Epsilon Germline Research Articles

Disodium cromoglycate inhibits S mu-->S epsilon deletional switch recombination and IgE synthesis in human B cells.

IgE synthesis requires interleukin 4 (IL-4) and a T-B cell interaction that involves the B cell antigen CD40 and its ligand expressed on activated T cells. IL-4 induces epsilon germline transcription whereas ligation of CD40 results in deletional S mu-->S epsilon switch recombination, expression of mature epsilon transcripts, and IgE synthesis and secretion. We demonstrate that disodium cromoglycate (DSCG), a drug commonly used for the prophylactic treatment of allergic disease, inhibits T cell-driven IgE synthesis by human B cells at concentrations readily achievable in the course of inhaled therapy for asthma. Inhibition of IgE synthesis by DSCG was not the result of drug toxicity because DSCG did not affect the viability of T and B cells or their proliferation to mitogens. DSCG did not interfere with CD40 ligand expression by T cells but clearly targeted the B cells because it inhibited IgE synthesis induced by anti-CD40 and IL-4 in populations of highly purified B cells. DSCG had no effect on the induction of epsilon germline transcripts by IL-4 but strongly inhibited CD40 mediated S mu-->S epsilon deletional switch recombination in IL-4-treated B cells as assayed by nested primer PCR. The effect of DSCG was not specific for CD40-mediated induction of IgE isotype switching because DSCG inhibited IgE synthesis as well as S mu-->S epsilon deletional switch recombination induced by hydrocortisone and IL-4 in B cells. Moreover, the effect of DSCG was not specific for IgE isotype switching because DSCG inhibited the synthesis of IgG4 by B cells sorted for lack of surface expression of IgG4 and stimulated with anti-CD40 and IL-4. DSCG caused only minimal inhibition (< 15%) of spontaneous IgE synthesis by lymphocytes from patients with the hyper-IgE syndrome and did not affect pokeweed mitogen-induced IgG and IgA synthesis by lymphocytes suggesting that it has little effect on B cells that have already undergone isotype switching. These results indicate that DSCG inhibits switching to IgE in B cells and suggest a novel potential mechanism for the prevention of allergic disease by DSCG.

CD58 (LFA-3) stimulation provides a signal for human isotype switching and IgE production distinct from CD40.

Induction of an IgE response involves several discrete steps: 1) induction of epsilon germ line transcription, 2) DNA recombination, and 3) mature RNA transcription/translation. Here we show that ligation of B cell CD58 by CD2, its natural ligand on T cells, or by mAb, provides a novel IL-4-dependent signal for the latter two steps. Highly purified human B cells were induced to produce IgE by costimulation with IL-4 and CD58 mAb. Although CD58 ligation alone was unable to induce epsilon germ-line transcription, in concert with IL-4-stimulated epsilon germ-line transcription it induced the appearance of productive epsilon transcripts and IgE production. The direct involvement of CD2 was demonstrated: B cells cultured with IL-4 plus murine T hybridoma cells transfected with human CD2 produced IgE. A CD40 Fc fusion protein had no effect on CD58-driven IgE production while inhibiting CD40-dependent responses. Furthermore, cells from patients with common variable immunodeficiency produced IgE in response to IL-4 plus CD40 mAb but not to IL-4 plus CD58 mAb. CD58-driven IgE synthesis was IFN-gamma independent and was not enhanced by exogenous IL-6. Functional differences between CD40 and CD58 IgE stimulation were demonstrated. Thus, the CD2:CD58 ligand/counterligand system provides an alternative pathway by which cell contact signaling may regulate IgE. Given the relative importance of CD2 triggering on mucosal T cells and the mucosal location of IgE production, this may be especially true on mucosal surfaces.

The transcription factor BSAP (NF-HB) is essential for immunoglobulin germ-line epsilon transcription.

