Epsilon Germline Transcription Research Articles

Extrafollicular IgD+ B cells generate IgE antibody secreting cells in the nasal mucosa

Increased IgE is a typical feature of allergic rhinitis. Local class-switch recombination has been intimated but B cell precursors and mechanisms remain elusive. Here we describe the dynamics underlying the generation of IgE-antibody secreting cells (ASC) in human nasal polyps (NP), mucosal tissues rich in ASC without germinal centers (GC). Using VH next generation sequencing, we identified an extrafollicular (EF) mucosal IgD+ naïve-like intermediate B cell population with high connectivity to the mucosal IgE ASC. Mucosal IgD+ B cells, express germline epsilon transcripts and predominantly co-express IgM. However, a small but significant fraction co-express IgG or IgA instead which also show connectivity to ASC IgE. Phenotypically, NP IgD+ B cells display an activated profile and molecular evidence of BCR engagement. Transcriptionally, mucosal IgD+ B cells reveal an intermediate profile between naïve B cells and ASC. Single cell IgE ASC analysis demonstrates lower mutational frequencies relative to IgG, IgA, and IgD ASC consistent with IgE ASC derivation from mucosal IgD+ B cell with low mutational load. In conclusion, we describe a novel mechanism of GC-independent, extrafollicular IgE ASC formation at the nasal mucosa whereby activated IgD+ naïve B cells locally undergo direct and indirect (through IgG and IgA), IgE class switch.

Corticosteroids inhibit anti-IgE activities of specialized proresolving mediators on B cells from asthma patients.

Specialized proresolving mediators (SPMs) promote the resolution of inflammation and exert beneficial effects in animal models of chronic inflammatory diseases, including asthma. Previously, we have shown that certain SPMs reduce IgE production in B cells from healthy individuals, which has a critical role in allergic asthma. Here, we investigated the effects of SPMs on B cell IgE production in asthma patients. Peripheral blood mononuclear cells from asthma patients were treated with 17-HDHA or RvD1, and IgE levels were measured. RvD1 and 17-HDHA dampened IgE production in B cells from most asthma patients, whereas B cells from a subset of patients taking oral steroids were refractory to SPM treatment. Molecular mechanisms underlying the interaction between corticosteroids and SPMs were investigated by treating B cells from nonasthmatic donors with corticosteroids in vitro. Corticosteroids blocked the inhibitory effects of 17-HDHA and RvD1 on B cell IgE production by abolishing the suppressive activity of these mediators on IgE class switching. Corticosteroids decreased the expression of transcriptional repressor Bcl-6 as well as its suppressive activity on epsilon germline transcription. We conclude that 17-HDHA and RvD1 can reduce IgE production in asthma patients not taking high doses of steroids but that corticosteroids interfere with the ability of B cells to respond to proresolving mediators.

Specialized proresolving mediators (SPMs) inhibit human B‐cell IgE production

Specialized proresolving mediators (SPMs) constitute a recently recognized class of bioactive molecules thatpromote the resolution of inflammation. We recently reported that the SPMs resolvin D1 (RvD1) and 17-hydroxydocosahexaenoic acid (17-HDHA) promote the differentiation of IgG-secreting B cells and enhance antibody-mediated immune responses. However, there is an important knowledge gap regarding whether or not SPMs regulate human B-cell IgE production, which is the key effector in diseases such as asthma and allergy. Therefore, we investigated whether a panel of diverse SPMs influences B-cell IgE production. An important finding was that 17-HDHA and RvD1 inhibit IgE production by human B cells and suppress the differentiation of naïve B cells into IgE-secreting cells by specifically blocking epsilon germline transcript. This effect is specific to human IgE, as the SPMs do not inhibit production of IgM and IgG and did not suppress other IL-4-upregulated genes. 17-HDHA and RvD1 act by stabilizing the transcriptional repressor B-cell lymphoma 6, which competes with STAT6 for binding at the epsilon germline transcript promoter. Overall, these new findings demonstrate that certain SPMs inhibit the differentiation of IgE-producing B cells, without being broadly immune suppressive, representing a novel class of potential therapeutics for IgE-driven diseases such as asthma and allergy.

