The importance of receptor binding in the potent immunogenicity of Escherichia coli heat-labile enterotoxin B subunit (EtxB) was tested by comparing its immunogical properties with those of a receptor binding mutant, EtxB(G33D). Subcutaneous immunization of EtxB(G33D) resulted in 160-fold reduction in antibody titer compared with wild-type EtxB, whereas its oral delivery failed to provoke any detectable secretory or serum anti-B subunit responses. Moreover, the two proteins induced strikingly different effects on lymphocyte cultures in vitro. EtxB, in comparison with EtxB(G33D), caused an increase in the proportion of B cells, many ofwhich were activated (CD25+); the complete depletion of CD8+ T cells; an increase in the activation of CD4+ T cells; and an increase in interleukin 2 and a decrease in interferon 'y. These data indicate that EtxB exerts profound effects on immune cells, suggesting that its potent immunogenicity is dependent not only on efficient receptor-mediated uptake, but also on direct receptor-mediated immunomodulation of lymphocyte subsets.
Gangliosides are glycosphingolipids found ubiquitously on the surface of mammalian cells. They contain a ceramide tail that is inserted into the membrane and exposed carbohydrate and sialic acid moieties. The non-toxic B subunit oligomer (EtxB) of Escherichia coli heat-labile enterotoxin (Etx) is a potent immunogen in vivo and has profound modulatory effects on EtxB-primed lymphocytes in vitro, properties which are dependent on its ability to bind to GM1 ganglioside receptors. Here, it is shown that cross-linking GM1 by EtxB causes a differential effect on mature CD4(+) and CD8(+) T cells from lymph node cultures proliferating in response to an unrelated antigen, ovalbumin. Addition of EtxB to such cultures led to the complete depletion of CD8(+) T cells compared with enhanced activation of CD4(+) cells [as measured by expression of CD25 (IL-2Ralpha)]. By contrast, addition of a mutant EtxB, EtxB(G33D), which does not bind to GM1, failed to trigger CD8(+) T cell depletion. When EtxB was added to isolated non-immune CD8(+) lymphocytes rapid (12-18 h) alterations in nuclear morphology and the appearance of sub-G0/G1 levels of DNA were induced; properties which are characteristic of cells undergoing apoptosis. EtxB(G33D) failed to trigger apoptosis, indicating that the induction of the apoptotic signal was dependent on the binding of GM1. These findings provide an insight into the potent immunogenicity and immunomodulatory properties of E. coli enterotoxins as well as heralding a novel method for the selective induction of apoptosis in mature CD8(+) T lymphocytes.
SUMMARYThe B subunits of cholera toxin (CtxB) and Escherichia coli heat-labile enterotoxin ( EtxB) are non-toxic lectins that bind and cross-link a ubiquitous cell glycolipid receptor, ganglioside GM1, and are recognized as potent mucosal and systemic immunogens. Here we examine the role of EtxB receptor occupancy in modulating the activation of B cells, in vitro, in primary lymphocyte cultures containing B and T cells. When 48-hr spleen cell cultures containing EtxB were compared with those in the presence of a non-receptor binding mutant, EtxB(G33D), a marked shift in the ratio of CD4+ T cells5B cells was noted. Evidence suggested that this was the result of either enhanced survival or proliferation of B cells associated with receptor occupancy by EtxB. Investigation revealed that EtxB induced only a minimal increase in proliferation above that of EtxB(G33D), in mixed cell cultures, and failed to induce any cell division of purified B cells or T cells. In contrast, receptor-binding by EtxB markedly up-regulated the expression of major histocompatability complex (MHC ) class II, B7, intracellular adhesion molecule-1 (ICAM-1), CD40 and CD25 on the B-cell surface. These results indicate that the polyclonal eÂects of EtxB on B cells are not associated with wide-scale proliferation, but more likely with maintenance of B-cell survival by activation of molecules essential for B-cell diÂerentiation. The findings also highlight the essential role of GM1-interaction with EtxB in the regulation of lymphocyte responses.
Antigen (Ag) binding to the BCR rapidly initiates two important events: a phosphorylation cascade that results in the production of secondary signaling intermediaries and the internalization of Ag-BCR complexes. Previous studies using anti-BCR antibodies (Ab) have suggested that BCR signaling is an essential requirement for BCR endocytosis and have further implicated lipid rafts as essential platforms for both BCR functions. However, published data from our laboratory indicate that lipid rafts and consequently raft-mediated signaling are dispensable for BCR-mediated internalization of Ag-specific BCR. Therefore, we investigated the relationship between BCR signaling and endocytosis by defining the role of early kinase signaling in the BCRmediated internalization of a model Ag (haptenated protein). The results demonstrate that Src kinases and Syk-mediated BCR signaling are not essential for BCR-mediated Ag internalization. Moreover, by comparing Ag and Ab, it was determined that while both localize to clathrin-coated pits, the internalization of Ab-BCR complexes is more susceptible to inhibition of signaling and highly sensitive to disruption of lipid rafts and the actin cytoskeleton compared to Ag-BCR complexes. Thus, these results demonstrate that the nature of the ligand ultimately determines the functional requirements and relative contribution of lipid rafts and other membrane structures to the internalization of BCR-ligand complexes.
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