Treatment of murine splenic B lymphocytes and certain B-lineage cell lines with mitogen (LPS) and the lymphokine IL-4 has been shown to induce expression of germ-line epsilon transcripts (l epsilon transcripts) and class switching to the C epsilon gene. Three protein complexes, one of which (complex 3) is constitutively expressed, have been shown to bind to a 179-base pair LPS/IL-4-responsive l epsilon promoter (Rothman, P., S. C. Li, B. Gorham, L. Glimcher, F. W. Alt, and M. Boothby. 1991. Mol. Cell. Biol. 11:5551). Complex 3 is indispensable for this inducible promoter activity. In this report, we have used electrophoretic mobility shift assays (EMSA) to demonstrate that the early B cell-specific transcription factor (BSAP) is involved in the formation of complex 3. In addition, BSAP is implicated functionally in l epsilon transcription because a BSAP binding site either from a sea urchin histone promoter (H2A-2.2) or from 5' of murine immunoglobulin S gamma 2a can substitute for the epsilon-associated site (epsilon(foot), as assayed by transient transfection assays of the l epsilon:CAT reporter constructs into the M12.4.1 B cell line. Like the sea urchin histone BSAP site, the complex 3 binding site (epsilon(foot)) functions as an upstream promoter element when assayed in the OVEC vector. These results indicate that BSAP is an essential protein required for LPS/IL-4 induction of the l epsilon promoter. In addition, experiments showing that a BSAP binding site from 5' of S gamma 2a also functions as an upstream promoter element in OVEC suggest a potential role for BSAP in regulation of the IgG2a isotype.

STF-IL-4: a novel IL-4-induced signal transducing factor.

The mechanism by which interleukin-4 (IL-4) regulates the expression of particular genes is unknown. We have determined that IL-4 induces a DNA binding factor (termed STF-IL-4) which has a strong affinity for an IFN-gamma activation site (GAS). Interestingly, STF-IL-4 also binds to the IL-4 responsive promoter for the Ig heavy chain germline epsilon transcript. The IL-4 dependent activation of STF-IL-4 is rapid, does not require protein synthesis and results in the sequential appearance of binding activity first in the cytoplasm and then later in the nucleus. Activation of STF-IL-4 is sensitive to tyrosine kinase inhibitors and the active factor is tyrosine phosphorylated. This pattern of activation is similar to the activation of interferon-induced transcription factors. STF-IL-4 appears to be a new member of a growing family of cytokine-induced transcriptional regulators.

IFN-gamma represses epsilon germline transcription and subsequently down-regulates switch recombination to epsilon.

Cytokines have the ability to regulate the isotypes of antibodies produced during an immune response. For instance, IL-4 has been shown to induce the production of IgE by B cells, while IFN-gamma has been shown to inhibit this induction. Recent work has revealed that IL-4 appears to induce class switching to epsilon through its ability to specifically induce germline epsilon transcripts. Germline epsilon transcription appears to target class-switch recombination to the epsilon locus. However, the mechanism by which IFN-gamma inhibits the IL-4 induction of IgE is unknown. We hypothesized that IFN-gamma and IL-4 may have antagonistic effects on the same stage of B cell differentiation. Northern blotting analyses show that IFN-gamma suppresses the IL-4 induction of germline epsilon transcripts. In transient transfection assays, the IL-4 induction of transcription imparted by the minimal 179 bp germline epsilon promoter is repressed by IFN-gamma. Utilizing a digestion circularization-polymerase chain reaction assay we show that IL-4 induces switch recombination to epsilon, while IFN-gamma suppresses switch recombination to epsilon. These studies support a model that, through their differential effects on a cis-controlling element that regulates germline epsilon transcription, IL-4 and IFN-gamma are able to modulate B cell switch recombination to epsilon in a coordinated manner.

IL-10 and viral IL-10 prevent IL-4-induced IgE synthesis by inhibiting the accessory cell function of monocytes.

