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.

Tracing Human IgE B Cell Antigen Receptor-Bearing Cells With a Monoclonal Anti-Human IgE Antibody That Specifically Recognizes Non-Receptor-Bound IgE.

Up to 30% of the population suffers from immunoglobulin E (IgE)-mediated allergies. Despite current stepwise gating approaches, the unambiguous identification of human IgE-producing cells by flow cytometry and immunohistology remains challenging. This is mainly due to the scarcity of these cells and the fact that IgE is not only expressed in a membrane-bound form on the surface of IgE-producing cells in form of the B cell antigen receptor (BCR), but is more frequently found on various cell types bound to the low and high affinity receptors, CD23 and FcϵRI, respectively. Here we sought to develop a sequential gating strategy for unambiguous detection of cells bearing the IgE BCR on their surface. To that aim we first tested the monoclonal anti-IgE antibody omalizumab for its ability to discriminate between IgE BCR and receptor-bound IgE using cells producing IgE or bearing IgE bound to CD23 as well as basophils exhibiting FcϵRI receptor-bound IgE. Using flow cytometry, we demonstrated that omalizumab recognized IgE producing cells with a high sensitivity of up to 1 IgE+ cell in 1000 human peripheral blood mononuclear cells (PBMCs). These results were confirmed by confocal microscopy both in cell suspensions as well as in nasal polyp tissue sections. Finally, we established a consecutive gating strategy allowing the clear identification of class-switched, allergen-specific IgE+ memory B cells and plasmablasts/plasma cells in human PBMCs. Birch pollen specific IgE+ memory B cells represented on average 0.734% of total CD19+ B cells in allergic patients after allergen exposure. Thus, we developed a new protocol for exclusive staining of non-receptor bound allergen-specific IgE+ B cell subsets in human samples.

YY1 control of mitochondrial-related genes does not account for regulation of immunoglobulin class switch recombination in mice.

Immunoglobulin class switch recombination (CSR) occurs in activated B cells with increased mitochondrial mass and membrane potential. Transcription factor Yin Yang 1 (YY1) is critical for CSR and for formation of the DNA loops involved in this process. We therefore sought to determine if YY1 knockout impacts mitochondrial gene expression and mitochondrial function in murine splenic B cells, providing a potential mechanism for regulating CSR. We identified numerous genes in splenic B cells differentially regulated when cells are induced to undergo CSR. YY1 conditional knockout caused differential expression of 1129 genes, with 59 being mitochondrial-related genes. ChIP-seq analyses showed YY1 was directly bound to nearly half of these mitochondrial-related genes. Surprisingly, at the time when YY1 knockout dramatically reduces DNA loop formation and CSR, mitochondrial mass and membrane potential were not significantly impacted, nor was there a significant change in mitochondrial oxygen consumption, extracellular acidification rate, or mitochondrial complex I or IV activities. Our results indicate that YY1 regulates numerous mitochondrial-related genes in splenic B cells, but this does not account for the impact of YY1 on CSR or long-distance DNA loop formation.

The Protective Effects of 2,3,5,4'-Tetrahydroxystilbene-2-O-ß-d-Glucoside in the OVA-Induced Asthma Mice Model.

Asthma is an inflammatory disease caused by an imbalance of Th1 and Th2 cells. In general, asthma is characterized by a stronger Th2 response. Most conventional asthma treatment focuses on improving airway flow or suppression of airway inflammation. To reduce the side effects of currently used asthma medicines, we have conducted studies on natural products that have no side effects. 2,3,5,4'-tetrahydroxystilbene-2-O-ß-d-glucoside (TSG), the main compound of Polygonum multiflorum (PM), has various biological activities, including anti-inflammation and anti-oxidation activities. However, the effect of TSG on asthma has not been studied yet. We examined the effects of TSG on Th2 immune responses using an OVA-induced asthma animal model. OVA-sensitized mice were treated with TSG. 24 h after the last intranasal challenge, airway hyperresponsiveness (AHR) was measured or serum and bronchoalveolar lavage fluid (BALF) were harvested. We measured typical Th1 and Th2 cytokines in serum and BALF. As a result, TSG suppressed Th2 responses, as shown by the lower levels of IL-4, IL-5, total IgE, OVA-specific IgE, and OVA-specific IgG1. On the other hand, TSG increased Th1 responses, as shown by the levels of IFN-gamma. Collectively, these results confirm the potential of TSG for asthma treatment through modulation of inflammatory responses. Considering that the cytotoxic effect of PM extract is due to the cis isomer of TSG, if the effect of TSG on asthma treatment is found to be non-toxic in clinical trials, it would be more effective to use it as a purified component than PM extract as an asthma treatment agent.

