IgE Isotype Switching Research Articles

Involvement of NF- [formula omitted]B in TGF- [formula omitted]-mediated suppression of IL-4 signaling

Control of immune response requires the coordinated integration of both stimulatory and inhibitory factors. Therefore, the cross-talk of different signaling pathways is critical in providing an integrated cellular response to multiple external signals. Both interleukin-4 (IL-4) and transforming growth factor (TGF-β) are pleiotropic cytokines and play critical roles in controlling immune responses. For example, IL-4 mediates important pro-inflammatory functions in asthma including induction of the IgE isotype switch and expression of vascular cell adhesion molecules. Whereas, TGF-β is secreted from B, T, and dendritic cells as well as macrophages, and negatively regulates their proliferation, differentiation, and activation by other cytokines. In this study, we examined the effect of TGF-β on IL-4 signaling using B cells as well as embryonic kidney cells. TGF-β inhibited IL-4-induced IgG1 production and gene expression of germline ε transcripts in B cells. In embryonic kidney cells, TGF-β signals suppressed IL-4-induced transcription, when we monitored using germline ε promoter DNA. Furthermore, activation of NF-κB resulted in a resistance to TGF-β-mediated suppression of IL-4 signaling. These results indicate that TGF-β-mediated regulation of IL-4 signaling may act by targeting NF-κB signaling.

C4b-Binding Protein (C4BP) Activates B Cells through the CD40 Receptor

We demonstrate that the α chain of human C4b binding protein (C4BP) binds directly to CD40 on human B cells at a site that differs from that used by CD40 ligand. C4BP induces proliferation, upregulation of CD54 and CD86 expression, and IL4-dependent IgE isotype switching in normal B cells but not in B cells from patients with CD40 or IKKγ/NEMO deficiencies. Furthermore, C4BP colocalized with B cells in the germinal centers of human tonsils. These observations suggest that C4BP is an activating ligand for CD40 and establish a novel interface between complement and B cell activation.

Vitamin E inhibits IL-4 gene expression in peripheral blood T cells.

IL-4 plays a pivotal role in the development of allergic inflammation via induction of IgE isotype switching, increase of IgE receptor expression, promoting Th2 cell differentiation, and stimulating several genes involved in atopic disorders. Previous studies in human and mouse models have shown that high vitamin E intake correlates with low IgE concentration and reduced prevalence of allergic reactions. We, therefore, investigated the mechanism of the vitamin E effect on T cells. We show here that the natural free radical scavenger vitamin E suppresses IL-4 protein levels in human peripheral blood T cells in a dose-dependent manner. The effect of vitamin E on IL-4 expression occurs at the mRNA level. Vitamin E has been implicated in inhibition of DNA binding and function of the redox-regulated transcription factors NF-kappaB and AP-1. Investigation of the molecular mechanism by which vitamin E suppresses IL-4 transcription shows that it blocks binding of transcription factors to two important IL-4 promoter binding sites for NF-kappaB and AP-1 and interferes with promoter activity upon T cell activation. Our data provide molecular evidence supporting the beneficial effect of dietary vitamin E on atopic disorders.

Adverse respiratory effects and allergic susceptibility in relation to particulate air pollution: flirting with disaster.

Adverse respiratory effects and allergic susceptibility in relation to particulate air pollution: flirting with disaster.

Efficacy of soluble IL-4 receptor for the treatment of adults with asthma

Background: IL-4 mediates important proinflammatory functions in asthma, including induction of the IgE isotype switch, increased expression of vascular cell adhesion molecule 1 and promotion of eosinophil transmigration across the endothelium, stimulation of mucus production, and TH2 lymphocyte differentiation, leading to release of IL-4, IL-5, IL-9, and IL-13. Objective: The current study evaluated the therapeutic potential of inhaled recombinant human soluble interleukin-4 receptor (IL-4R) as an IL-4 antagonist. Methods: This study was a randomized, double-blind, placebo-controlled study in 62 subjects involving 12 once weekly nebulizations of 0.75, 1.5, or 3.0 mg of IL-4R or placebo. During screening, subjects documented dependence on inhaled corticosteroids by an exacerbation in asthma induced by one or two 50% dose reductions at 2-week intervals. After restabilization for 2 weeks on the dose above which their asthma flared, inhaled steroids were discontinued, patients were randomized, and study medication was started on day 0. Results: IL-4R was well tolerated. Efficacy was demonstrated by a decline in FEV1 observed in the placebo group (–0.4 L and –13% predicted), which did not occur in the group receiving 3.0 mg of IL-4R (–0.1 L and –2% predicted; P = .05 over the 3-month treatment period). Daily patient-measured morning FEV1 also demonstrated a significant decline in the placebo group (–0.5 L and –18% predicted), which did not occur in the group receiving 3.0 mg of IL-4R (–0.1 L and –4% predicted; P = .02 over the 3-month treatment period). The efficacy of IL-4R was further confirmed by the absence of increase in asthma symptom scores in the group receiving 3.0 mg of IL-4R (Δ 0.1) compared with that seen in the placebo group (Δ 1.4 over 1 month; P = .07). Study discontinuation for asthma exacerbation was not significantly different between groups (placebo, 56%; 3.0 mg of IL-4R, 47%; P = not significant). Conclusion: These promising data suggest that IL-4R is safe and effective in the treatment of moderate persistent asthma. (J Allergy Clin Immunol 2001;107:963-70.)

