Epsilon Heavy Chain Research Articles

Clinical and morphological features of allergic enterocolitis in young children

The purpose was to study the clinical and morphological features of allergic enterocolitis in young children.Material and methods. 29 patients aged from 2 months to 2 years with severe allergic enterocolitis, who underwent endoscopic examination with targeted biopsy of the intestinal mucosa, were treated. For comparison of morphological, histochemical and immunohistochemical features, intestinal mucosal biopsies of children of the same age with gastrointestinal disorders, caused by protracted or chronic course of non-specific non-ulcer process in the intestine, were used. Immunohistochemical studies were performed on serial paraffin sections in accordance with standard protocols using Ab68 (Macrophage Marker) Ab-3 (Clone KP1), IgA (Heavy Chain) Ab-2, IgE (Epsilon-Heavy Chain) Ab-1 monoclonal antibodies and Imaging Ultra Vision Quanto Detection System HRP DAB (USA).Results. The debut of gastrointestinal symptoms in young children with allergic enterocolitis was observed in the first months of life and was characterized by vomiting or persistent regurgitations, intense griping, diarrhea with large amounts of mucus and/or blood, and delayed physical development. Presence of inflammatory process of intestinal mucosa with eosinophilic infiltration was morphologically established. According to immunohistochemical study, expression of CD68-macrophages and increased expression of IgA and IgE in the duodenal mucosa were detected.Conclusions. The diagnosis of allergic enterocolitis at an early age is complicated by the absence of specific clinical gastrointestinal symptoms and non-informative allergic examination (IgE-independent mechanism of inflammation), which may require morphological study to verify the diagnosis. Morphologically, in the examined patients, not only the presence of inflammatory process of the mucous membrane of the intestine with expressed eosinophilic infiltration is established, but also the immunohistochemistry data revealed the increased expression of CD68-macrophages, IgA and IgE in the mucous membrane of the duodenum, indicating the activation of both cellular and local humoral immunity.

281 Use of Monoclonal Antibody Omalizumab in the Treatment of Urticaria Chronic

BackgroundThe chronic Urticaria is a real problem of health and a frequent problem in the consultation of the allergist, which the treatment is not to satisfactory. Some Urticaria can be for autoimmunity where the antibody involved is IgE. Omalizumab is an monoclonal antibody against the C3 domain of the epsilon heavy chain of the antibody IgE (domain C3 of the IgE), involved, in allergic problems, which has proved a great utility in asthma off difficult control.MethodFive patients of both genders were studied aged between 30 and 45 years, carriers of chronic Urticaria at least of ten years duration. Theirs control was not satisfactory, with the treatment habitual. They were not used glucocorticoids. The clinical evaluation and test of laboratory stated Chronic Urticaria idiopathic. The total IgE was below 100 U. I. The monoclonal antibody Omalizumab applying it for 6 month accorded to habitual schedule.According to dose schedule monoclonal antibody Omalizumab apply the antibody to them, calculating the dose habitual schedule applying for 6 month. According to dose schedule the monoclonal antibody Omalizumab apply to them, it applying for 6 month.ResultsAll the patient improved their Urticaria between weeks 3 and fort of application of the drug, getting the control of the symptoms between the month 2 and 3 in the 5 patients, without requiring other drugs for their control, and remained asymptomatic for 3 and forth months discontinuity the product up to 6 months, not reactivity the Urticaria, the older case takes now 1 year without activity of his disease.ConclusionsOmalizumab must be considered to be another therapeutic alternative in patients with idiopathic Urticaria.

Catalytic folding of the Cε3 domain by its high affinity receptor

The interaction of immunoglobulin E (IgE) with its cellular receptor FcεRIα is a central regulator of allergy. Structural studies have identified the third domain (Cε3) of the constant region of epsilon heavy chain as the receptor binding region. The isolated Cε3 domain is a “molten globule” that becomes structured upon binding of the FcεRIα ligand. In this study, fluorescence and nuclear magnetic resonance spectroscopies are used to characterise the role of soluble FcεRIα in the folding of the monomeric Cε3 domain of IgE. Soluble FcεRIα is shown to display characteristic properties of a catalyst for the folding of Cε3, with the rate of Cε3 folding being dependent on the concentration of the receptor.

Cross reactivity between fibrinogen and epsilon heavy chain antiserum in immunofixation electrophoresis

Sir,Pseudoparaproteins are benign proteins that are not normally present, or present at low concentrations, in serum and could be misinterpreted upon protein electrophoresis as a monoclonal parapro...