In the present study it is demonstrated that IL-10 and viral (v)-IL-10 inhibit IL-4-induced IgG4 and IgE synthesis in cultures of PBMC from healthy nonatopic donors. The inhibition occurred at the mRNA level. IL-10 strongly reduced IL-4-induced expression of both germline and productive epsilon transcripts in PBMC. The inhibitory effects were completely neutralized, or IgG4 and IgE synthesis was even enhanced, by anti-IL-10 mAb, demonstrating the specificity of the reaction. IL-4-induced IgG4 and IgE synthesis by PBMC was monocyte dependent. IL-4 failed to induce IgG4 or IgE synthesis by monocyte-depleted PBMC, but the production of these isotypes was completely restored by reconstitution with autologous monocytes. However, monocytes preincubated with IL-10 for 24 h failed to provide the accessory signals required for IL-4-induced IgG4 and IgE synthesis, indicating that the inhibitory effects of IL-10 are indirectly mediated via monocytes. This notion was further supported by the finding that IL-10 and v-IL-10 failed to inhibit IL-4-induced IgE synthesis in the absence of monocytes, i.e., when highly purified B cells were cultured in the presence of anti-CD40 mAb or cloned activated CD4+ T cells. Moreover, IL-10 failed to inhibit IL-4-induced germline epsilon mRNA synthesis in highly purified B cells. The inhibitory effects of IL-10 could not be restored by exogenous TNF-alpha or IL-6, indicating that the inhibitory effects were not mediated through inhibition of production of these cytokines. This is compatible with the observation that monocytes preincubated with IL-10 did not inhibit IgG4 or IgE synthesis in a monocyte- and T cell-independent culture system, in which purified B cells were cultured in the presence of anti-CD40 mAb and IL-4. Collectively, these data indicate that IL-10 mediates a potent, monocyte-dependent, inhibitory effect on IL-4-induced IgG4 and IgE production by human PBMC.

Regulation of the expression of human C epsilon germline transcript. Identification of a novel IL-4 responsive element.

Transcriptional regulation for Ig H chain germline transcripts induced by cytokines is a topic of recent interest for the understanding of the mechanism of class switch recombination. Among human B cell lines examined, we have found that a human IgM-producing B cell line, DND39 (EBV negative) expressed germ-line transcripts of epsilon constant gene (C epsilon) when stimulated with IL-4. In our study, the regulatory element responsible for the expression of IL-4-induced human C epsilon germ-line transcript was determined using DND39 cells. To identify the IL-4 responsive promoter/enhancer element, deletion analysis of the upstream region of the germ-line exon (I epsilon) of the C epsilon germ-line transcript which is located 5' to the switch region, was performed by using a luciferase gene as a reporter. Deletion analysis showed that a DNA fragment which lies between -215 and -154 bp upstream from the most 3' transcriptional initiation site of human I epsilon gene is fully responsible for the induction of germ-line transcripts by IL-4. According to a mutational analysis, the DNA fragment between -163 and -152 bp, was identified to be a novel IL-4 responsive element in a human C epsilon gene. Electrophoretic gel mobility shift assay showed the presence of IL-4-induced nuclear factor that specifically bound to this IL-4 responsive element. This novel IL-4 responsive element and an IL-4-induced DNA binding protein may play an important role for the induction of C epsilon germ-line transcript as well as class switching to IgE.

Replacement of germ-line epsilon promoter by gene targeting alters control of immunoglobulin heavy chain class switching.

Recent work has shown that the ability of cytokines to direct immunoglobulin heavy chain class-switch recombination to particular heavy chain constant (C) region (CH) genes correlates with the induction of specific germ-line CH transcripts. To test the role of germ-line transcripts in class switching, we have used homologous recombination to mutate the immunoglobulin heavy chain locus of the 18.81A20 murine pre-B-cell line. In the parent cell line, the combination of interleukin-4 (IL-4) and lipopolysaccharide (LPS) induces germ-line epsilon locus transcription prior to class switching to epsilon. The heavy chain locus of the mutated cell line contains the immunoglobulin heavy chain enhancer and variable region gene promoter in place of the LPS/IL-4-responsive germ-line epsilon promoter. The mutant cell line constitutively transcribes the epsilon locus in the absence of IL-4. Strikingly, the mutant cell line also switches to epsilon in the absence of IL-4. This result demonstrates that, at least in the 18.81A20 cell line, germ-line epsilon transcription plays a direct role in class switching to the epsilon locus. In addition, the ability to change the pattern of class switching by altering transcriptional activity indicates that transcription of germ-line CH is mechanistically important in regulation of class switching.

