytotoxicity since L-NIO did not influence the degree of macrophage death caused by LPS. TxA and TxB therefore appear to exert cytotoxic effects on cultured macrophages by different mechanisms. TNF-alpha is involved in TxA and LPS-mediated cytotoxicity but not in the toxicity caused by TxB. NO is not involved in the killing action of any of these toxins.