Berberine and limonin suppress IgE production by human B cells and peripheral blood mononuclear cells from food-allergic patients

BackgroundCurrently, there is no satisfactory treatment for IgE-mediated food allergy. Food Allergy Herbal Formula 2 (FAHF-2) and butanol-purified FAHF-2 (B-FAHF-2) have been shown to protect against peanut-induced anaphylaxis and inhibit IgE synthesis in a murine model. ObjectiveTo determine which herbs and compounds in FAHF-2 and B-FAHF-2 suppress IgE production. MethodsThe effect of FAHF-2 and B-FAHF-2 on IgE production was determined using a human B-cell line (U266). Individual compounds were isolated and identified using column chromatography, liquid chromatographic mass spectrometry, and nuclear magnetic resonance techniques. The potency of compounds on IgE suppression were investigated using U266 cells and verified using human peripheral blood mononuclear cells (n = 25) from peanut-allergic patients. Epsilon germline transcript expression was determined. Phosphorylated IκBα level was analyzed using the In-Cell Western assay. The mRNA expression of signal transducer and activator of transcription-3, T-box transcription factor TBX21, interferon-γ, forkhead box P3, GATA-binding protein 3, interleukin-10, and interleukin-5 also were analyzed using real-time polymerase chain reaction. ResultsFAHF-2 and B-FAHF-2 inhibited IgE production by U266 cells. B-FAHF-2 was 9 times more effective than FAHF-2. Two compounds that inhibited IgE production were isolated from Philodendron chinensis and identified as berberine and limonin. Berberine was more potent and inhibited IgE production by peripheral blood mononuclear cells by 80% at 0.62 μg/mL. Berberine significantly inhibited ε-germline transcript expression by peripheral blood mononuclear cells. Phosphorylated IκBα level was significantly suppressed and mRNA expressions of T-box transcription factor TBX21 and signal transducer and activator of transcription-3 were significantly increased by berberine. ConclusionBerberine and limonin mediated IgE suppression. The mechanism by which berberine modulates ε-germline transcript expression might be through regulating the phosphorylated IκBα level and the expressions of signal transducer and activator of transcription-3 and T-box transcription factor TBX21. Trial RegistrationClinicaltrials.gov identifier NCT00602160.

Local Class Switching In Nasal Mucosa In Non-allergic Rhinitis

BACKGROUND: Non-allergic rhinitis (NAR) can be symptomatically similar to allergic rhinitis (AR) except that patients do not exhibit allergen sensitivity with skin prick testing (SPT). Recent studies investigated the presence of epsilon germline transcripts in the lungs and nasal cavities in patients with active allergic disorders. This marker is a reflection of active isotype class switching to IgE and, therefore, is categorically diagnostic of an ongoing allergic disorder. Given that IgE can be produced locally and lead to symptoms of NAR, we sought to determine whether the presence of epsilon germline transcription indicates local production of IgE in NAR.

Inhibition of IgE production by docosahexaenoic acid is mediated by direct interference with STAT6 and NFκB pathway in human B cells