Suppressive effect of ethanol extract from mango (Mangifera indica L.) peel on IgE production in vitro and in vivo.

Immunoglobulin E (IgE) is involved in the onset of allergic reaction, and the suppression of IgE production leads to alleviation of allergic symptoms. We found that mango peel ethanol extract (MPE) significantly suppresses IgE production by human myeloma cell line U266 cells, suggesting that MPE has an anti-allergic effect by inhibiting the production of IgE. Although mangiferin is contained in mango, which suppresses IgE production by U266 cells, it was not contained in MPE. We investigated the suppressive effect of MPE in 2,4-dinitrofluorobenzene (DNFB)-induced allergic contact dermatitis model mice. The elevation of serum IgE level was significantly suppressed by oral administration of MPE. Intake of MPE also suppressed the expression level of IL-4 in the DNFB-challenged ears, suggesting that MPE suppresses the IL-4-mediated maturation into IgE-producing cells. Our findings indicate that MPE has a potential to alleviate the increase in serum IgE level that is feature of type I allergy.

Transcription factor YY1 can control AID-mediated mutagenesis in mice.

Activation-induced cytidine deminase (AID) is crucial for controlling the immunoglobulin (Ig) diversification processes of somatic hypermutation (SHM) and class switch recombination (CSR). AID initiates these processes by deamination of cytosine, ultimately resulting in mutations or double strand DNA breaks needed for SHM and CSR. Levels of AID control mutation rates, and off-target non-Ig gene mutations can contribute to lymphomagenesis. Therefore, factors that control AID levels in the nucleus can regulate SHM and CSR, and may contribute to disease. We previously showed that transcription factor YY1 can regulate the level of AID in the nucleus and Ig CSR. Therefore, we hypothesized that conditional knock-out of YY1 would lead to reduction in AID localization at the Ig locus, and reduced AID-mediated mutations. Using mice that overexpress AID (IgκAID yy1f/f ) or that express normal AID levels (yy1f/f ), we found that conditional knock-out of YY1 results in reduced AID nuclear levels, reduced localization of AID to the Sµ switch region, and reduced AID-mediated mutations. We find that the mechanism of YY1 control of AID nuclear accumulation is likely due to YY1-AID physical interaction which blocks AID ubiquitination.

[Effect of "Gubentongluo Formula" on the IgA Class Switch Recombination of B Lymohocytes in Peyer's Patches in Mice with IgA Nephropathy].

OBJECTIVE: To explore the underlying mechanism of "Gubentongluo Formula" in treatment of IgA nephropathy (IgAN). METHODS: After the IgAN model was successfully induced at week 12, the Kunming mice were randomly divided into three groups: normal control group (n = 15), IgAN group (n = 15) and Traditional Chinese Medicine (TCM) group. The mice in normal control and IgAN group were intragastriclly administrated with normal saline for 8 weeks; meanwhile, the mice in TCM group were intragastriclly administrated with "Gubentongluo Formula" 1.35 mL/ (g · d). The levels of 24 h urine protein were determined at Week 0, 12 and 20. At week 20, the changes of renal pathology were detected; the mRNA expressions of transforming growth factor-ß (TGF-ß) and small mothers against decapentaplegic (Smad) 3 in Peyer's patches (PPs) were detected by fuorescent quantitative reverse transcription-polymerase chain reaction; the protein expressions of TGF-ß and Smad 3 in PPs were detected by immunohistochemistry technique; the levels of (IgA + B)/B lymphocytes in PPs were determined by flow cytometry. RESULTS: Compared with those results of normal control group, the levels of 24 h urine protein, IgA deposition in glomerular mesangial area, and expressions of protein and mRNA of TGF-ß and Smad3 in IgAN group were significantly increased (P < 0.01). Besides, the levels of (IgA+B)/B lymphocytes were significantly elevated in IgAN group (P < 0.01). All these indicators were improved in TCM group. Compared with IgAN group, the differences were statistically significant (P < 0.01). Compared with those results of control group, the levels of (IgA + B)/B lymphocytes showed no significant difference in TCM group (P > 0.05), but other indicators showed significant differences (P < 0.01). CONCLUSION: "Gubentongluo Formula" could effectively improve proteinuria and suppress IgA deposition in glomerular mesangial area in IgAN mice, due to affect IgA class switch recombination of B lymphocytes in PPs through regulating TGF-ß/Smad3 pathway.