Genetically engineered negative signaling molecules in the immunomodulation of allergic diseases

This review summarizes current knowledge regarding the control of human mast cell and basophil signaling and recent developments using a new therapeutic platform consisting of a human bifunctional gamma and epsilon heavy chain (Fc gamma-Fc epsilon) protein to inhibit allergic reactivity. Crosslinking of Fc gamma RIIb to Fc epsilon RI on human mast cells and basophils by a genetically engineered Fc gamma-Fc epsilon protein (GE2) leads to the inhibition of mediator release upon Fc epsilon RI challenge. GE2 protein was shown to inhibit cord blood-derived mast cell and peripheral blood basophil mediator release in vitro in a dose-dependent fashion, including inhibition of human IgE reactivity to cat. IgE-driven mediator release from lung tissue was also inhibited by GE2. The mechanism of inhibition in mast cells included alterations in IgE-mediated Ca mobilization, spleen tyrosine kinase phosphorylation and the formation of downstream of kinase-growth factor receptor-bound protein 2-SH2 domain-containing inositol 5-phosphatase (dok-grb2-SHIP) complexes. Proallergic effects of Langerhan's like dendritic cells and B-cell IgE switching were also inhibited by GE2. In vivo, GE2 was shown to block passive cutaneous anaphylaxis driven by human IgE in mice expressing the human Fc epsilon RI and inhibit skin test reactivity to dust mite antigen in a dose-dependent manner in rhesus monkeys. The balance between positive and negative signaling controls mast cell and basophil reactivity, which is critical in the expression of human allergic diseases. This approach using a human Fc gamma-Fc epsilon fusion protein to co-aggregate Fc epsilon RI with the Fc gamma RII holds promise as a new therapeutic platform for the immunomodulation of allergic diseases and potentially other mast cell/basophil-dependent disease states.

Co-aggregation of FcγRII with FcϵRI on Human Mast Cells Inhibits Antigen-induced Secretion and Involves SHIP-Grb2-Dok Complexes

Signaling through the high affinity IgE receptor FcepsilonRI on human basophils and rodent mast cells is decreased by co-aggregating these receptors to the low affinity IgG receptor FcgammaRII. We used a recently described fusion protein, GE2, which is composed of key portions of the human gamma1 and the human epsilon heavy chains, to dissect the mechanisms that lead to human mast cell and basophil inhibition through co-aggregation of FcgammaRII and FcepsilonRI. Unstimulated human mast cells derived from umbilical cord blood express the immunoreceptor tyrosine-based inhibitory motif-containing receptor FcgammaRII but not FcgammaRI or FcgammaRIII. Interaction of the mast cells with GE2 alone did not cause degranulation. Co-aggregating FcepsilonRI and FcgammaRII with GE2 1) significantly inhibited IgE-mediated histamine release, cytokine production, and Ca(2+) mobilization, 2) reduced the antigen-induced morphological changes associated with mast cell degranulation, 3) reduced the tyrosine phosphorylation of several cellular substrates, and 4) increased the tyrosine phosphorylation of the adapter protein downstream of kinase 1 (p62(dok); Dok), growth factor receptor-bound protein 2 (Grb2), and SH2 domain containing inositol 5-phosphatase (SHIP). Tyrosine phosphorylation of Dok was associated with increased binding to Grb2. Surprisingly, in non-stimulated cells, there were complexes of phosphorylated SHIP-Grb2-Dok that were lost upon IgE receptor activation but retained under conditions of Fcepsilon-Fcgamma co-aggregation. Finally, studies using mast cells from Dok-1 knock-out mice showed that IgE alone triggers degranulation supporting an inhibitory role for Dok degranulation. Our results demonstrate how human FcepsilonRI-mediated responses can be inhibited by co-aggregation with FcgammaRIIB and implicate Dok, SHIP, and Grb2 as key intermediates in regulating antigen-induced mediator release.

