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Interactions of cholera toxin with isolated hepatocytes. Effects of low pH, chloroquine and monensin on toxin internalization, processing and action.

ABSTRACT: The major steps in cholera-toxin action, i.e. binding, internalization, generation of A1 peptide and activation of adenylate cyclase, were examined in isolated hepatocytes. The binding of toxin involves a single class of high-affinity sites (KD congruent to 0.1 nM; Bmax. congruent to 10(7) sites/cell). At 37 degrees C, cell-associated toxin is progressively internalized, as judged by the loss of its accessibility to antibodies against whole toxin, A and B subunits (about 50, 75 and 30% of initially bound toxin after 40 min respectively). Two distinct pathways are involved in this process: endocytosis of the whole toxin, and selective penetration of the A subunit into the plasma membrane. Exposure of hepatocytes to an acidic medium (pH 5) results in a rapid and marked disappearance of the A subunit from the cell surface. Generation of A1 peptide and activation of adenylate cyclase by the toxin occur after a lag phase (10 min at 37 degrees C), and increase with time in a parallel manner up to 2-3% A1 peptide generated; they are unaffected by exposure of cells to an acidic medium. Chloroquine and monensin, which elevate the pH in acidic organelles, inhibit by 2-4-fold both the generation of A1 peptide and the activation of adenylate cyclase. Unexpectedly, these drugs also inhibit the internalization of the toxin. These results suggest that an acidic pH facilitates the penetration of A subunit into the plasma membrane and presumably the endosomal membrane as well, and that endocytosis of cholera toxin is required for generation of A1 peptide and activation of adenylate cyclase.

SUBMITTER: Janicot M

PROVIDER: S-EPMC1149365 | biostudies-other | 1988 Aug

REPOSITORIES: biostudies-other

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Project description:The calcium-binding protein S100A4 has been described to promote pathological inflammation in experimental autoimmune and inflammatory disorders and in allergy and to contribute to antigen presentation and antibody response after parenteral immunization with an alum-adjuvanted antigen. In this study, we extend these findings by demonstrating that mice lacking S100A4 have a defective humoral and cellular immune response to mucosal (sublingual) immunization with a model protein antigen [ovalbumin (OVA)] given together with the strong mucosal adjuvant cholera toxin (CT), and that this impairment is due to defective adjuvant-stimulated antigen presentation by antigen-presenting cells. In comparison to wild-type (WT) mice, mice genetically lacking S100A4 had reduced humoral and cellular immune responses after immunization with OVA plus CT, including a complete lack of detectable germinal center reaction. Further, when stimulated in vitro with OVA plus CT, S100A4-/- dendritic cells (DCs) showed impaired responses in several CT-stimulated immune regulatory molecules including the co-stimulatory molecule CD86, inflammasome-associated caspase-1 and IL-1?. Coculture of OVA-specific OT-II T cells with S100A4-/- DCs that had been pulse incubated with OVA plus CT resulted in impaired OT-II T cell proliferation and reduced production of Th1, Th2, and Th17 cytokines compared to similar cocultures with WT DCs. In accordance with these findings, transfection of WT DCs with S100A4-targeting small interfering RNA (siRNA) but not mock-siRNA resulted in significant reductions in the expression of caspase-1 and IL-1? as well as CD86 in response to CT. Importantly, also engraftment of WT DCs into S100A4-/- mice effectively restored the immune response to immunization in the recipients. In conclusion, our results demonstrate that deficiency in S100A4 has a strong impact on the development of both humoral and cellular immunity after mucosal immunization using CT as adjuvant.

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Interactions of cholera toxin with isolated hepatocytes. Effects of low pH, chloroquine and monensin on toxin internalization, processing and action.

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