Nutrition can modify the onset or severity of diseases and recent changes in eating habits are supposed to promote immunoglobulin (Ig) E-dependent disorders. The n-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) possesses immunomodulatory properties and has been shown to influence chronic and allergic inflammatory disorders in vivo. Here, we examined the impact of DHA on primary human B cells to elucidate its potential role in direct regulation of IgE production and the underlying mechanisms of action. Therefore, cells were stimulated with anti-CD40/interleukin (IL)-4 in the presence of DHA. Subsequently, Ig production, generation of antibody secreting cells, epsilon-germline transcript (ɛGLT) and activation induced desaminase (AID) expression as well as IgE relevant signaling pathways were analyzed. Our results reveal that DHA inhibits IgE production (75±14%) and, depending on concentration, the differentiation of IgE secreting cells (59±27%). The reduction of IgE is accompanied by a direct inhibition of the switching process indicated by decreased ɛGLT and AID transcription. DHA causes both a reduced CD40 dependent nuclear factor κB-p50 translocation into the nucleus and a decreased IL-4 receptor expression which was associated with a reduction of IL-4 driven signal transducer and activator of transcription 6 phosphorylation. Taken together, DHA inhibits IgE production of human B cells by direct interference with both the CD40 and the IL-4 signaling pathway. The data provide one explanation for the anti-allergic role of DHA at the molecular level.

Transmembrane activator, calcium modulator, and cyclophilin ligand interactor drives plasma cell differentiation in LPS-activated B cells

Transmembrane activator, calcium modulator, and cyclophilin ligand interactor (TACI) expression on B cells is upregulated by Toll-like receptor (TLR) 4. We sought to examine whether TACI synergizes with TLR4 in driving immunoglobulin production by B cells and to examine the mechanism of this synergy. Purified mouse naive B cells were stimulated with the TACI ligand a proliferation-inducing ligand (APRIL) and with suboptimal concentrations of the TLR4 ligand LPS in the presence or absence of IL-4. Immunoglobulin secretion was measured by means of ELISA. Surface IgG1-positive B cells and CD138+ plasmacytoid cells were enumerated by means of FACS. Expression of gamma1 and epsilon germline transcripts, activation-induced cytidine deaminase, and gamma1 and epsilon mature transcripts was measured by means of RT-PCR. APRIL synergized with LPS in driving B-cell proliferation and IgM, IgG1, IgG3, IgE, and IgA production. This was mediated by TACI because it was preserved in B-cell maturation antigen-/-, but not TACI-/-, B cells. APRIL and LPS synergized to promote isotype switching, as evidenced by increased expression of activation-induced cytidine deaminase and gamma1 and epsilon mature transcripts and generation of surface IgG1-positive cells. More importantly, APRIL and LPS strongly synergized to drive the plasma cell differentiation program, as evidenced by an increase in CD138+ cells and expression of B lymphocyte induced maturation protein-1 (Blimp-1), interferon regulatory factor-4 (IRF-4), and the spliced form of X-box binding protein-1 (XBP-1). TACI-/- mice had impaired IgM and IgG1 antibody responses to immunization, with a suboptimal dose of the type I T cell-independent antigen 2, 4, 6- Trinitrophenol (TNP)-LPS. These observations suggest that TACI cooperates with TLR4 to drive B-cell differentiation and immunoglobulin production in vitro and in vivo.

Andolast Acts at Different Cellular Levels to Inhibit Immunoglobulin E Synthesis

The anti-asthmatic agent andolast is thought to inhibit the release of allergic mediators, but its mechanism of action is not fully understood. We investigated whether the compound inhibits immunoglobulin E (IgE) synthesis and tested the hypothesis that andolast affects immunoglobulin class switching. Interleukin (IL)-4 and the interaction of CD40 expressed on B cells with its ligand on T cells are necessary for IgE synthesis. Thus, peripheral blood mononuclear cells (PBMCs) from 40 asthmatic, 16 non-asthmatic allergic, and 9 normal donors were stimulated with IL-4 and/or anti-CD40 antibody. T cells from 9 additional allergic donors were activated with anti-CD3/CD28 antibodies to express IL-4 mRNA. After incubation in the absence or presence of test compounds, immunoglobulin concentrations were measured by enzyme immunoassay, and mRNA levels were analyzed by RT-PCR. Andolast significantly inhibited IgE synthesis by stimulated PBMCs from both asthma patients and combined allergic/normal donors. In mechanistic studies, andolast was found to act at different cellular levels. Firstly, it reduced by about 45 percent (p<0.05) the levels of IL-4 mRNA in T cells stimulated with anti-CD3/CD28. Secondly, andolast reduced by about 36 percent (p<0.05) the expression of epsilon germline transcripts in PBMCs stimulated with IL-4/anti-CD40. Thirdly, the effect of andolast on immunoglobulin synthesis was selective in that the production of IgG4 antibodies was not significantly inhibited. Our findings, while supporting the evidence that andolast is effective for the treatment of asthma, provide new insights into its mechanism of action.