Lactoferrin causes IgA and IgG2b isotype switching through betaglycan binding and activation of canonical TGF-ß signaling.

Lactoferrin (LF), a pleiotropic iron-binding glycoprotein, is known to modulate the humoral immune response. However, its exact role in Ig synthesis has yet to be elucidated. In this study, we investigated the effect of LF on Ig production by mouse B cells and its underlying mechanisms. LF, like transforming growth factor (TGF)-ß1, stimulated B cells to produce IgA and IgG2b, while downregulating other isotypes. Using limiting dilution analysis, LF was shown to increase the frequency of IgA-secreting B-cell clones. This was paralleled by an increase in Ig germ-line α (GLα) transcripts, indicating that LF plays a role as an IgA switch factor. Interestingly, LF directly interacted with betaglycan (TGF-ß receptor III, TßRIII) and in turn induced phosphorylation of TßRI and Smad3 through formation of the TßRIII/TßRII/TßRI complex, leading to IgA isotype switching. Peroral administration of LF increased intestinal/serum IgA production as well as number of IgA plasma cells in lamina propria. Finally, we found that LF has an adjuvant activity when nontoxigenic Salmonella typhimurium was inoculated perorally, conferring protection against intragastrical infection of toxigenic S. typhimurium. These results suggest that LF has an important effect on the mucosal/systemic IgA response and can contribute to protection against intestinal pathogens.

Tyrosine kinase inhibitors impair B-cell immune responses in CML through off-target inhibition of kinases important for cell signaling.

Tyrosine kinase inhibitors (TKIs) have significant off-target multikinase inhibitory effects. We aimed to study the impact of TKIs on the in vivo B-cell response to vaccination. Cellular and humoral responses to influenza and pneumococcal vaccines were evaluated in 51 chronic phase chronic myeloid leukemia (CML) patients on imatinib, or second-line dasatinib and nilotinib, and 24 controls. Following vaccination, CML patients on TKI had significant impairment of IgM humoral response to pneumococcus compared with controls (IgM titer 79.0 vs 200 U/mL, P = .0006), associated with significantly lower frequencies of peripheral blood IgM memory B cells. To elucidate whether CML itself or treatment with TKI was responsible for the impaired humoral response, we assessed memory B-cell subsets in paired samples collected before and after imatinib therapy. Treatment with imatinib was associated with significant reductions in IgM memory B cells. In vitro coincubation of B cells with plasma from CML patients on TKI or with imatinib, dasatinib, or nilotinib induced significant and dose-dependent inhibition of Bruton's tyrosine kinase and indirectly its downstream substrate, phospholipase-C-γ2, both important in B-cell signaling and survival. These data indicate that TKIs, through off-target inhibition of kinases important in B-cell signaling, reduce memory B-cell frequencies and induce significant impairment of B-cell responses in CML.

Essential role of macrophages in the initiation of allergic rhinitis in mice sensitized intranasally once with cedar pollen: regulation of class switching of immunoglobulin in B cells by controlling interleukin-4 production in T cells of submandibular lymph nodes.