Inhibition of Interleukin-4- and CD40-induced IgE Germline Gene Promoter Activity by 2′-Aminoethoxy-modified Triplex-forming Oligonucleotides

Elevated levels of IgE are intimately associated with a number of allergic diseases, such as allergic rhinitis or asthma. Therefore, prevention of IgE production in human B-cells represents an attractive therapeutic target. IL-4-induced IgE germline gene transcription represents a crucial early step during IgE isotype switch differentiation. Gene induction is orchestrated by the coordinated action of the transcription factors STAT6 (signal transducer and activator of transcription), NF-kappaB, PU.1, and C/EBP. This study shows that 2'-aminoethoxy-modified oligonucleotides, which partially overlap with the STAT6 and the adjacent PU.1/NF-kappaB binding site, inhibit DNA binding of all three proteins with high affinity in a dose- and time-dependent fashion in vitro. Loss of protein binding correlated strongly with increasing DNA triplex formation. Importantly, the oligomers also effectively displaced pre-bound recombinant NF-kappaB p50 from double-stranded DNA in vitro. Functionally, the oligonucleotides led to a selective inhibition of IL-4-induced reporter gene activity from a construct driven by the IgE germline gene promoter in human B-cells. These data confirm the critical role of this cytokine-responsive regulatory region in IgE germline gene induction and further support the concept of specific modulation of gene expression by DNA triplex formation induced with chemically modified oligonucleotides.

Th2 cytokines and asthma — Interleukin-4: its role in the pathogenesis of asthma, and targeting it for asthma treatment with interleukin-4 receptor antagonists

Interleukin-4 (IL-4) mediates important pro-inflammatory functions in asthma including induction of the IgE isotype switch, expression of vascular cell adhesion molecule-1 (VCAM-1), promotion of eosinophil transmigration across endothelium, mucus secretion, and differentiation of T helper type 2 lymphocytes leading to cytokine release. Asthma is a complex genetic disorder that has been linked to polymorphisms in the IL-4 gene promoter and proteins involved in IL-4 signaling. Soluble recombinant IL-4 receptor lacks transmembrane and cytoplasmic activating domains and can therefore sequester IL-4 without mediating cellular activation. We report the results of initial clinical trials, which demonstrate clinical efficacy of this naturally occurring IL-4 antagonist as a therapeutic agent in asthma.

Tumour-necrosis-factor-receptor-associated factor 6, NF-κB-inducing kinase and IκB kinases mediate IgE isotype switching in response to CD40

Tumour-necrosis-factor-receptor-associated factor 6, NF-κB-inducing kinase and IκB kinases mediate IgE isotype switching in response to CD40

Tumour-necrosis-factor-receptor-associated factor 6, NF-κB-inducing kinase and IκB kinases mediate IgE isotype switching in response to CD40

The process of IgE switching requires the prior transcription of the unrearranged Cepsilon gene, which leads to its recombination with the VDJ region. The activation of NF-kappaB by CD40 is a key process in facilitating this transcription by promoting the activation of the Cepsilon promoter. The present study explores the uncharacterized signalling pathways employed by CD40 in activating NF-kappaB by the overexpression of genes encoding wild-type and dominant-negative forms of the signalling components tumour-necrosis-factor-receptor-associated factor 6 (TRAF-6), NF-kappaB-inducing kinase (NIK), IkappaB kinase (IKK)-1 and IKK-2 in the BJAB B-cell line. The overexpression of TRAF-6 or NIK was sufficient to activate NF-kappaB and the Cepsilon promoter, whereas their dominant-negative counterparts decreased the ability of CD40 to activate NF-kappaB and the Cepsilon promoter. The overexpression of wild-type IKK-1 or IKK-2 seemed to cause toxic effects on the cells, whereas the dominant-negative forms were selective in their blockade of NF-kappaB and the Cepsilon promoter. These results suggest that CD40 employs TRAF-6, which presumably recruits NIK, which in turn employs IKK-1/IKK-2 to activate NF-kappaB and the Cepsilon promoter, the prologue to IgE switching. Thus the findings define a crucially important pathway in the generation of allergic states.

