Effect of downregulation of germline transcripts on immunoglobulin A isotype differentiation.
In this study we determined the role of immunoglobulin (Ig) germline transcripts in the isotype switch differentiation of the cloned lymphoma B cell line CH12.LX. In initial studies, we showed that addition of transforming growth factor beta (TGF-beta) and interleukin 4 (IL-4), either alone or in combination, augment switching from membrane (m)IgM+ to mIgA+ cells, and that increased switching is preceded and paralleled by an increase in the steady-state level of alpha germline transcripts (alpha GLT). Interestingly, TGF-beta and IL-4 affect switching in different ways, as shown by the fact that IL-4 increases and TGF-beta decreases the number of dual-positive (mIgM+/mIgA+) cells; in addition, TGF-beta and IL-4 have different effects on the time course of induction of alpha GLT. In subsequent studies, we established that we could downregulate alpha GLT levels in CH12.LX B cells by transfecting an expression vector that can be induced to produce transcripts antisense to the I alpha exon. Using this approach we downregulated alpha GLT in CH12.LX B cells undergoing switching in the presence of TGF-beta and IL-4 and showed that such downregulation led to decreased switching, as evidenced by decreased appearance of dual-positive B cells as well as decreased IgA synthesis relative to IgM synthesis. This result was corroborated by the fact that incubation of CH12.LX cells with phosphorothio-oligo antisense DNA to I alpha sequence also led to a decrease in the number of dual-positive cells and in the IgA/IgM secretion ratio. In summary, IgA isotype differentiation in CH12.LX B cell, particularly the steps necessary for the elaboration of mIgM+/mIgA+ switch intermediate cells, is inhibited by downregulation of alpha GLT; it is therefore apparent that alpha GLT plays a key role in the initial stage of isotype switch differentiation.
Wakatsuki
Y
Y
Mucosal Immunity Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Strober
W
W
D001402:B-Lymphocytes / Q000276:immunology*; D001483:Base Sequence; D002454:Cell Differentiation; D015536:Down-Regulation; D005434:Flow Cytometry; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors; D006801:Humans; D007070:Immunoglobulin A / Q000096:biosynthesis / Q000235:genetics*; D007132:Immunoglobulin Isotypes / Q000096:biosynthesis / Q000235:genetics*; D007075:Immunoglobulin M / Q000096:biosynthesis / Q000235:genetics; D007134:Immunoglobulin Switch Region; D015847:Interleukin-4 / Q000494:pharmacology; D008969:Molecular Sequence Data; D014158:Transcription, Genetic; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D007070:Immunoglobulin A; D007132:Immunoglobulin Isotypes; D007075:Immunoglobulin M; D016212:Transforming Growth Factor beta; D015847:Interleukin-4