Regulation of germ-line epsilon transcription and induction of epsilon switching in cloned EBV-transformed and malignant human B cell lines by cytokines and CD4+ T cells.

IL-4 induces germ-line epsilon transcript synthesis in normal human B cells, but a second signal provided by CD4+ T cells is required for subsequent production of productive epsilon mRNA and IgE synthesis. In the present study we demonstrate that IL-4 specifically induces germ-line epsilon transcripts in most EBV lymphoblastoid (LCL) and EBV+ and EBV- Burkitt's lymphoma (BL) cells without inducing IgE synthesis. However, cocultivation of cloned sIgM+, sIgE- EBV-LCL or BL cells with activated CD4+ T cell clones in the presence of IL-4 resulted in IgE production in 13 to 24% of the wells. Analysis of rearranged DNA in IgE-producing cloned BL cells indicated that epsilon switching occurred through a recombination deletion event. IgE production is a stable feature of the cloned switched EBV-LCL and BL cells because these cells continued to produce relatively high levels of IgE in the absence of IL-4 and CD4+ T cells. Induction of IgE synthesis was blocked by IFN-gamma, IFN-alpha, and transforming growth factor beta (TGF-beta), but only TGF-beta inhibited IL-4-induced germ-line epsilon mRNA expression. These results suggest that the inhibitory effects of TGF-beta are mediated via inhibition of germ-line epsilon expression, whereas IFN-alpha and IFN-gamma block other steps in the regulatory processes resulting in induction of IgE synthesis by established human B cell line cells. Our results indicate that the same set of signals that induce normal B cells to switch to IgE synthesis also induce high frequency epsilon switching in cloned established EBV transformed and malignant B cell lines including classical cell lines such as JY and BL-2.

Interleukin 5 (IL-5) provides a signal that is required in addition to IL-4 for isotype switching to immunoglobulin (Ig) G1 and IgE.

We have examined the contributions of Interleukin 4 (IL-4), IL-5, and other stimuli to the expression of Immunoglobulin G1 (IgG1) and IgE in murine B lymphoblasts activated with anti-Ig. The combination of IL-4 and -5 induced B lymphoblasts to proliferate and to secrete IgM and IgG1. However, an additional stimulus was required along with IL-4 and -5 for induction of IgE secretion. This stimulus was provided by lipopolysaccharides (LPS) or cytokines produced by TC-1 or EL4 cells. In the absence of IL-5, exceptionally high concentrations of IL-4 (greater than 1,000 U/ml) were required to elicit IgG1 and IgE secretion from B lymphoblasts cultured with either LPS or TC-1-conditioned media (CM). To investigate regulation of expression of gamma 1 and epsilon genes by IL-4, -5, and LPS, the requirements for induction of gamma 1 and epsilon germline and productive transcripts were examined. Germline gamma 1, but not epsilon, transcripts were detected in RNA from B lymphoblasts treated with IL-4 and -5 for 48 h. In contrast, both germline gamma 1 and epsilon transcripts could be detected in B lymphoblasts cultured with IL-4 and LPS, and steady state levels of germline gamma 1 transcripts were four- to sevenfold higher in blasts cultured with LPS and IL-4, compared with blasts cultured with IL-4 and -5. LPS enhanced steady state levels of germline transcripts induced by IL-4, but LPS did not promote substantial accumulation of productive gamma 1 and epsilon transcripts. In contrast, IL-5 did not affect steady state levels of germline transcripts stimulated by IL-4, but did markedly increase levels of productive gamma 1 and epsilon transcripts. Thus, lymphokines regulate two distinct events in isotype switching: induction of germline transcripts (IL-4), and production of VDJ-C gamma 1 and VDJ-C epsilon mRNA (IL-5), which leads to secretion of IgG1 and IgE.