Regulation of epsilon germline transcription and switch region mutations by IgH locus 3′ enhancers in transgenic mice (35.3)

Abstract Germline (GL) transcription is regulated by specific promoters and Immunoglobulin heavy chain (IgH) 3′ locus enhancers and is necessary for Ig class switch recombination (CSR). We have generated different transgenic lines containing the GL epsilon promoter, switch (S)_psilon region and constant (C)_psilon region with or without the DNase I-sensitive regions (HS)3A-HS1,2 or HS3B-HS4 3′ IgH enhancer pairs. The enhancerless construct was expressed in B cells activated by interleukin (IL)-4 and cluster of differentiation (CD)40, thus resembling regulation of the endogenous gene. Both enhancer-containing transgenes efficiently increased expression in B cells and were strongly up-regulated by stimuli. In addition, S epsilon regions of the transgene containing HS3B-HS4 were mutated in activated, sorted B cells. Such mutations are known to precede CSR and are dependent on activation induced cytidine deaminase (AID). Our findings show that all elements necessary for recruitment of the recombination machinery are present in the transgene containing HS3 and HS4. These enhancers probably provide something more specific than mere increased accessibility of switch regions. We propose that transcription factors binding the enhancers help to target the recombination machinery to the switch regions. This work was supported by grants from the Swedish Research Council, the Swedish Institute and the Karolinska Institutet

Induction of IgE and allergic‐type responses in fur mite‐infested mice

High serum IgE levels are characteristic of allergic diseases and immune responses to nematode parasites. A murine allergy model based on infestation with the fur mites Myocoptes musculinus and Myobia musculi was investigated. Analysis of mite infestation in various knockout mice revealed that IgE production in response to these ectoparasites was dependent on T cells, IL-4, and CD40L. Secretion of IL-4 by CD4+ T cells obtained from peripheral LN draining mite-infested skin sites was increased with progressing mite infestation and correlated with the serum IgE induction. A time course analysis of the mRNA expression of switched IgE, activation-induced cytidine deaminase (AID), and epsilon germ-line transcripts (epsilonGLT) suggested that switching to IgE in response to fur mites occurred initially in skin-draining LN. In addition, mite infestation induced mast cell degranulation in the skin as well as mast cell infiltration into skin-draining LN. Analysis of the immune response generated in mite-infested mice is a valuable model for the investigation of allergic disorders and provides information for better understanding of mechanisms involved in IgE induction and regulation in a physiological way of allergen-exposure resembling atopic sensitization in humans.

The extract of syngeneic keratinocytes enhances IgE production from BALB/c mouse splenic lymphocytes in vitro

