In this study an in vitro model was developed with the aim of investigating the modulatory effect of cholera toxin (CT) and its counterpart the heat labile toxin of Escherichia coli (LT) on TNF-alpha release induced by murine macrophages and primary human monocytes. Previous studies have demonstrated that the enzymatic activity of CT and LT molecules can inhibit TNF-alpha release by macrophages. The results obtained in this study showed that CT and LT are both, in a dose dependent manner, able either to induce or inhibit TNF-alpha release by murine macrophages and primary human monocytes. The results also showed that recombinant B subunits of CT and LT in the absence of their A subunit induce high levels of TNF-alpha release by macrophages and, in addition, increase the level of TNF-alpha release induced by LPS. The ability of both B subunits (CTB and LTB) in inducing TNF-alpha release by macrophages is not related to the level of LPS contamination, since direct measurements of LPS made in the samples employed in this study showed only traces of LPS (3.4 x 10(-8) EU/ml) which is in our system does not induce TNF-alpha release by macrophages. In contrast to the results obtained with the B subunits, incubation of cells with the A subunit of CT (CTA) inhibit TNF-alpha release induced by native CT, native LT, recombinant LTB and LPS. This inhibitory effect must be related to the activity of the A subunit since viability tests performed in terms of metabolic rate demonstrated that high concentrations of CTA are not toxic to the cells. The data presented herein demonstrate that the A subunits of CT and LT have an inhibitory effect on TNF-alpha release in macrophages, whereas their B subunits have a stimulatory effect on TNF-alpha. The results also suggest that the dose dependent bi-modal effect of native CT and native LT on TNF-alpha release by macrophages is a result of the combined effect of their individual A and B subunits.
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