Modulation of IL-4 induced germline epsilon RNA synthesis in human B cells by tumor necrosis factor-alpha, anti-CD40 monoclonal antibodies or transforming growth factor-beta correlates with levels of IgE production.

To determine the role of germline epsilon transcription in IgE synthesis, the effects of cytokines on germline epsilon RNA synthesis in IL-4 dependent epsilon switching in B cells was investigated. Induction of germline epsilon transcription in highly purified B cells seems to be a specific property of IL-4, since none of the other cytokines tested [IL-1 alpha, beta, IL-2, IL-3, IL-5, IL-6, IL-7, IL-9, IL-10, G-CSF, GM-CSF, M-CSF, IFN-gamma, IFN-alpha, tumor necrosis factor (TNF)-alpha, and transforming growth factor (TGF)-beta] were effective. TGF-beta, IFN-gamma, and IFN-alpha inhibit IL-4 dependent IgE synthesis, but only TGF-beta blocked germline epsilon RNA synthesis in purified B cells, indicating that this may be the mechanism by which TGF-beta inhibits IgE synthesis, and that IFN-gamma and IFN-alpha act on other stages of the regulatory process resulting in IgE production. IL-5, IL-6, and TNF-alpha enhance IL-4 dependent IgE synthesis, but only TNF-alpha enhanced IL-4 induced germline epsilon RNA synthesis. Finally, anti-CD40 mAbs and the non-IL-4 producing CD4+ T cell clone A3, which in the presence of IL-4 induce IgE synthesis by purified B cells, both strongly enhanced germline epsilon transcription. These data, together with the observation that epsilon switching in cultures initiated with single sIgM+, sIgE- B cells in all instances was preceded by germline epsilon RNA synthesis, indicate that there is a strong relationship between germline epsilon transcription and IgE synthesis.

The effect of cytokines and mitogens on the induction of C epsilon germline transcripts in a human Burkitt lymphoma B cell line.

IL-4 is known to induce a 1.8 kb constant epsilon (C) transcript in human peripheral blood mononuclear cells (PBMC). This is a C germline or 'sterile' transcript which is encoded by a germline exon (I) located 5' of the epsilon switch (S) region and the four C germline exon including 3' untranslated region. Among the human B cell lines examined, one Burkitt lymphoma B cell line, DND39 (Epstein-Barr virus negative, sIgM+, CD40+), was found to respond to IL-4 and express the C germline transcript. Induction of C germline transcript in DND39 cells was entirely dependent on the presence of IL-4. Phorbol myristate acetate enhanced its expression in DND39 cells as well as in the purified B cells in combination with IL-4. Pokeweed mitogen (PWM) enhanced the expression of C germline transcripts induced by IL-4 in PBMC but the enhancing activity was not observed when purified B cells or DND39 cells were used. Supernatants obtained from PWM-stimulated PBMC strongly enhanced the expression of C germline transcript in DND39 cells when co-stimulated with IL-4. None of the known cytokines tested mimicked this effect, suggesting that PWM may induce in T cells or monocytes a novel cytokine production that works synergistically with IL-4 and enhances the expression of IL-4-induced C germline transcripts. A similar enhancing effect was observed when anti-CD40 mAb was added to the IL-4-stimulated DND39 cells. Interestingly, transforming growth factor-beta strongly suppressed it. Taken together, DND39 cells resemble normal B cells in terms to responsiveness to IL-4 and should be a useful tool to study the molecular mechanisms of regulation of C germline transcription and class switching to IgE.

