The cholera toxin B chain (CTB) has been reported to suppress T cell-dependent autoimmune diseases and to potentiate tolerance of the adaptive immune system. We have analyzed the effects of CTB on macrophages in vitro and have found that preincubation with CTB (10 μg/ml) suppresses the proinflammatory reaction to LPS challenge, as demonstrated by suppressed production of TNF-α, IL-6, IL-12(p70), and NO (p < 0.01) in cells of macrophage lines. Pre-exposure to CTB also suppresses LPS-induced TNF-α and IL-12(p70) formation in human PBMC. Both native and recombinant CTB exhibited suppressive activity, which was shared by intact cholera toxin. In cells of the human monocyte line Mono Mac 6, exposure to CTB failed to suppress the production of IL-10 in response to LPS. Control experiments excluded a role of possible contamination of CTB by endotoxin or intact cholera toxin. The suppression of TNF-α production occurred at the level of mRNA formation. Tolerance induction by CTB was dose and time dependent. The suppression of TNF-α and IL-6 production could be counteracted by the addition of Abs to IL-10 and TGF-β. IFN-γ also antagonized the actions of CTB on macrophages. In contrast to desensitization by low doses of LPS, tolerance induction by CTB occurred silently, i.e., in the absence of a measurable proinflammatory response. These findings identify immune-deviating properties of CTB at the level of innate immune cells and may be relevant to the use of CTB in modulating immune-mediated diseases.

Model systems of human type 1 diabetes have revealed an important role of cellular immune reactions involving macrophages and T cells in the destruction of autologous insulin-producing pancreatic β cells. Recently, the cholera toxin B chain (CTB) was found to suppress T cell-dependent autoimmune diseases including autoimmune diabetes of nonobese diabetic mice. Therefore, we tested the hypothesis that CTB exerts much of its immunomodulatory activity by targeting macrophages. These studies are reviewed here. Cells of the human monocyte line Mono Mac 6 were exposed to CTB and subsequently tested for proinflammatory immunoreactivity in response to challenge with endotoxin (LPS from Escherichia coli, 10 ng/ml for 5 h). Incubation of monocytes with CTB (10 μg/ml) suppressed a later proinflammatory response to LPS as demonstrated by suppression of TNFα release from 6.7 ± 0.7 ng/ml in cultures without CTB preexposure to 1.8 ± 1.1 ng/ml in CTB-pretreated cells (p < 0.001). In contrast, the release of IL-10 remained inducible after CTB pretreatment. RT-PCR analysis showed that the suppression of TNFα production occurred at the level of mRNA formation. Control experiments excluded a role of possible contamination of CTB by endotoxin or the intact cholera toxin. Tolerance induction was maximal after 5 h of CTB exposure and persisted for 24 h. The suppressive effect of CTB was dose-dependent and no more recognizable at ≤1 μg/ml. Incubation with IL-10- and TGFβ-neutralizing antibodies during CTB pretreatment prevented tolerization of macrophages. IFNγ (1,200 U/ml) was found to antagonize actions of CTB. In contrast to desensitization by low doses of LPS, tolerance induction by CTB occurred ‘silently’, i.e. in the absence of a measurable proinflammatory response. In view of the potent instructive role of the innate immune system on T cell responses these findings are important in understanding how CTB prevents the development of autoimmune diabetes and improves tolerance to islet autoantigens.

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