IgG Anti-IgE Autoantibodies in Visceral Leishmaniasis

Procedures for IgG depletion in visceral leishmaniasis (VL) and schistosomiasis sera using Sepharose-protein G beads also deplete IgE. In this study, the presence of IgG anti-IgE autoantibodies in sera from patients with VL (n = 10), and hepatic-intestinal schistosomiasis (n = 10) and from healthy individuals (n = 10) was investigated. A sandwich ELISA using goat IgG anti-human IgE to capture serum IgE and goat anti-human IgG peroxidase conjugate to demonstrate the binding of IgG to the IgE captured was performed. VL sera had higher titers (p < 0.05) of IgG anti-IgE autoantibodies (OD = 2.01 +/- 0.43) than sera from healthy individuals (OD = 1.35 +/- 0.16) or persons infected with Schistosoma mansoni (OD = 1.34 +/- 0.18). The immunoblotting carried out with eluates from Sepharose-protein G beads used to deplete IgG from these sera and goat anti-human IgE peroxidase conjugate, showed a similar pattern of bands, predominating the 75 kDa epsilon-heavy chain and also polypeptides resulting from physiological enzymatic digestion of IgE. A frequent additional band immediately above 75 kDa was observed only in VL sera.

Immunoglobulin E-Mediated Hypersensitivity in Food-Producing Animals

Type I hypersensitivity has been described as a cause of allergic reactivity to inhalants, injectables, endoparasites, and ectoparasites in food animal species. In addition, IgE is credited with showing some host-sparing effect when produced in response to certain gastrointestinal and other parasites. Recently, the sophistication of diagnostic procedures has increased with the elucidation of epsilon heavy chain sequences, expressed protein, development of chimeric IgE antibodies, and production of species-specific anti-IgE reagents. Application of ELISA and Western blotting has replaced the passive cutaneous anaphylaxis test for demonstration of antigen-specific IgE in serum. Regulation of the IgE response is complex, and its dependence on induction of T helper cell type 2 cytokines is now established. The next frontier in IgE research, as for many inherited diseases, lies in understanding the genetic make-up of the animal and which genes are important in controlling the IgE response.

Membrane immunoglobulins are stabilized by interchain disulfide bonds occurring within the extracellular membrane-proximal domain.

Membrane-bound immunoglobulins have, in addition to the transmembrane and cytoplasmic portions, an extracellular membrane-proximal domain (EMPD), absent in the secretory forms. EMPDs of immunoglobulin isotypes alpha, gamma, and epsilon contain cysteines whose role has so far not been elucidated. Using a genetic strategy, we investigated the ability of these cysteines to form disulfide bridges. Shortened versions of human membrane immunoglobulins, depleted of cysteines known to form intermolecular disulfide bonds, were constructed and expressed on the surface of a B-cell line. The resulting membrane proteins contain a single chain fragment of variable regions (scFv) linked to the dimerizing domain from the immunoglobulin heavy chains (CH3 for alpha and gamma or CH4 for epsilon isotypes), followed by the corresponding EMPD and the transmembrane and cytoplasmic domains. The two functional membrane versions of the epsilon chain, containing the short and long EMPD, were analyzed. Our results show that the single cysteine within alpha1L and gamma1 EMPD and the short version of epsilon EMPD form an interchain disulfide bond. Conversely, the cysteine resident in the epsilon transmembrane domain remains unreacted. epsilon-long EMPD contains four cysteines; two are involved in interchain bonds while the remaining two are likely forming an intrachain bridge. Expression of a full-length membrane epsilon heavy chain mutant, in which Cys(121) and Cys(209) within domain CH2 (involved in interchain bridges) were mutated to alanines, confirmed that, within the complete IgE, EMPD cysteines form interchain disulfide bonds. In conclusion, we unveil evidence for additional covalent stabilization of membrane-bound immunoglobulins.

The IgE Antigen Receptor: A Key Regulator for the Production of IgE Antibodies

Immunoglobulins in general form a substantial component of serum proteins, and play a role in homeostatic mechanisms, a first line of defense against pathogenic organisms and in immunological memory. In the secreted form, immunoglobulins represent the effector arm of the humoral immune system. However, immunoglobulins are not only secreted, but can also be expressed on the surface of a B lymphocyte (membrane immunoglobulin), and, in this physical state, most likely convey signals to steer the B cell along its differentiation pathway. A step forward in the understanding of the role of membrane immunoglobulins other than membrane IgM or IgD was achieved with two mouse lines with mutations in the epsilon heavy chain gene. In IgE^ΔM1M2 mice serum IgE is reduced to less than 10% of normal mice, while IgE^KVKΔtail mice show a reduction of 50%, reflecting a serious impairment of the IgE-mediated immune response. We think that the cytoplasmic tail of IgE is involved in a signal transduction which leads to the expression of high quantities and qualities of secreted IgE immunoglobulins.