Effect of cytokines on switching to IgA and alpha germline transcripts in the B lymphoma I.29 mu. Transforming growth factor-beta activates transcription of the unrearranged C alpha gene.

H chain isotype switch recombination is preceded by the appearance of RNA initiating 5' of the specific switch region that will undergo recombination. In an effort to understand the potential function of germline transcripts in switch recombination and whether the regulation of germline transcripts correlates with the regulation of switching, we are studying this process in the murine B lymphoma cell line I.29 mu, which switches, after treatment with bacterial LPS, primarily to IgA and less frequently to IgE. Levels of alpha germline transcripts initiating upstream of alpha switch (S alpha) sequences are elevated in clones of this line that switch well, compared with clones that switch less frequently. Transforming growth factor-beta (TGF-beta) has been shown to increase alpha germline transcripts and switching to IgA expression in LPS-stimulated murine splenic B cells. We now demonstrate that TGF-beta increases LPS-induced switching to IgA by 10-fold at optimal doses and increases the level of alpha germline transcripts 5- to 9-fold in I.29 mu cells. Nuclear run-on analysis shows that this increase is at the level of transcription. Thus, TGF-beta appears to direct switching to IgA by inducing transcription from the unrearranged S alpha-C alpha DNA segment. Germline alpha RNA is quite stable in I.29 mu cells, having a half-life of about 5 h, and we find no evidence for further stabilization in the presence of TGF-beta. Levels of epsilon germline transcripts are decreased by TGF-beta treatment. IL-4, which modestly increases switching in I.29 mu cells, slightly increases transcription of alpha germline RNA. IFN-gamma, which reduces switching to IgA in these cells, also reduces the level of alpha germline transcripts. IFN-gamma also reduces the level of epsilon germline transcripts induced by IL-4. Our results support the hypothesis that the regulation of transcription of particular switch sequences by cytokines regulates the specificity of recombination. We also present evidence that IL-4 may provide other signals, distinct from transcriptional targeting, that increase LPS-induced switching to IgA.

Identification of a conserved lipopolysaccharide-plus-interleukin-4-responsive element located at the promoter of germ line epsilon transcripts.

Treatment of splenic B lymphocytes and certain B-lineage cell lines with the mitogen lipopolysaccharide (LPS) and the lymphokine interleukin-4 (IL-4) induces expression of germ line immunoglobulin C epsilon transcripts and class switching to the C epsilon gene. We show that LPS-plus-IL-4 induction of germ line epsilon transcripts (termed I epsilon transcripts) occurs at the transcriptional level in an Abelson murine leukemia virus-transformed pre-B-cell line. A 1.1-kb region of DNA surrounding the I epsilon promoter endows inducible transcription to a heterologous reporter gene stably transfected into these cells; such inducible expression depends on combined treatment with LPS and IL-4. Analyses of constructs transiently introduced into a B-cell lymphoma line demonstrated that LPS-plus-IL-4-inducible expression can be conferred by a 179-bp segment of DNA spanning the I epsilon transcriptional initiation site. Mutational analyses demonstrated that this expression depended on DNA sequences within a conserved region directly upstream from the I epsilon transcriptional initiation region. One nuclear protein that is constitutively expressed in normal B cells binds to the downstream end of the conserved sequence; its binding specificity correlates with the functional effect of several mutations. Two additional proteins, which are induced by IL-4 treatment of splenic B cells, bind to the transcription initiation sites of I epsilon. These proteins are indistinguishable in binding assays from proteins previously shown to bind an enhancer region of the class II major histocompatibility complex gene A alpha.