The increase of serum IgE levels is closely associated with atopic dermatitis. We have previously revealed that cellular extract of PAM212 cells (PAM-extract), BALB/c mouse keratinocyte cell line, induced a remarkable increase of serum IgE levels, in vivo, when subcutaneously injected into BALB/c mice. However, precise mechanism of IgE-increasing activity was unclear. To elucidate the mechanism of IgE-increase in sera of BALB/c mice induced by PAM-extract, we explored the direct influence of PAM-extract on immunoglobulin production and class-switching in the culture of splenic lymphocytes and purified B-cells, in vitro. Splenic lymphocytes or purified B-cells obtained from BALB/c mice were cultured with various combinations of IL-4, anti-CD40 antibody, and PAM-extract for 7 days. IgE and IgG concentrations of culture supernatants were measured by ELISA. Epsilon germ-line transcriptions were assessed by RT-PCR from the cultured cells. IgE and IgG concentrations in culture supernatant of splenic lymphocytes were increased by an addition of PAM-extract in the presence of both IL-4 and anti-CD40 antibody. Epsilon germ-line transcript was also induced in parallel to the increase of IgE production. Similar results were obtained when purified B-cells were employed in stead of whole splenic lymphocytes. The enhancement of IgE production in vitro was also observed, when splenic lymphocytes of CBAj mouse were cultured with cellular extract of KCMH-1 cells, CBAj mouse keratinocyte cell line. The cellular extract of keratinocyte promotes immunoglobulin class-switching to IgE and IgE production from mouse splenic B-cells in an IL-4- and CD40-stimuli-dependent manner. Such enhancement may account for the increase of serum IgE in patients with dermatitis in association with a Th2 microenvironment.

Oral administration of pulverized Konjac glucomannan prevents the increase of plasma immunoglobulin E and immunoglobulin G levels induced by the injection of syngeneic keratinocyte extracts in BALB/c mice

Immunoglobulin (Ig) E plays a key role in the pathogenesis of atopic diseases, such as asthma, atopic dermatitis and allergic rhinitis. Oral administration of pulverized Konjac glucomannan (PKGM) has recently been demonstrated to prevent both plasma IgE elevation and developing dermatitis in NC/Nga mice, a model of atopic dermatitis. To clarify the direct effect of PKGM on the increase of plasma IgE, we employed the system of BALB/c mouse that increases IgE level without developing dermatitis in response to continuous injection of the extract of syngeneic keratinocytes, PAM 212 cells (PAM extract). Three weeks after the start of feeding with either control or PKGM diet, mice were injected subcutaneously with PAM extract bi-weekly for 10 weeks. The levels of plasma Igs were measured by enzyme-linked immunosorbent assay every 2 weeks after the injection. The levels of epsilon germline transcription and the amounts of mRNA for IL-4, IFN-gamma, GATA-3 and T bet gene in the spleen were evaluated by real-time RT-PCR at the end of the experiment. On the one hand, PKGM prevented the increase of plasma IgE and IgG (IgG1, IgG2b) induced by PAM extract, and on the other hand, it enhanced the levels of plasma IgG3. However, it did not affect the level of plasma IgM. PKGM also reduced the levels of plasma ovalbumin (OVA)-specific IgE in OVA-sensitized mice. Moreover, PKGM attenuated the induction of epsilon germline transcription and expression levels of mRNA for IL-4, IFN-gamma and GATA-3 in the spleen of PAM extract-injected mice. PKGM also attenuated the induction of epsilon germline transcription and mRNA for IFN-gamma and T bet in the spleen of phosphate-buffered saline-injected control mice. These results suggested that oral administration of PKGM prevents the elevation of plasma IgE by suppressing IgE class switching in B cells and/or the commitment development of naive lymphocytes to both T-helper type 1 (Th1) and Th2.