Pollution and the immune response: atopic diseases--are we too dirty or too clean?

The effect of pollutants on the immune system has filled tomes and is almost always controversial whether the topic is disinfectants in drinking water or particulates and lung cancer. Even whether outdoor and indoor air quality is improving or declining has been a source of considerable investigation, consultation and often litigation. Few areas, however, yield so much uncertainty and debate as the role of airborne pollutants in atopy. Atopy is defined as an immunoglobulin E- (IgE) mediated allergic response to environmental antigens and can be manifested as rhinitis, asthma or atopic dermatitis. While there is some debate over whether there has been a real increase in atopy over the last decades, it is indisputable that there has been a dramatic change over the last 200 years. While isolated cases of anaphylaxis had been noted before, allergies were unheard of until the early 19th century. Only 54 years after the first description of hay fever in England in 1819, it was considered an epidemic and a clear association with urbanization had been reported.1 What aspect of urbanization is involved has been much debated. There has been much discussion amongst both experimental immunologists and epidemiologists over whether pollutants are responsible for increased allergic sensitization and/or severity.2,3 In the last few years new studies have supported an alternative explanation, namely the decline in infectious diseases in industrialized countries.4,5 Does this ‘hygiene theory’ toll the death knell for the ‘pollution theory’? In this review I will argue that there is a compelling case to be made for both theories and that even this is not the entire picture.

Differences in IgE isotype switching induced by anti-CD40 monoclonal antibody and cytokines among subtypes of chronic B lymphoid leukemias

Objective Immunologic differences among the chronic B lymphoid leukemias defined by the French-American-British (FAB) classification were evaluated with respect to IgE isotype switching induced by anti-CD40 monoclonal antibody (mAb) and cytokines. Materials and Methods We immunocytochemically studied IgE isotype switching of leukemic B cells from 25 cases and three cell lines established from the leukemias after stimulation with anti-CD40 mAb, plus each of the following cytokines: interleukin 2 (IL-2); IL-4; IL-10; tumor necrosis factor α (TNF-α); and transforming growth factor β (TGF-β). Also, genomic Cϵ and Cϵ transcripts were analyzed by polymerase chain reaction and reverse transcriptase polymerase chain reaction. Results Leukemic cells from hairy cell leukemia variant and its cell line, with deletion of the Cϵ gene, did not undergo IgE isotype switching in response to any of the stimuli. In contrast, a cell line (FH-5) established from chronic lymphocytic leukemia cells, bearing the Cϵ gene, underwent the highest level of IgE isotype switching on stimulation with anti-CD40 mAb and IL-4. This response was correlated with the production of Cϵ transcripts. IL-4, IL-10, and TNF-α induced higher levels of IgE isotype switching than the others. No IgE isotype switching was observed in any of the non-Hodgkin's lymphomas examined, except mantle cell lymphoma and lymphoplasmacytic lymphoma. Percentages of CD40 + cells in five cases with follicular lymphoma were significantly lower than the other leukemias. Conclusions IgE isotype switching induced by anti-CD40 mAb with cytokines other than IL-4 was first demonstrated, whereas none of the non-Hodgkin's lymphomas except mantle cell lymphoma and lymphoplasmacytic lymphoma showed IgE isotype switching in response to any of the stimuli. Cells of follicular lymphoma were suggested to be different from cells of the other leukemias.

IL-13 genetics: Pale rider or horse of a different color?

IL-13 genetics: Pale rider or horse of a different color?

Molecular mechanisms of IgE regulation

IgE antibody plays an important role in allergic diseases. IgE synthesis by B cells requires two signals. The first signal is delivered by the cytokines IL-4 or IL-13, which target the Cϵ gene for switch recombination. The second signal is delivered by interaction of the B cell surface antigen CD40 with its ligand (CD40L) expressed on activated T cells. This activates deletional switch recombination. We review the molecular mechanisms of IL-4 and CD40 signaling that lead to IgE isotype switching and discuss the implications for intervening to abort or suppress the IgE antibody response. (J Allergy Clin Immunol 2000;105:S547-58.)