Identification of a Conserved Lipopolysaccharide-Plus-Interleukin-4-Responsive Element Located at the Promoter of Germ Line ε Transcripts

Treatment of splenic B lymphocytes and certain B-lineage cell lines with the mitogen lipopolysaccharide (LPS) and the lymphokine interleukin-4 (IL-4) induces expression of germ line immunoglobulin C epsilon transcripts and class switching to the C epsilon gene. We show that LPS-plus-IL-4 induction of germ line epsilon transcripts (termed I epsilon transcripts) occurs at the transcriptional level in an Abelson murine leukemia virus-transformed pre-B-cell line. A 1.1-kb region of DNA surrounding the I epsilon promoter endows inducible transcription to a heterologous reporter gene stably transfected into these cells; such inducible expression depends on combined treatment with LPS and IL-4. Analyses of constructs transiently introduced into a B-cell lymphoma line demonstrated that LPS-plus-IL-4-inducible expression can be conferred by a 179-bp segment of DNA spanning the I epsilon transcriptional initiation site. Mutational analyses demonstrated that this expression depended on DNA sequences within a conserved region directly upstream from the I epsilon transcriptional initiation region. One nuclear protein that is constitutively expressed in normal B cells binds to the downstream end of the conserved sequence; its binding specificity correlates with the functional effect of several mutations. Two additional proteins, which are induced by IL-4 treatment of splenic B cells, bind to the transcription initiation sites of I epsilon. These proteins are indistinguishable in binding assays from proteins previously shown to bind an enhancer region of the class II major histocompatibility complex gene A alpha.

Anti-CD40 monoclonal antibodies or CD4+ T cell clones and IL-4 induce IgG4 and IgE switching in purified human B cells via different signaling pathways.

IL-4 induces IgE and IgG4 synthesis, but in addition to IL-4, a second signal provided by CD4+ T cells is required. Here we demonstrate that the signal provided by CD4+ T cells can be replaced by anti-CD40 mAb. Highly purified surface (sIgM+) human B cells cultured with soluble anti-CD40 mAb in the presence of IL-4 produced IgM, total IgG, IgG4, and relatively high levels of IgE, indicating that production of these isotypes represented H chain switching and was not the result of a selective outgrowth of isotype committed B cells. No IgA was produced in these cultures. However, the T cell signal was different from the signal provided by anti-CD40 mAb, because in contrast to CD4+ T cells, anti-CD40 mAb failed to induce germ-line epsilon transcripts. However, anti-CD40 mAb strongly enhanced germ-line epsilon mRNA expression induced by IL-4. In addition, IFN-gamma, IFN-alpha, and anti-CD23 mAb, which block IL-4-induced IgE production by PBMC, or B cells cocultured with CD4+ T cell clones, failed to inhibit IgG4 and IgE synthesis induced by anti-CD40 mAb. Finally, anti-CD40 mAb and CD4+ T cell clones had strong synergistic effects on IgG4 and IgE synthesis. These results indicate that different B cell activation pathways can result in IgG4 and IgE switching in the presence of IL-4.

Induction of germ-line and mature C epsilon transcripts in human B cells stimulated with rIL-4 and EBV.

EBV and rIL-4 induce T cell-independent IgE production by normal human B cells. We demonstrate here that EBV and IL-4 induced the synthesis of IgE by surface IgE-negative B cell precursors isolated by cell sorting. This result suggests that the induction of IgE by EBV and IL-4 results not merely from the expansion of a precommitted surface IgE-positive B cell population but more likely from IL-4-directed switching to IgE. At the molecular level, IL-4 and EBV induced the appearance of 2.0- and of 1.8-kilobase (kb) RNA bands, both of which hybridized with an 0.88-kb HinfI fragment spanning part of the C epsilon 1 exon and the entire C epsilon 2 exon. The 1.8-kb band but not the 2.0-kb band also hybridized with a cloned genomic 0.7-kb SmaI fragment located approximately 2 kb upstream of C epsilon. Thus, EBV and IL-4 induced germline (1.8-kb) as well as mature (2.0-kb) C epsilon transcripts. IL-4 by itself induced germ-line C epsilon transcripts but not mature C epsilon transcripts in purified normal B cells. IL-4 failed to induce IgE synthesis in established EBV B cell lines and failed to induce 2.0-kb mature C epsilon transcripts but induced 1.8-kb germ-line C epsilon transcripts. These data show that IL-4 is sufficient for the induction of C epsilon germ-line transcription. In contrast, the transcription of mature epsilon mRNA requires an additional activating signal, provided by infection with EBV. Established EBV transformation results in a dissociation between germ-line C epsilon transcription and the ability to undergo IgE switching in response to IL-4.