094 IFN-gamma-mediated inhibition of human IgE synthesis by IL-21 is associated with a polymorphism in the IL-21 R gene

Introduction IL-21 is a cytokine produced by CD4+ T cells that has been reported to regulate human, as well as, mouse T and NK cell function and to inhibit antigen-induced IgE production by mouse B cells. Methods Human splenic or peripheral B cells were activated in vitro with an anti-CD40 monoclonal antibody (mAb) and IL-4, in the presence or absence of IL-21, and the production of IgE, as well as the expression of IL-4-induced germline epsilon trans-cripts by these cells were analyzed by isotype-specific ELISA and Northern blotting analysis, respectively. The polymorphism of the IL-21R gene was determined by DNA sequencing analysis. Results Although rIL-21 does not affect rIL-4-induced germline epsilon mRNA expression in purified human spleen or peripheral blood B cells, it strongly enhances IgE production by both CD 19+, CD27- naive, and CD19+.CD27+ memory B cells, stimulated with anti-CD40 mAb and rIL-4. However, rIL-21 inhibits allergen-induced IgE production in peripheral blood mononuclear cells (PBMC), as well as rIL-4-induced production of this isotype in cultures of PBMC or total splenocytes, by an IFN- gamma-dependent mechanism. The presence of a polymorphism (T-83C), encoding a variant IL-21R, in donors heterozygous for this mutation was not only associated with a lower sensitivity to the IgE production inhibiting effects rIL-21, but also with lower rIL-21-induced IFN-gamma production levels, as compared to donors carriyng the wild-type allele of the IL-21R. Conclusion These results show that IL-21 differentially regulâtes IL-4-induced human IgE production, via its growth-promoting capacity on IgE-committed B cells, as well as via its ability to induce the production of IFN-gamma, most likely by T and NK cells, whereas the outcome of these IL-21-mediated effects is dependent on the presence of a polymorphism in the IL-21R.

Inhibition of Interleukin-4-induced Class Switch Recombination by a Human Immunoglobulin Fcγ-Fcϵ Chimeric Protein

Immunoglobulin E (IgE) is important in mediating human allergic diseases. We tested the hypothesis that a human Ig Fc gamma-Fc epsilon bifunctional chimeric protein, GE2, would inhibit IgE class switch recombination (CSR) by co-aggregating B-cell CD32 and CD23. Indeed, GE2 directly inhibited epsilon germ-line transcription, subsequent CSR to epsilon and IgE protein production. This CSR inhibition was dependent on CD23 binding and the phosphorylation of extracellular signal-related kinase (ERK), and it was mediated via suppression of interleukin-4-induced STAT6 phosphorylation. Treatment with PD98059, a specific inhibitor of mitogen-activated protein kinase kinase 1 (MAPKK1 (MEK1)) and MEK2 reversed the ability of GE2 to decrease CSR and STAT6 phosphorylation. GE2 stimulation induced ERK phosphorylation, whereas it did not alter the phosphorylation of c-Jun N-terminal kinase or p38 MAPK. The ability of GE2 to block human isotype switching to epsilon, in addition to its already demonstrated ability to inhibit mast cell and basophil function, suggests that it will provide an important novel benefit in the treatment of IgE-mediated diseases.

1?,25-dihydroxyvitamin?D3 inhibits anti-CD40 plus IL-4-mediated IgE productionin vitro

In the present study, we examined whether anti-CD40+IL-4-mediated B cell proliferation and immunoglobulin synthesis is affected by vitamin D (VD) and its low-hypercalcemic analogue EB1089 in Bcells from healthy donors. Analysis of vitamin D receptor (VDR) expression showed that only anti-CD40+IL-4-stimulated, but not resting B cells express VDR. Studies on B cell proliferation revealed that anti-CD40+IL-4-mediated proliferation of B cells was not affected by VD or EB1089. By contrast, IgE synthesis was markedly inhibited by both, VD and EB1089, starting at concentrations from 10(-10) M for VD and 10(-12) M for EB1089, with maximal inhibition at 10(-6) M (VD 85.5+/-9.7%; EB1089 77.3+/-10.8%). The production of the other Ig (IgA and IgG) was not significantly inhibited by VD after anti-CD40+IL-4 stimulation, and IgM production was only slightly reduced (18.7+/-7.9%). These observations were confirmed by intracellular staining of the different isotypes in B cells after anti-CD40+IL-4 stimulation, which showed a strong reduction of IgE(+) cells in the presence of VD. Analyses of molecules that are known to affect IgE production (CD23 and IL-6) revealed that these are not involved in VD-dependent inhibition of IgE production. By contrast, epsilon germ-line transcription was inhibited by VD (41.2+/-26.1%; n=5), as was NF-kappaB (p50 and p65) protein expression in stimulated cells. These data show that VD and its analogue EB1089 inhibit IgE production of anti-CD40+IL-4-stimulated B cells in vitro. The involved mechanism includes epsilon germ-line transcription, NF-kappaB activation and switch recombination suggesting that complex mechanisms of VD action in anti-CD40+IL-4-stimulated B cells are responsible.

