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.
Lycke
N Y
NY
Department of Medical Microbiology and Immunology, University of Göteborg, Sweden.
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