Structure and expression of germline epsilon transcripts in human B cells induced by interleukin 4 to switch to IgE production.

Interleukin 4 (IL-4)-induced IgE production coincides with the appearance of the 2.2-kb productive epsilon-mRNA, but is preceded by synthesis of a 1.7-kb epsilon-RNA. Analysis of cDNA copies of the 5' end of this RNA indicated that the 1.7-kb epsilon-RNA is a germline epsilon immunoglobulin heavy chain transcript with an exon mapping 5' to the switch region. Transcription through switch regions has been implicated in the control of class switching. However, IL-4 or cloned CD4+ T cells were able to induce germline epsilon transcripts without inducing IgE synthesis, for which both signals were required. These results indicate that induction of human germline epsilon-RNA does not necessarily result in IgE synthesis, and that additional regulatory mechanisms are involved in class switching.

Structure and expression of germ line immunoglobulin heavy-chain epsilon transcripts: interleukin-4 plus lipopolysaccharide-directed switching to C epsilon.

We have isolated cDNA clones complementary to a truncated immunoglobulin heavy-chain C epsilon RNA transcript previously found to be induced in B lymphoid cells by treatment with lipopolysaccharide (LPS) combined with interleukin-4 (IL-4). We demonstrate that this transcript initiates from a promoter upstream of the germ line epsilon class-switch recombination region (S epsilon region). The major germ line C epsilon transcript contains a small 5' exon contributed by sequences upstream of the S epsilon region spliced to the normal C epsilon exons. Treatment of splenic B lymphoid cells with LPS plus IL-4 induces the expression of transcripts from the germ line epsilon transcription unit followed by expression of normal immunoglobulin epsilon heavy-chain mRNA. Furthermore, we demonstrate that similar treatment of transformed precursor B cell lines induces the expression of germ line epsilon transcripts followed by class switching to epsilon expression in these lines. This is the first demonstration of switching to epsilon in cells of the pre-B stage. The general structure of the germ line epsilon transcript and transcription unit is similar to that previously characterized for germ line gamma 2b transcripts. However, expression of these two germ line transcription units in B-lineage cells is inversely regulated by IL-4 (plus LPS) treatment, correlating with the effects of these treatments on switching to these loci.

Structure and Expression of Germ Line Immunoglobulin Heavy-Chain ε Transcripts: Interleukin-4 Plus Lipopolysaccharide-Directed Switching to Cε

We have isolated cDNA clones complementary to a truncated immunoglobulin heavy-chain C epsilon RNA transcript previously found to be induced in B lymphoid cells by treatment with lipopolysaccharide (LPS) combined with interleukin-4 (IL-4). We demonstrate that this transcript initiates from a promoter upstream of the germ line epsilon class-switch recombination region (S epsilon region). The major germ line C epsilon transcript contains a small 5' exon contributed by sequences upstream of the S epsilon region spliced to the normal C epsilon exons. Treatment of splenic B lymphoid cells with LPS plus IL-4 induces the expression of transcripts from the germ line epsilon transcription unit followed by expression of normal immunoglobulin epsilon heavy-chain mRNA. Furthermore, we demonstrate that similar treatment of transformed precursor B cell lines induces the expression of germ line epsilon transcripts followed by class switching to epsilon expression in these lines. This is the first demonstration of switching to epsilon in cells of the pre-B stage. The general structure of the germ line epsilon transcript and transcription unit is similar to that previously characterized for germ line gamma 2b transcripts. However, expression of these two germ line transcription units in B-lineage cells is inversely regulated by IL-4 (plus LPS) treatment, correlating with the effects of these treatments on switching to these loci.