1alpha,25-dihydroxyvitamin D3 inhibits anti-CD40 plus IL-4-mediated IgE production in vitro.

In the present study, we examined whether anti-CD40+IL-4-mediated B cell proliferation and immunoglobulin synthesis is affected by vitamin D (VD) and its low-hypercalcemic analogue EB1089 in Bcells from healthy donors. Analysis of vitamin D receptor (VDR) expression showed that only anti-CD40+IL-4-stimulated, but not resting B cells express VDR. Studies on B cell proliferation revealed that anti-CD40+IL-4-mediated proliferation of B cells was not affected by VD or EB1089. By contrast, IgE synthesis was markedly inhibited by both, VD and EB1089, starting at concentrations from 10(-10) M for VD and 10(-12) M for EB1089, with maximal inhibition at 10(-6) M (VD 85.5+/-9.7%; EB1089 77.3+/-10.8%). The production of the other Ig (IgA and IgG) was not significantly inhibited by VD after anti-CD40+IL-4 stimulation, and IgM production was only slightly reduced (18.7+/-7.9%). These observations were confirmed by intracellular staining of the different isotypes in B cells after anti-CD40+IL-4 stimulation, which showed a strong reduction of IgE(+) cells in the presence of VD. Analyses of molecules that are known to affect IgE production (CD23 and IL-6) revealed that these are not involved in VD-dependent inhibition of IgE production. By contrast, epsilon germ-line transcription was inhibited by VD (41.2+/-26.1%; n=5), as was NF-kappaB (p50 and p65) protein expression in stimulated cells. These data show that VD and its analogue EB1089 inhibit IgE production of anti-CD40+IL-4-stimulated B cells in vitro. The involved mechanism includes epsilon germ-line transcription, NF-kappaB activation and switch recombination suggesting that complex mechanisms of VD action in anti-CD40+IL-4-stimulated B cells are responsible.

Retrovirally Delivered Random Cyclic Peptide Libraries Yield Inhibitors of Interleukin-4 Signaling in Human B Cells

Inteins are polypeptide sequences found in a small set of primarily bacterial proteins that promote the splicing of flanking pre-protein sequences to generate mature protein products. Inteins can be engineered in a "split and inverted" configuration such that the protein splicing product is a cyclic polypeptide consisting of the sequence linking two intein subdomains. We have engineered a split intein into a retroviral expression system to enable the intracellular delivery of a library of random cyclic peptides in human cells. Cyclization of peptides could be detected in cell lysates using mass spectrometry. A functional genetic screen to identify 5-amino acid-long cyclic peptides that block interleukin-4 mediated IgE class switching in B cells yielded 13 peptides that selectively inhibited germ line epsilon transcription. These results demonstrate the generation of cyclic peptide libraries in human cells and the power of functional screening to rapidly identify biologically active peptides.

CD40-mediated p38 mitogen-activated protein kinase activation is required for immunoglobulin class switch recombination to IgE.