The production of allergen-specific IgE antibodies (Abs) in allergen-sensitized patients or animals has a mutual relationship with the immunologic response leading to allergic rhinitis. We recently reported that, after an intranasal injection of cedar pollen into mice, an interleukin-4 (IL-4)-dependent increase in serum nonspecific IgE Abs was a prerequisite for the production of serum allergen-specific IgE Abs. Here, we explored which lymphoid organs were responsive to the intranasally injected allergen and how IL-4 and IgE Abs were produced in the lymphocytes. Time-dependent changes in the total cell numbers and in in vitro IgE Ab production in various lymphoid organs revealed that the submandibular lymph nodes were the main responsible organ. After treatment with allergen (for IgE production) or allergen and complete Freund's adjuvant (for IgG production), we separated submandibular lymph node cells into macrophage-, lymphocyte-, and granulocyte-rich populations by discontinuous Percoll density-gradient centrifugation. Unexpectedly, bulk cells, but not the lymphocyte- or macrophage-rich populations, produced significant amounts of IL-4, IgE, and IgG; whereas production was restored by addition of Mac-1(+) cells from the macrophage-rich to the lymphocyte-rich fraction. Furthermore, a combination of the lymphocyte-rich population (for IgG [or IgE]) production) and the macrophage-rich population (for IgE [or IgG]) production) produced a large amount of IgE (or IgG). These results indicate that, in the initiation of allergic rhinitis, macrophages in the submandibular lymph nodes are essential not only for IL-4 or immunoglobulin production, but also for class switching of immunoglobulin in lymphocytes.

YY1 controls immunoglobulin class switch recombination and nuclear activation-induced deaminase levels.

Activation-induced deaminase (AID) is an enzyme required for class switch recombination (CSR) and somatic hypermutation (SHM), processes that ensure antibody maturation and expression of different immunoglobulin isotypes. AID function is tightly regulated by tissue- and stage-specific expression, nuclear localization, and protein stability. Transcription factor YY1 is crucial for early B cell development, but its function at late B cell stages is unknown. Here, we show that YY1 conditional knockout in activated splenic B cells interferes with CSR. Knockout of YY1 did not affect B cell proliferation, transcription of the AID and IgM genes, or levels of various switch region germ line transcripts. However, we show that YY1 physically interacts with AID and controls the accumulation of nuclear AID, at least in part, by increasing nuclear AID stability. We show for the first time that YY1 plays a novel role in CSR and controls nuclear AID protein levels.

Imatinib mesylate directly impairs class switch recombination through down-regulation of AID: its potential efficacy as an AID suppressor.

Activation-induced cytidine deaminase (AID) is essential for class switch recombination and somatic hypermutation. Its deregulated expression acts as a genomic mutator that can contribute to the development of various malignancies. During treatment with imatinib mesylate (IM), patients with chronic myeloid leukemia often develop hypogammaglobulinemia, the mechanism of which has not yet been clarified. Here, we provide evidence that class switch recombination on B-cell activation is apparently inhibited by IM through down-regulation of AID. Furthermore, expression of E2A, a key transcription factor for AID induction, was markedly suppressed by IM. These results elucidate not only the underlying mechanism of IM-induced hypogammaglobulinemia but also its potential efficacy as an AID suppressor.

Oral-nasopharyngeal dendritic cells mediate T cell-independent IgA class switching on B-1 B cells.

Native cholera toxin (nCT) as a nasal adjuvant was shown to elicit increased levels of T-independent S-IgA antibody (Ab) responses through IL-5- IL-5 receptor interactions between CD4+ T cells and IgA+ B-1 B cells in murine submandibular glands (SMGs) and nasal passages (NPs). Here, we further investigate whether oral-nasopharyngeal dendritic cells (DCs) play a central role in the induction of B-1 B cell IgA class switch recombination (CSR) for the enhancement of T cell-independent (TI) mucosal S-IgA Ab responses. High expression levels of activation-induced cytidine deaminase, Iα-Cµ circulation transcripts and Iµ-Cα transcripts were seen on B-1 B cells purified from SMGs and NPs of both TCRß⁻/⁻ mice and wild-type mice given nasal trinitrophenyl (TNP)-LPS plus nCT, than in the same tissues of mice given nCT or TNP-LPS alone. Further, DCs from SMGs, NPs and NALT of mice given nasal TNP-LPS plus nCT expressed significantly higher levels of a proliferation-inducing ligand (APRIL) than those in mice given TNP-LPS or nCT alone, whereas the B-1 B cells in SMGs and NPs showed elevated levels of transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI) expression. Interestingly, high frequencies of IgA+ B-1 B cells were induced when peritoneal IgA⁻ IgM+ B cells were stimulated with mucosal DCs from mice given nasal TNP-LPS plus nCT. Taken together, these findings show that nasal nCT plays a key role in the enhancement of mucosal DC-mediated TI IgA CSR by B-1 B cells through their interactions with APRIL and TACI.

Effect of vitamin D(3) supplementation on peripheral B cell differentiation and isotype switching in patients with multiple sclerosis.

