Cholera toxin promotes B cell isotype switching by two different mechanisms. cAMP induction augments germ-line Ig H-chain RNA transcripts whereas membrane ganglioside GM1-receptor binding enhances later events in differentiation.
In a recent study we provided evidence that isotype switching in LPS-stimulated murine B cells was greatly enhanced by cholera toxin (CT). We found that CT acted synergistically with IL-4 to promote IgG1 differentiation at the gene transcriptional level by strongly enhancing the expression of germ-line gamma 1-RNA transcripts. In this study we ask which mechanisms are responsible for the isotype-switching effect of CT on B cells and which second messenger systems are involved in this process. We found that at least two different mechanisms are involved: 1) increased intracellular cAMP levels stimulated by the A subunit potentiates isotype switching early in differentiation by augmenting the formation of sterile germ-line gamma 1-RNA transcripts and 2) the binding of the nontoxic B subunit to the membrane GM1-ganglioside receptor promotes later stages of differentiation. Although the whole toxin gave up to a ninefold increase in IgG1 differentiation the cAMP-independent effect of rCTB gave at most a fivefold increase in IgG1 differentiation as compared to that seen with IL-4 alone. However, on a molar basis whole CT was at least a 1000-fold more efficient at promoting B cell switch-differentiation as compared to rCTB. Moreover, IL-4 did not stimulate cAMP in murine B cells and its effect on LPS-stimulated B cell differentiation was not decreased by inhibitors of cAMP-dependent protein kinases. However, CT's effect on B cell switch differentiation was blocked by inhibitors of protein kinases and could be partially mimicked by dibutyryl cAMP. In contrast to CT, the enhancing effect of rCTB on IgG1-differentiation was not affected by blocking of the protein kinases and the combination of rCTB and dBcAMP was as potent as the intact CT in promoting IL-4-stimulated IgG1 differentiation. Finally, the IL-4 pathway, but not the CT pathway, was sensitive to phorbol esters: In IL-4 plus LPS-stimulated B cell cultures IgG1 production was almost completely blocked by PMA. This inhibition was not associated with a decreased B cell proliferation or expression of germ-line gamma 1-RNA transcripts. The addition of CT, and to a significantly lesser extent rCTB, to these cultures enhanced IgG1-differentiation and expression of germ-line gamma 1-RNA transcripts to the same extent as in cultures without PMA. The existence of dual mechanisms operating together on B cell differentiation help to explain the strong adjuvant function by CT on IgG and IgA antibody responses after oral and parenteral immunizations.
Department of Medical Microbiology and Immunology, University of Göteborg, Sweden.
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D000276:Adjuvants, Immunologic / Q000494:pharmacology*; D000818:Animals; D000916:Antibody Diversity / Q000235:genetics; D001402:B-Lymphocytes / Q000187:drug effects / Q000276:immunology*; D002454:Cell Differentiation / Q000187:drug effects / Q000276:immunology; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000502:physiology*; D005677:G(M1) Ganglioside / Q000378:metabolism*; D007074:Immunoglobulin G / Q000032:analysis* / Q000096:biosynthesis; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007132:Immunoglobulin Isotypes / Q000032:analysis*; D015847:Interleukin-4 / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic / Q000502:physiology*; D011994:Recombinant Proteins / Q000494:pharmacology; D013154:Spleen / Q000276:immunology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic
D000276:Adjuvants, Immunologic; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007132:Immunoglobulin Isotypes; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; C023552:choleragen receptor; D015847:Interleukin-4; D005677:G(M1) Ganglioside; D002772:Cholera Toxin; D000242:Cyclic AMP; D013755:Tetradecanoylphorbol Acetate