Evidence for a role of ganglioside GM1 in antigen presentation: binding enhances presentation of Escherichia coli enterotoxin B subunit (EtxB) to CD4(+) T cells.

Successful antigen presentation by antigen-presenting cells is governed by a number of factors including the efficiency of antigen capture by cell-surface receptors, targeting to compartments of antigen processing, surface expression of MHC II-peptide complexes and presence of co-stimulatory signals. Ganglioside GM1 is an important component of membrane glycosphingolipids, and has been implicated in cell differentiation, apoptosis and signal transduction pathways. Using the B subunit of Escherichia coli enterotoxin (EtxB), a potent immunogen that binds GM1 with high affinity, and a non-binding mutant of EtxB, EtxB(G33D), we demonstrate that GM1 is intimately involved in several aspects of antigen presentation. Thus, GM1-mediated presentation of EtxB by B cells and CD11c(+) dendritic cells (DC) significantly enhanced the proliferation and cytokine expression of EtxB-specific CD4(+) T cells. Investigation regarding potential mechanisms revealed that EtxB binding directly augments the expression of MHC class II on B cells, and fractionation of B cells demonstrated that EtxB binding to GM1 results in rapid internalization and targeting to class II-rich compartments. GM1-mediated uptake of antigens and access to class II compartments in B cells can be exploited to significantly enhance the presentation of ovalbumin-conjugated to EtxB. These results demonstrate that GM1 can play an important role in antigen presentation via the MHC II pathway.

Immune modulation by the cholera-like enterotoxins: from adjuvant to therapeutic.

Cholera toxin and its close relative, Escherichia coli heat-labile enterotoxin, are potent immunogens and mucosal adjuvants. The recent findings that their B subunits can promote tolerance highlights the complexity of their interactions with the immune system. Here, Neil Williams and colleagues review the mechanisms by which these molecules modulate leukocyte populations and seek to explain the paradox.

Modulation of B-cell activation by the B subunit of Escherichia coli enterotoxin: receptor interaction up-regulates MHC class II, B7, CD40, CD25 and ICAM-1.

The 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 cells: B 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 effects 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 differentiation. The findings also highlight the essential role of GM1-interaction with EtxB in the regulation of lymphocyte responses.

Prevention of autoimmune disease due to lymphocyte modulation by the B-subunit of Escherichia coli heat-labile enterotoxin.

We demonstrate that the receptor binding moiety of Escherichia coli heat-labile enterotoxin (EtxB) can completely prevent autoimmune disease in a murine model of arthritis. Injection of male DBA/1 mice at the base of the tail with type II collagen in the presence of complete Freund's adjuvant normally leads to arthritis, as evidenced by inflammatory infiltration and swelling of the joints. A separate injection of EtxB at the same time as collagen challenge prevented leukocyte infiltration, synovial hyperplasia, and degeneration of the articular cartilage and reduced clinical symptoms of disease by 82%. The principle biological property of EtxB is its ability to bind to the ubiquitous cell surface receptor GM1 ganglioside, and to other galactose-containing glycolipids and galactoproteins. The importance of receptor interaction in mediating protection from arthritis was demonstrated by the failure of a non-receptor-binding mutant of EtxB to elicit any protective effect. Analysis of T cell responses to collagen, in cultures of draining lymph node cells, revealed that protection was associated with a marked increase in interleukin 4 production concomitant with a reduction in interferon gamma levels. Furthermore, in protected mice there was a significant reduction in anti-collagen antibody levels as well as an increase in the IgG1/IgG2a ratio. These observations show that protection is associated with a shift in the Th1/Th2 balance as well as a general reduction in the extent of the anti-type II collagen immune response. This suggests that EtxB-receptor-mediated modulation of lymphocyte responses provides a means of preventing autoimmune disease.

Cross-linking of cell surface ganglioside GM1 induces the selective apoptosis of mature CD8+ T lymphocytes.

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.

Potent immunogenicity of the B subunits of Escherichia coli heat-labile enterotoxin: receptor binding is essential and induces differential modulation of lymphocyte subsets.

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 of which 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 gamma. 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.

Immunoregulatory role of H-2 and intra-H-2 alleles on antibody responses to recombinant preparations of B-subunits of Escherichia coli heat-labile enterotoxin (rEtxB) and cholera toxin (rCtxB).