Signaling through CD40 activates multiple kinases and signal pathways that drive diverse CD40-mediated biologic functions. The specific pathways activated by CD40 signaling involving CD40-dependent Ig class switch recombination (CSR) have not been defined. We sought to dissect CD40-activated signaling required for CD40-mediated Ig CSR by using the specific signal pathway inhibitors, with the emphasis on CD40-activated p38 mitogen-activated protein kinase (p38 MAPK) signaling in CD40-mediated CSR to IgE. Human B cells were costimulated with IL-4 plus anti-CD40 in the presence or absence of specific signal pathway inhibitors. Ig production, kinase phosphorylation, IgH epsilon germline transcripts and Smu-Sepsilon recombination were examined, and their relationships were analyzed. CD40-dependent IgE induction was inhibited by the specific p38 MAPK inhibitor SB203580 but not by the extracellular signal-regulated protein kinase-specific inhibitor PD98059 or the phosphatidylinositol 3-kinase-specific inhibitor LY294002. CD40 activation of p38 MAPK correlated with CD40-dependent IgE production, and IgE suppression by SB203580 correlated with the inhibition of CD40-activated p38 MAPK phosphorylation. Suppression of IgE production by SB203580 was not due to inhibition of cell proliferation because SB203580 did not suppress IL-4 plus alpha-CD40-induced cell proliferation. SB203580, but neither PD98059 nor LY294002, inhibited CD40-dependent Smu-Sepsilon recombination, as determined by using a digestion circularization PCR assay. The inhibitory effects of SB203580 on IgE production and Smu-Sepsilon recombination were directly related to its ability to suppress production of Ig epsilon germline transcripts. These results demonstrate that p38 MAPK is required for CD40-mediated class switching to IgE.

IL-10 enhances B-cell IgE synthesis by promoting differentiation into plasma cells, a process that is inhibited by CD27/CD70 interaction.

Interleukin-10 (IL-10) is a major regulatory cytokine of inflammatory responses that is considered to play an important role in specific immunotherapy. However, whether IL-10 enhances or inhibits B-cell IgE production has remained a matter of contention. To clarify the effect of IL-10 on IgE synthesis in the presence of IL-4 and CD40 signalling, we examined B-cell proliferation, germline epsilon transcripts and plasma cell differentiation. In addition, the effect of CD27 signalling on IgE synthesis in the presence of IL-10, IL-4 and CD40 signalling was investigated. IL-10 facilitated the production of IgE in mononuclear cells and highly purified B-cells, enhanced B-cell proliferation and, most importantly, promoted the generation of plasma cells. However, IL-10 did not enhance expression of germline epsilon transcripts. The addition of CD27 signalling through the use of CD32-CD27 ligand (CD70) double transfectants significantly diminished the B-cell proliferation, IgE synthesis and plasma cell differentiation enhanced by IL-10. IL-10 enhances B-cell IgE production by promoting differentiation into plasma cells. CD27/CD70 interactions under IL-10 and sufficient CD40 cosignalling exert the opposite effect on IgE synthesis. The results of this study indicate that precautions are critical when planning immunotherapy using IL-10 in IgE-related allergic diseases.

CD45 Controls Interleukin-4-mediated IgE Class Switch Recombination in Human B Cells through Its Function as a Janus Kinase Phosphatase

CD45 plays a critical regulatory role in receptor signaling through its protein tyrosine phosphatase and Janus kinase (JAK) phosphatase activities. To investigate whether CD45 also plays a regulatory role in Ig class switching in human B cells, we examined the effects of CD45 triggering on Ig class switching to IgE and its relationship with CD45 JAK phosphatase activity. Anti-CD45 triggering of CD45 significantly inhibited interleukin-4 + anti-CD40-induced switch recombination in a switch recombination vector assay in stably transfected Ramos 2G6 human B cells, as well as Ig epsilon germ-line transcription and Smu-Sepsilon switch recombination in primary human B cells. These negative regulatory effects on Ig class switching were concomitant with the ability of CD45 to dephosphorylate the induced phosphorylation of JAK1, JAK3, and signal transducer and activator of transcription 6, but not on stress-activated/mitogen-activated protein kinases. We also showed that phosphorylated JAK1 and JAK3 were directly dephosphorylated by recombinant CD45 in vitro. These results indicate that CD45 is able to function as JAK phosphatase in human B cells and that this activity is directly associated with the negative regulation of the class switch recombination to IgE. CD45 may be an appropriate target drug for modulating IgE in allergic diseases.