Molecular cloning and phylogenetic analysis of a cDNA encoding the cat ( Felis domesticus) Ig epsilon constant region

A feline splenic cDNA library was screened with a 32P-labelled cDNA probe encoding the canine IgE epsilon heavy chain subunit. A cDNA sequence of 1614 nucleotides encoding the complete feline IgE heavy chain, as well as a portion of a variable region, was identified. A search of the GenBank database revealed an identity of 82% at the nucleotide level and 76% at the amino acid level between the feline epsilon heavy chain sequence and the canine homologue. In a separate study, feline genomic DNA, isolated from whole feline embryo cells, was subjected to PCR amplification using primers based on known partial genomic DNA sequences for the feline Cε gene. Following removal of an intron from the 683 bp PCR product, the coding sequence yielded an ORF of 506 bp. The DNA sequence of this PCR clone differed by a single nucleotide from the cDNA clone. This difference is silent, and therefore the proteins encoded by the two sequences are identical over the regions cloned and sequenced. Phylogenetic analysis of the constant regions of nine immunoglobulin epsilon genes revealed that the feline cDNA is most similar to the canine homologue.

Partial sequences of feline and caprine immunoglobulin epsilon heavy chain cDNA and comparative binding studies of recombinant IgE fragment-specific antibodies across different species

Parts of the feline and caprine IgE epsilon heavy chain cDNA (third and fourth constant domains, IgEf3/4) were cloned, sequenced, and expressed to raise antibodies (Abs). The DNA and derived protein sequences of the feline recombinant IgEf (rIgEf) shared high homology with the analogous canine parts (81% at the nucleotide and 71% at the protein levels) and the caprine with the ovine ones (95%/84%), respectively. The polyclonal Abs raised in chickens against the feline and caprine rIgEf3/4 were subjected to a comparative binding study utilizing an ELISA including rIgEf and specific Abs to these rIgEf from dog and horse (rIgEf2 and rIgEf3/4) and sheep (rIgEf3/4). All but the ovine-specific rIgEf3/4 Ab were polyclonal, which had been raised in chickens, and bound to most applied rIgEf; the ovine-specific monoclonal mouse Ab recognized only in addition to ovine rIgEf3/4 the closely related caprine rIgEf3/4. Significant, positive correlations were detected between binding reactions of the polyclonal Abs in ELISA and percentage protein sequence homology ( p<0.01). Thus, the newly described feline and caprine IgE nucleotide sequences and corresponding Abs represent useful tools for further species-specific and comparative allergy and disease-associated research.

The novel human IgE epsilon heavy chain, epsilon tailpiece, is present in plasma as part of a covalent complex

Several splice variants of the secreted human epsilon heavy chain have previously been identified by reverse transcription-PCR. The heavy chain of one isoform, IgE tailpiece, differs from the originally identified IgE, IgE classic, by the replacement of the 2 carboxy-terminal amino acids by 8 novel amino acids including a carboxy-terminal cysteine residue. Recombinant human epsilon tailpiece and epsilon classic heavy chains were expressed and secreted as H 2L 2 monomers in Sp2/0 murine myeloma cells. We have investigated the in vitro function and in vivo occurrence of epsilon tailpiece heavy chains using receptor binding assays, granule release assays, flow cytometry, half-life studies, immunoprecipitation, SDS-PAGE, two-dimensional SDS-PAGE, and Western blotting. IgE tailpiece and IgE classic exhibited similar in vivo half-lives in BALB/c mice, bound the human high- and low-affinity IgE receptors with similar affinities and triggered equivalent levels of high affinity IgE receptor induced degranulation. In humans, IgE classic is present as a 190 kD circulating protein in vivo. In contrast, we found that in plasma epsilon tailpiece was primarily present as part of covalent complexes of approximately 300 and 338 kD. Dissociation of the complexes revealed that two species of epsilon tailpiece heavy chains were present therein and surprisingly, these in vivo derived epsilon tailpiece heavy chains were approximately 5 and 10 kD smaller than the recombinant expressed epsilon tailpiece or epsilon classic heavy chains. These results show that epsilon tailpiece is present in novel covalent complexes in humans.

Basis of the 1:1 stoichiometry of the high affinity receptor Fc epsilon RI-IgE complex.