The failure of STAT6-deficient mice to develop airway eosinophilia and airway hyperresponsiveness is overcome by interleukin-5.

While signal transducer and activator of transcription protein 6 (STAT6) is important in interleukin-4 (IL-4)-induced commitment of CD4(+) T cells to the T helper cell, type 2 (Th2) phenotype and IgE isotype switching in B cells, its role in other IL-4-mediated events and their impact upon the allergic response is less evident. In the present study we demonstrate the critical role of STAT6 in the development of allergic airway eosinophilia and airway hyperresponsiveness (AHR) after allergen sensitization and challenge. STAT6-deficient (STAT6-/-) mice did not develop a Th2 cytokine response or an allergen-specific IgE response. Further, STAT6-/- mice had a reduced constitutive and allergen-induced expression of CD23 as well as lower mucus production in the airway epithelium. Critically, we show that IL-5 alone can reconstitute airway eosinophilia and AHR in sensitized and challenged STAT6-/- mice. This emphasizes the essential nature of the IL-4-dependent signaling of T cells to the Th2 phenotype and secretion of IL-5, resulting in the airway eosinophilia and AHR. These observations underscore the importance of targeting this pathway in new antiallergic asthma drug development.

Role of JAK3 in CD40-Mediated Signaling

CD40 is a member of the tumor necrosis factor receptor family and plays an important role in B-cell survival, growth, differentiation, and isotype switching. Recently, CD40 has been shown to associate with JAK3, a member of the family of Janus Kinases, which are nonreceptor protein kinases involved in intracellular signaling mediated by cytokines and growth factors. To investigate the role of JAK3 in CD40-mediated signaling, we studied the effect of CD40 stimulation on B-cell proliferation, IgE isotype switching, and upregulation of surface expression of CD23, ICAM-1, CD80, and LT-α in JAK3-deficient patients. Our studies show that stimulation of B cells with monoclonal antibody to CD40 in the presence of interleukin-4 (IL-4) or IL-13 resulted in similar responses in JAK3-deficient patients and normal controls. This suggests that JAK3 is not essential for CD40-mediated B-cell proliferation, isotype switching, and upregulation of CD23, ICAM-1, CD80, and LT-α surface expression.

Role of JAK3 in CD40-Mediated Signaling

CD40 is a member of the tumor necrosis factor receptor family and plays an important role in B-cell survival, growth, differentiation, and isotype switching. Recently, CD40 has been shown to associate with JAK3, a member of the family of Janus Kinases, which are nonreceptor protein kinases involved in intracellular signaling mediated by cytokines and growth factors. To investigate the role of JAK3 in CD40-mediated signaling, we studied the effect of CD40 stimulation on B-cell proliferation, IgE isotype switching, and upregulation of surface expression of CD23, ICAM-1, CD80, and LT-α in JAK3-deficient patients. Our studies show that stimulation of B cells with monoclonal antibody to CD40 in the presence of interleukin-4 (IL-4) or IL-13 resulted in similar responses in JAK3-deficient patients and normal controls. This suggests that JAK3 is not essential for CD40-mediated B-cell proliferation, isotype switching, and upregulation of CD23, ICAM-1, CD80, and LT-α surface expression.

Combined nasal challenge with diesel exhaust particles and allergen induces In vivo IgE isotype switching.

In this study we undertook to provide evidence for local in vivo isotype switching to IgE following nasal challenges. Detection of deleted switch circular DNA (switch circles) by a novel nested polymerase chain reaction-based approach was employed as definitive molecular evidence of Ig isotype switching. Nasal challenge in humans with diesel exhaust particles (DEP) plus ragweed antigen has been shown to enhance local IgE production, stimulate local cytokine production, and markedly increase mucosal IgE antibody to ragweed. Four days after combined intranasal DEP plus ragweed challenge, we detected and characterized clones of deleted switch circular DNA (Sepsilon /Smu) representing switching from mu to epsilon from nasal lavage cells. No switch circular DNA was detected in nasal lavage cells following challenge with DEP alone nor with ragweed allergen alone. These results indicate that the combination of mucosal stimulation with DEP and ragweed allergen is capable of driving in vivo isotype switching to IgE in humans with ragweed allergy. These results are the first direct demonstration of in vivo IgE isotype switching in humans.