BACKGROUND: Vitamin D has been proposed as a promoter of immune homeostasis in multiple sclerosis (MS). During the past decade, the focus of the effects of vitamin D has been on dendritic cells and on T cells. Since there is an increasing interest in the role of B cells in the pathophysiology of MS, we studied the role of vitamin D on B cells in vivo in patients with MS. OBJECTIVE: We explored the effects of 12 weeks high-dose vitamin D(3) supplementation on peripheral B cell differentiation, immunoglobulin production and levels of B cell activating factor (BAFF) in 15 patients with MS. METHODS: Circulating B cell subsets were characterized by flow cytometry. Plasma immunoglobulin levels were assessed by nephelometry. Plasma BAFF levels were assessed by enzyme-linked immunosorbent assay (ELISA). RESULTS: Although a significant increase serum 25-hydroxyvitamin D was induced, we found no significant shift in B cell differentiation, isotype switching, or plasma BAFF levels. CONCLUSION: In patients with MS, supplementation of high doses vitamin D(3) does not have substantial effects on phenotypic markers of B cell differentiation in circulating B cells. Future studies may unravel more subtle changes in the B cell compartment, either in the circulation or in the central nervous system.

Toll-like receptor 4-dependent adjuvant activity of Kakkon-to extract exists in the high molecular weight polysaccharide fraction.

Kakkon-to, a traditional herbal medicine (Kampo formula), has been used historically in China and Japan for the treatment of infectious diseases such as influenza and the common cold. However, the biological mechanism of its therapeutic action has not yet been elucidated. In this study, we investigated the immunological function of Kakkon-to and found that the high molecular weight fraction of the extract activated macrophages in vitro. This fraction was found to be composed primarily of saccharides and in vitro intensively stimulated mouse peritoneal macrophages that produce Th1 inflammatory cytokines such as tumor necrosis factor α (TNFalpha), interleukin-1beta (IL-1beta), interferon-gamma (IFN-gamma), and interleukin-6 (IL-6). The fraction did not activate macrophages from C3H/HeJ lacking Toll-like receptor 4 (TLR4) or MyD88-deficient mice, indicating that macrophage activation by the fraction was mediated by TLR4. The route of administration of the fraction into mice regulated the kinetics of TNFalpha production in immune organs. Intravenous administration induced TNFalpha production in the four target organs of spleen, liver, lung, and Peyer’s patch; however, the most abundant production occurred in the liver and peaked at 30-60 min post administration. Peritoneal administration induced similar kinetics but the most abundant production occurred in the spleen. In contrast, oral administration induced TNFalpha production in the liver, lung, and Peyer’s patch, but not in the spleen. Although liver and lung are TNFalpha-abundant organs, production peaks in these organs occurred later than in Peyer’s patch. We also found that the fraction induced antibody production as an adjuvant against a specific antigen ovalbumin (OVA) when administered simultaneously and subcutaneously in a dose-dependent manner. Interestingly, the fraction induced IgG-class antibody in response to low doses of the antigen, which induced only IgM-class antibody when administered alone, suggesting that the fraction induces a class switch of immunoglobulin as an adjuvant in vivo. The high molecular weight fraction of Kakkon-to extract could be applicable as a potent immunostimulating drug and adjuvant.

Molecular insight into the immune up-regulatory properties of the leaf extract of Ashwagandha and identification of Th1 immunostimulatory chemical entity.

Withania somnifera, commonly called Ashwagandha in the Indian traditional system of medicine has been reported for several pharmacological activities. This study demonstrates, for the first time, the potential role of the chemically standardized leaf extract of W. somnifera (WSL) and it's identified component in activating immune system. WSL enhanced Th1 cytokine IFN-gamma expression in Con A primed splenocytes in vitro. When given orally for 2 weeks to BALB/c mice immunized with emulsion of OVA in Freund's adjuvant (OVA-FCA), it caused dose-dependent proliferation of T cells and improved their ability to secrete IL-2 and IFN-gamma, but moderately down-regulated Th2 cytokine IL-4. Flow cytometric analysis of lymphocyte surface markers of T cells CD3(+), CD4(+) and CD8(+), and B cells CD19(+) indicated prominent enhancement in proliferation and differentiation of lymphocytes. Further, the effect of WSL in immunized mice elicited up-regulation of beta-integrins LFA (CD11a) and Mac-1 (CD11b) in splenocytes. Co-stimulatory molecules CD80 and CD86 that are important secondary signals for the activation of immune system elicited remarkable enhanced expression when observed in spleen-derived macrophages isolated from WSL treated mice. Chemical standardization of WSL suggested that the withanolide 2,3 dihydro-3-sulphonile withanone is a major constituent of WSL responsible for skewing to Th1 immune polarization by stimulating the expression of IFN-gamma and B cell switch over to secrete IgG2a while simultaneously enhancing the expression of co-stimulatory molecules and integrins. These studies demonstrate the possible usefulness of WSL and its major constituent WSL-2 as Th1 immune adjuvants for chronic infectious ailments where patients suffer from weakened Th1 immunity.