The immunoregulatory role of H-2 and intra-H-2 alleles on antibody responses to recombinant preparations of B-subunits of Escherichia coli heat-labile enterotoxin (rEtxB) and cholera toxin (rCtxB) is reported. Oral delivery of rEtxB to congenic mice of several different H-2 haplotypes resulted in H-2 dependent serum IgG responses (H-2d > H-2b = H-2q > H-2a > H-2k) and a similar spectrum of intestinal IgA responses in those strains tested. Responses to rEtxB and rCtxB were found to be differentially modulated by the H-2 locus, with significant differential effects in H-2b and H-2d congenic strains (H-2d > H-2b for rEtxB; H-2b > H-2d for rCtxB). Additionally, it was found that when rEtxB was fed to mice previously primed (orally) with either rEtxB or rCtxB only those mice primed with rEtxB exhibited a booster response. A second booster immunisation with rEtxB in rCtxB-primed mice produced an H-2 dependent spectrum of responses characteristic of those elicited by rEtxB, with the antibodies predominantly directed against rEtxB and not rCtxB. These results indicate that the differential response to rEtxB and rCtxB is set at the T- and B-cell level. Also, immunoregulation of antibody responses to rEtxB by intra-H-2 I-E in mice transgenic for the entire IEka gene was investigated. No significant difference between responses in transgene-positive and -negative mice was found, suggesting that antigen presentation does not involve I-E, but occurs in the context of I-A.(ABSTRACT TRUNCATED AT 250 WORDS)

Current progress in the development of the B subunits of cholera toxin and Escherichia coli heat-labile enterotoxin as carriers for the oral delivery of heterologous antigens and epitopes.

The development of non-living carrier systems for delivery of protective antigens or epitopes to the immune system represents both a fundamental and an applied aspect of vaccinology. A wide range of carrier systems, ranging from inert supports to proteins that exert direct immunomodulating effects on the immune response, are being studied. In this overview we describe the current progress in the development of the B-subunits of cholera toxin and Escherichia coli heat-labile enterotoxin as potential protein carriers for the oral delivery of chemically and genetically attached antigens and epitopes.

Immune responses to intramammary infusion with soluble (ovalbumin) and particulate (S uberis) antigens in the preparturient bovine udder.

Pregnant non-lactating cows were immunised by intramammary infusion with killed Streptococcus uberis into one quarter and ovalbumin into another, at one week (group 2) or one week and two weeks (group 1) before the expected date of parturition. A small IgG1 and IgG2 antibody response to ovalbumin was detected in the serum of these cows. There was also a small increase in IgG1 and IgA serum antibody activity to S uberis. In whey the response was restricted to IgA with activity to S uberis. The IgA antibody response to S uberis in group 1 was significantly greater in the quarter immunised with bacteria than that of the control quarters for up to two months after calving. In contrast, the serum IgA response was short or absent in a number of animals.

Fate and uptake of soluble and particulate antigens in the preparturient bovine mammary gland.

Pregnant heifers were infused intramammarily with ovalbumin in one quarter and killed S. uberis in another quarter. The animals were slaughtered at time intervals after infusion, and tissues were collected from different parts of the gland and from the local lymph nodes. Ovalbumin and bacteria reached the lymph nodes as early as 1 and 3 h after infusion respectively. Ovalbumin was also present in blood 5-10 min after infusion and in non-infused quarters. Both ovalbumin and bacteria were phagocytosed by the epithelium of the mammary gland. However, bacteria were seen in the tissues in small numbers at all stages after infusion. Larger numbers were phagocytosed by neutrophils in the lumen of ducts, 18 h after infusion. Phagocytosis of both ovalbumin and bacteria was not present in the connective tissue of the gland. The results indicate increased permeability of the preparturient gland to soluble proteins, but limited uptake of bacteria. Furthermore, a mechanism of transfer of antigens among quarters has been suggested.

The humoral immune response of mice to intra-mammary immunization with ovalbumin.

Groups of lactating mice were immunized intra-mammarily on the second day of lactation with 20 micrograms, 150 micrograms or 400 micrograms of ovalbumin (OVA). This resulted in the appearance of IgG in serum, and IgA and IgG in milk. In serum, no IgA antibodies were detected 16 days after immunization in any of the groups. The serum response of IgG was variable and not related directly to the immunizing dose. Both IgA and IgG antibodies were absent in milk 5 days after immunization and IgG antibody level in milk increased significantly throughout lactation as measured 10 and 15 days after inoculation. No IgA antibodies appeared in the milk of the 20 micrograms and 150 micrograms group; however, responses appeared in milk with the highest dose (400 micrograms), but the number of responders for IgG increased in milk but not in blood. The results suggest that intra-mammary immunization can provoke a local IgA response in milk, and that serum is not a major source of IgG in that fluid. Moreover, the kinetics of the IgA and IgG responses differ.