A thiol antioxidant regulates IgE isotype switching by inhibiting activation of nuclear factor-κB

The binding site for nuclear factor-κB (NF-κB) is present at the promoter region of the germline Cϵ gene, but there is little information on whether this factor is involved in regulating IgE synthesis by human B cells. Accordingly, we studied the role of NF-κB in germline Cϵ transcription by using two human Burkitt's lymphoma B cell lines, DND39 and DG75. In both cell lines, n-acetyl-l-cysteine (NAC), a potent thiol antioxidant, inhibited the triggering of the nuclear expression of NF-κB by IL-4 and by anti-CD40 monoclonal antibody. Although IL-4 activated signal transducers and activators of transcription (STAT) 6 in addition to NF-κB, NAC treatment or the transfection of decoy oligodeoxynucleotides for NF-κB or STAT6 only partly blocked IL-4–induced germline Cϵ transcription. However, these two decoy oligodeoxynucleotides together almost completely abrogated IL-4–induced germline Cϵ transcription. Of note, CD40-mediated enhancement of IL-4–driven germline Cϵ transcription was markedly decreased by NAC or by a decoy oligodeoxynucleotide for NF-κB. The effect of NAC was also examined on deletional switch recombination underlying the isotype switch to IgE. NAC inhibited the generation of Sμ/Sϵ switch fragments in normal human B cells costimulated with IL-4 and anti-CD40 monoclonal antibody. It also abolished IL-4–induced upregulation of CD40 but promoted upregulation of CD23. These results suggest that coordination of NF-κB and STAT6 may be required for induction of germline Cϵ transcription by IL-4, and that CD40-mediated NF-κB activation may be important in regulating both enhancement of germline Cϵ transcription and class switching to IgE. (J Allergy Clin Immunol 1997;100:S33-8.)

Homodimerization of the human interleukin 4 receptor alpha chain induces Cepsilon germline transcripts in B cells in the absence of the interleukin 2 receptor gamma chain.

The cytokines interleukin (IL)-4 and IL-13 play a critical role in inducing Cepsilon germline transcripts and IgE isotype switching in human B cells. The IL-4 receptor (IL-4R) in B cells is composed of two chains, the IL-4-binding IL-4Ralpha chain, which is shared with the IL-13R, and the IL-2Rgamma (gammac) chain, which is shared with IL-7R, IL-9R, and IL-15R. IL-4 induces Cepsilon germline transcripts and IgE isotype switching in B cells from patients with gammac chain deficiency. Induction of Cepsilon germline transcripts by IL-4 in B cells that lack the gammac chain may involve signaling via the IL-13R. Alternatively, the IL-4Ralpha chain may transduce intracellular signals that lead to Cepsilon gene transcription independently of its association with other chains. We show that ligand-induced homodimerization of chimeric surface receptors consisting of the extracellular and transmembrane domains of the erythropoietin receptor and of the intracellular domain of IL-4Ralpha induces Janus kinase 1 (Jak1) activation, STAT6 activation, and Cepsilon germline transcripts in human B cell line BJAB. Disruption of the Jak1-binding proline-rich Box1 region of IL-4Ralpha abolished signaling by this chimeric receptor. Furthermore, B cells transfected with a chimeric CD8alpha/IL-4Ralpha receptor, which is expressed on the cell surface as a homodimer, constitutively expressed Cepsilon germline transcripts. These results suggest that homodimerization of the IL-4Ralpha chain is sufficient to transduce Jak1-dependent intracellular signals that lead to IgE isotype switching.

Chromosome Mapping and Expression of the Human Interleukin-13 Receptor

Interleukin-13 (IL-13) is a cytokine secreted by activated T cells and shares most but not all biological activities with interleukin-4 (IL-4). Both cytokines play an important role as a switch factor directing synthesis of IgE; they act on monocytes and endothelial cells, but unlike IL-4, IL-13 does not act on T cells. These cytokines have both common and distinct components in their respective receptors. Based on sequence similarity shared by cytokine receptor family members, we have identified a cDNA encoding the human IL-13 receptor (IL-13R). This cDNA was used to examine the pattern of IL-13R mRNA expression by Northern blot analyses of poly(A)+RNA purified from different human tissues and cell lines. Among several myeloma cell lines analyzed, the U266 cell line was the only one found to express IL-13R transcripts. This cell line is also the only one described as producing IgE. The IL-13R gene was mapped to chromosome Xq24 byin situhybridization. Interestingly, this locus is near that of the CD40 ligand gene, the product of which is also involved, like IL-13, in proliferation and IgE isotype switching of human B cells. The human IL-13R gene maps between two cytokine receptor genes located on the chromosome arm Xq region: the interleukin-2 receptor γ chain gene (Xq13.1) and the interleukin-9 receptor gene (Xq28). The lack of nucleotide sequence similarity suggests unrelated evolutionary pathways between these receptor genes.