Augmentation of antibody responses by retinoic acid and costimulatory molecules.

Antibody production is crucial for a successful vaccine response. Beyond the ability of vitamin A (VA) and its active metabolite, all-trans-retinoic acid (RA) to restore growth in VA-deficient animals, supplementation with VA and/or treatment with RA can augment antibody responses in both VA-deficient and VA-adequate animals. RA alone, and in combination with stimuli that are ligands for the Toll-like receptor family, can augment the adaptive immune response leading to a heightened primary antibody response, and a stronger recall response upon restimulation. Mechanisms may include regulation of cell populations, type 1/type 2 cytokines, and B cell-related transcription factors, leading to accelerated B cell maturation.

Positive selection of the peripheral B cell repertoire in gut-associated lymphoid tissues.

Gut-associated lymphoid tissues (GALTs) interact with intestinal microflora to drive GALT development and diversify the primary antibody repertoire; however, the molecular mechanisms that link these events remain elusive. Alicia rabbits provide an excellent model to investigate the relationship between GALT, intestinal microflora, and modulation of the antibody repertoire. Most B cells in neonatal Alicia rabbits express V(H)n allotype immunoglobulin (Ig)M. Within weeks, the number of V(H)n B cells decreases, whereas V(H)a allotype B cells increase in number and become predominant. We hypothesized that the repertoire shift from V(H)n to V(H)a B cells results from interactions between GALT and intestinal microflora. To test this hypothesis, we surgically removed organized GALT from newborn Alicia pups and ligated the appendix to sequester it from intestinal microflora. Flow cytometry and nucleotide sequence analyses revealed that the V(H)n to V(H)a repertoire shift did not occur, demonstrating the requirement for interactions between GALT and intestinal microflora in the selective expansion of V(H)a B cells. By comparing amino acid sequences of V(H)n and V(H)a Ig, we identified a putative V(H) ligand binding site for a bacterial or endogenous B cell superantigen. We propose that interaction of such a superantigen with V(H)a B cells results in their selective expansion.

Differential activation of signal transducer and activator of transcription 6 in B cells from allergic children and their non-allergic siblings.

BACKGROUND: The germline (GL) epsilon promoter is regulated by IL-4 and is essential for class switching to IgE. IL-4-induced gene expression is largely mediated through activation of latent transcription factor STAT6 (signal transducer and activator of transcription 6). OBJECTIVE: We investigated whether increased levels of IgE in allergic individuals may be associated with alteration in the level or activation of STAT6 and subsequent increase in GL epsilon promoter activity. METHODS: Electrophoretic mobility shift assay and Western blotting assays were used to investigate the level of expression and activation of STAT6 in Epstein-Barr virus (EBV)-transformed B cell lines from children with birch pollen allergy and their non-allergic siblings. The activity of the GL epsilon promoter was tested in a transient transfection assay. RESULTS: STAT6 was expressed at the same level in all B cell lines tested. In two out of five sibling pairs STAT6 was activated by IL-4 more efficiently in the allergic individuals but in the three other pairs the opposite was found. In transient transfections, no difference in IL-4-induced GL epsilon promoter function was detected, although basal promoter activity varied between allergic and healthy siblings in two out of five pairs. CONCLUSIONS: We demonstrate for the first time that upon IL-4 signalling STAT6 transcription factor activation differs in B cells from different individuals. Although we did not find any association between STAT6 activation and allergy, we do not exclude a possibility that stronger activation of this transcription factor is associated with an expression of allergic phenotype.