Browsing by keyword "Immunoglobulin epsilon-Chains"
Now showing items 1-5 of 5
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Activation of the mouse Ig germline epsilon promoter by IL-4 is dependent on AP-1 transcription factorsInduction of germline (GL) epsilon transcripts, an essential step preceding Ig isotype switching to IgE, requires activation of transcription factors by IL-4 and a B cell activator, e.g., CD40 ligand or LPS. We demonstrate that AP-1 (Fos and Jun), induced transiently by CD40 ligand or LPS, binds a DNA element in the mouse GL epsilon promoter. AP-1 synergizes with Stat6 to activate both the intact GL epsilon promoter and a minimal heterologous promoter driven by the AP-1 and Stat6 sites of the mouse GL epsilon promoter. By contrast, C/EBP beta, which trans-activates the human GL epsilon promoter, inhibits IL-4 induction of the mouse promoter, probably by attenuating the synergistic interaction between AP-1 and Stat6. Furthermore, AP-1 does not trans-activate the human GL epsilon promoter. Thus, induction of GL epsilon transcripts in mice and humans may be regulated differently. In addition, although mouse GL epsilon transcripts have a half-life of approximately 100 min, the RNA level continues to increase for up to 24 h, and the promoter appears to be active for at least 2 days after B cell activation. Altogether, these data suggest that induction of AP-1 activity, although transient, is required for activation of the mouse GL epsilon promoter by IL-4-induced Stat6.
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Characterization of an interleukin 4 (IL-4) responsive region in the immunoglobulin heavy chain germline epsilon promoter: regulation by NF-IL-4, a C/EBP family member and NF-kappa B/p50A large body of data indicate that antibody class switching is directed by cytokines by inducing or repressing transcription from unrearranged, or germline, CH genes. Interleukin 4 (IL-4) induces transcription of the germline C epsilon genes in activated B cells and subsequently, cells in this population will undergo switch recombination to immunoglobulin E. Furthermore, the data suggest that transcription of germline C epsilon genes is required for class switching. In this paper we define DNA elements required for induction of transcription of the germline C epsilon genes by IL-4. To do this, segments of DNA from the 5' flank of the initiation sites for germline epsilon RNA were ligated to a luciferase reporter gene and transfected into two mouse B cell lines, one of which can be induced to switch to IgE. By analysis of a series of 5' deletion constructs and linker-scanning mutations, we demonstrate that a 46-bp segment (residing at -126/-79 relative to the first RNA initiation site) contains an IL-4 responsive region. By electrophoretic mobility shift assays, we find that this segment binds three transcription factors: the recently described NF-IL4, one or more members of the C/EBP family of transcription factors, and NF-kappa B/p50. Mutation of any of the binding sites for these three factors abolishes or reduces IL-4 inducibility of the epsilon promoter. A 27-bp segment within this IL-4 response region containing binding sites for NF-IL4 and a C/EBP factor is sufficient to transfer IL-4 inducibility to a minimal c-fos promoter.
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Differential regulation of mouse germline Ig gamma 1 and epsilon promoters by IL-4 and CD40Before Ig class switching, RNA transcription through the specific S regions undergoing recombination is induced by cytokines and other activators that induce and direct switching. The resulting germline (GL) transcripts are essential for switch recombination. To understand the differential regulation of mouse IgG1 and IgE, we compared the promoters for GL gamma1 and epsilon transcripts. We addressed the question of why the promoter that regulates GL epsilon transcription is more responsive to IL-4 than the gamma1 promoter and also why GL epsilon transcription is more dependent on IL-4 than is gamma1 transcription. We found that the IL-4-responsive region of the GL epsilon promoter is more inducible than that of the gamma1 promoter, although each promoter contains a binding site for the IL-4-inducible transcription factor Stat6, located immediately adjacent to a binding site for a basic region leucine zipper (bZip) family protein. However, the arrangement and sequences of the sites differ between the epsilon and gamma1 promoters. The GL epsilon promoter binds Stat6 with a 10-fold higher affinity than does the gamma1 promoter. Furthermore, the bZip elements of the two promoters bind different transcription factors, as the GL epsilon promoter binds and is activated by AP-1, whereas the gamma1 promoter binds and is activated by activating transcription factor 2. C/EBPbeta and C/EBPgamma also bind the gamma1 bZip element, although they inhibit rather than activate transcription. However, inhibition of promoter activity by C/EBPbeta does not require the bZip element and may instead occur via inhibiting the activity of NF-kappaB.
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Overexpression of BSAP/Pax-5 inhibits switching to IgA and enhances switching to IgE in the I.29 mu B cell lineB 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.
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TRIF signaling is essential for TLR4-driven IgE class switchingThe 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.
