Jabara, Haifa H.
Fitzgerald, Katherine A.
Golenbock, Douglas T.
Geha, Raif S.
UMass Chan AffiliationsDepartment of Medicine, Division of Infectious Diseases and Immunology
Document TypeJournal Article
KeywordsAdaptor Proteins, Vesicular Transport
Immunoglobulin Class Switching
Mice, 129 Strain
Mice, Inbred BALB C
Mice, Inbred C57BL
Myeloid Differentiation Factor 88
Reverse Transcriptase Polymerase Chain Reaction
Toll-Like Receptor 4
Transcription Factor RelA
Immunology and Infectious Disease
Immunology of Infectious Disease
MetadataShow full item record
AbstractThe TLR4 ligand LPS causes mouse B cells to undergo IgE and IgG1 isotype switching in the presence of IL-4. TLR4 activates two signaling pathways mediated by the adaptor molecules MyD88 and Toll/IL-IR domain-containing adapter-inducing IFN-beta (TRIF)-related adaptor molecule (TRAM), which recruits TRIF. Following stimulation with LPS plus IL-4, Tram(-/-) and Trif(-/-) B cells completely failed to express Cepsilon germline transcripts (GLT) and secrete IgE. In contrast, Myd88(-/-) B cells had normal expression of Cepsilon GLT but reduced IgE secretion in response to LPS plus IL-4. Following LPS plus IL-4 stimulation, Cgamma1 GLT expression was modestly reduced in Tram(-/-) and Trif(-/-) B cells, whereas Aicda expression and IgG1 secretion were reduced in Tram(-/-), Trif(-/-), and Myd88(-/-) B cells. B cells from all strains secreted normal amounts of IgE and IgG1 in response to anti-CD40 plus IL-4. Following stimulation with LPS plus IL-4, Trif(-/-) B cells failed to sustain NF-kappaB p65 nuclear translocation beyond 3 h and had reduced binding of p65 to the Iepsilon promoter. Addition of the NF-kappaB inhibitor, JSH-23, to wild-type B cells 15 h after LPS plus IL-4 stimulation selectively blocked Cepsilon GLT expression and IgE secretion but had little effect on Cgamma1 GLT expression and IgG secretion. These results indicate that sustained activation of NF-kappaB driven by TRIF is essential for LPS plus IL-4-driven activation of the Cepsilon locus and class switching to IgE.
SourceJ Immunol. 2014 Mar 15;192(6):2651-8. doi: 10.4049/jimmunol.1300909. Epub 2014 Feb 14.Link to article on publisher's site
Permanent Link to this Itemhttp://hdl.handle.net/20.500.14038/34957
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Induction of immunoglobulin isotype switching in cultured I.29 B lymphoma cells. Characterization of the accompanying rearrangements of heavy chain genesStavnezer, Janet; Sirlin, S.; Abbott, J. (1985-03-01)The murine B cell lymphoma I.29 contains cells expressing surface IgM or IgA with identical heavy chain variable regions (9, 25, and D. Klein and J. Stavnezer, unpublished data). Purified IgM+ cells from the lymphoma have been adapted to culture and induced to switch to IgA, IgE, or IgG2 by treatment with lipopolysaccharide (LPS) or by treatment with a monoclonal anti-I.29 antiidiotype plus LPS. Clones of IgM+ cells have been obtained and induced to switch. Under optimal conditions, 30% of the cells in the culture expressed IgA 8 d after the inducers were added, and by 15 d 90% of the cells were IgA+. In actively switching cultures, up to 50% of the cells whose cytoplasm stained positively with anti-IgA stained simultaneously with anti-IgM, which indicates that the appearance of IgA+ cells in the cultures was due to isotype switching and not to clonal outgrowth. Examination by Southern blotting experiments of the Ig heavy chain genes in I.29 cells before and after switching revealed that isotype switching was accompanied by DNA recombinations that occurred within or immediately 5' to the tandemly repeated switch sequences. Within 3 d after the addition of inducers of switching, the nonexpressed chromosome underwent a variety of deletions or expansions within the S mu region, and a portion of the S alpha regions had undergone a 0.9-kb deletion. In cultures that contained at least 12% IgA+ cells, rearranged, expressed alpha genes, produced by recombination between the S mu region within the expressed mu gene and the S alpha region, were detected.
Overexpression of BSAP/Pax-5 inhibits switching to IgA and enhances switching to IgE in the I.29 mu B cell lineQiu, G.; Stavnezer, Janet (1998-09-15)B cell-specific activator protein (BSAP)/Pax-5 is a paired domain DNA-binding protein expressed in the developing nervous system, testis, and in all B lineage cells, except terminally differentiated plasma cells. BSAP regulates transcription of several genes expressed in B cells and also the activity of the 3' IgH enhancer. As it has binding sites within or 5' to the switch regions of nearly all Ig heavy chain C region genes and also is known to increase transcription of the germline epsilon RNA, BSAP has been hypothesized to be involved in regulation of Ab class switch recombination. To directly examine the effects of BSAP on isotype switching, we use a tetracycline-regulated expression system to overexpress BSAP in the surface IgM+ I.29 mu B cell line, a mouse cell line that can be induced to undergo class switch recombination. We find that overexpression of BSAP inhibits switching to IgA in I.29 mu cells stimulated with LPS + TGF-beta 1 + nicotinamide, but enhances switching to IgE in cells stimulated with LPS + IL-4 + nicotinamide. Parallel to its effects on switching, overexpression of BSAP inhibits germline alpha RNA expression and the transcriptional activity of the germline alpha promoter, while enhancing activity of the germline epsilon promoter. Proliferation of I.29 mu cells is not affected in this system. The possible mechanisms and significance of the effect of BSAP on isotype switching are discussed.
Rearrangements and deletions of immunoglobulin heavy chain genes in the double-producing B cell lymphoma I.29Stavnezer, Janet; Marcu, K. B.; Sirlin, S.; Alhadeff, B.; Hammerling, U. (1982-08-01)The B cell lymphoma I.29 consists of a mixture of cells expressing membrane-bound immunoglobulin M (IgM) (lambda) and IgA (lambda) of identical idiotypes. Whereas most of the cells express either IgM or IgA alone, 1 to 5% of the cells in this tumor express IgM and IgA simultaneously within the cytoplasm and on the cell membrane (R. Sitia et al., J. Immunol. 127:1388-1394, 1981; R. Sitia, unpublished data). When IgM+ cells are purified from the lymphoma and passaged in mice or cultured, a portion of the cells convert to IgA+. These properties suggest that some cells of the I.29 lymphoma may undergo immunoglobulin heavy chain switching, although it is also possible that the mixed population was derived by a prior switching event in a clone of cells. We performed Southern blotting experiments on genomic DNAs isolated from populations of I.29 cells containing variable proportions of IgM+ and IgA+ cells and on a number of cell lines derived from the lymphoma. The results were consistent with the deletion model for heavy chain switching, as the IgM+ cells contained rearranged mu genes and alpha genes in the germ line configuration on both the expressed and nonexpressed heavy chain chromosomes, whereas the IgA+ cells had deleted both mu genes and contained one rearranged and one germ line alpha gene. In addition, segments of DNA located within the intervening sequence 5' to the mu gene, near the site of switch recombination, were deleted from both the expressed and the nonexpressed chromosomes. Although mu genes were deleted from both chromosomes in the IgA+ cells, the sites of DNA recombination differed on the two chromosomes. On the expressed chromosome, Smu sequences were recombined with S alpha sequences, whereas on the nonexpressed chromosome, Smu sequences were recombined with S gamma 3 sequences.