Melo Filho A A, Souza M H, Lyerly D M, Cunha F Q, Lima A A, Ribeiro R A
Department of Physiology and Pharmacology, Federal University of Ceará, Fortaleza, Brazil.
Toxicon. 1997 May;35(5):743-52. doi: 10.1016/s0041-0101(96)00172-9.
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.
艰难梭菌是与抗生素相关性结肠炎有关的细菌，它产生两种外毒素，即毒素A（TxA）和毒素B（TxB）。尽管这些毒素对多种哺乳动物细胞类型具有细胞毒性已广为人知，但其涉及的机制尚未完全明确。本研究的目的是检测TxA和TxB对培养的腹膜巨噬细胞的细胞毒性，并研究肿瘤坏死因子-α（TNF-α）和一氧化氮（NO）是否参与这一过程。作为对照，还研究了大肠杆菌脂多糖（LPS）的作用。TxA、TxB和LPS对巨噬细胞单层具有剂量依赖性细胞毒性，其中TxB的毒性最强。所有毒素均刺激巨噬细胞释放TNF-α。TxB在诱导这种反应方面再次最为活跃。释放的TNF-α似乎参与了LPS和TxA的作用，但不参与TxB的作用，因为抗TNF-α单克隆抗体抑制了前两者的细胞毒性，而对后者没有影响。NO不参与TxA和TxB的作用，因为这些毒素即使在存在γ干扰素的情况下也不会在巨噬细胞中诱导这种介质的产生。此外，NO合酶抑制剂L-亚氨基乙基-L-鸟氨酸（L-NIO）不会改变TxA或TxB引起的巨噬细胞死亡。尽管LPS能够诱导产生大量NO，但NO并不介导LPS的细胞毒性，因为L-NIO不影响LPS引起的巨噬细胞死亡程度。因此，TxA和TxB似乎通过不同机制对培养的巨噬细胞发挥细胞毒性作用。TNF-α参与TxA和LPS介导的细胞毒性，但不参与TxB引起的毒性。NO不参与这些毒素中的任何一种的杀伤作用。