A soluble fragment of the high-affinity IgE receptor Fc epsilon RI alpha-chain (sFc epsilon RI alpha) binds to the Fc fragment of IgE (IgE-Fc) as a 1:1 complex. IgE-Fc consists of a dimer of the C epsilon 2, C epsilon 3 and C epsilon 4 domains of the epsilon-heavy chain of IgE. This region of IgE has been modelled on the crystal structure of the Fc region of IgG1, which exhibits twofold rotational symmetry. This implies that IgE should be divalent with respect to its ligands. X-ray scattering studies reveal however that the twofold rotational symmetry of IgE-Fc is perturbed by a bend in the linker region between the C epsilon 2 and C epsilon 3 domains. The 1:1 stoichiometry could then arise from the conformational asymmetry or from steric occlusion of one of the sites by the overhanging C epsilon 2 domains. To test this hypothesis we have expressed a recombinant epsilon-chain fragment containing C epsilon 3 and C epsilon 4. This product, Fc epsilon 3-4, is secreted from cells as a disulphide linked dimer and binds with higher affinity than either IgE or IgE-Fc to cell surface Fc epsilon RI. Titration experiments, together with molecular mass measurements of the Fc epsilon 3-4/sFc epsilon RI alpha complex, reveal that Fc epsilon 3-4 binds only a single receptor molecule. This excludes the possibility that steric hindrance by C epsilon 2 accounts for the unexpected stoichiometry.

Independent pathways for de-repression of the mouse Ig heavy chain germ-line ε promoter: an IL-4 NAF/NF-IL-4 site as a context-dependent negative element

The activation of germ-line promoters in the Ig heavy chain loci is regulated by cytokines as part of the regulation of B cell commitment to production of new antibody isotypes. Activation of the germ-line promoter of the epsilon heavy chain locus (Gepsilon) and production of IgE are induced by IL-4 and each is virtually undetectable in the absence of IL-4 or the homologous cytokine IL-13. Basal expression of the Gepsilon promoter is repressed by the non-histone chromosomal protein HMG-I(Y), which also contributes to promoter inducibility, and IL-4 stimulates phosphorylation of the C-terminus of HMG-I(Y) through a rapamycin-sensitive pathway. IL-4 treatment of mouse B cells also induces a Gepsilon DNA binding activity with the properties of IL-4 NAF, which is rapidly induced and requires phosphotyrosine for DNA binding activity. This protein binds to a different site from HMG-I(Y), but the IL-4 NAF/NF-IL-4 binding site also is a negative element more active in repression of basal transcription of the Gepsilon promoter. This site acts as a negative element when transferred to the thymidine kinase promoter, but does not confer inducibility. In contrast to HMG-I(Y), IL-4 NAF/NF-IL-4 activation is refractory to rapamycin but sensitive to genistein. These findings indicate that two independent signal transduction pathways diverge from the IL-4 receptor and suggest that normal expression of Gepsilon RNA or IgE is low in part because the germ-line promoter is kept in a state of repression which requires de-repression through several cooperative pathways. These pathways target conserved nucleotide sequence motifs whose precise function depends on the promoter context in which they are situated.

Differential regulation of alternative 3' splicing of epsilon messenger RNA variants.

Alternative 3' splicing of the one active human epsilon heavy chain gene results in variants of epsilon mRNA encoding distinct IgE proteins. The same relative amounts of these epsilon mRNA variants were produced by non-atopic donor B cells when driven in a variety of T-dependent or T-independent systems. The most abundant variants were those for classic secreted epsilon a novel secreted form (CH4-M2"). In contrast, cells from subjects with high levels of serum IgE secondary to parasitic infection or atopy spontaneously produced higher relative levels of the CH4-M2' epsilon mRNA variant, lower relative amounts of both the membrane and CH4-M2" secreted variants, and very low levels of the CH4'-CH5 variant. The existence of and corresponding changes in levels of the CH4-M2'-encoded secreted protein were demonstrated. IL-10 induced this same differential expression of epsilon splice variants in vitro when used to costimulate IL-4 plus CD40-driven B cells and could differentially enhance the production of CH4-M2' protein by established IgE-secreting cell lines. Inhibition of IgE by cross-linking the low affinity IgE receptor (CD23) decreased the levels of epsilon mRNA and resulted in a distinct pattern of epsilon mRNA characterized by a dramatic decrease in CH4-M2' splice variant. IL-6, IL-2, or IFN-gamma did not change the epsilon mRNA pattern. Overall, the absolute and relative amounts of the different epsilon mRNA splice variants produced appear to be controlled in a differentiation-related fashion.

Role of two-dimensional electrophoretic analysis in the diagnosis and characterization of IgD monoclonal gammopathy.

Over a period of 5 years, an isolated light chain (kappa = 9, lambda = 12) was detected in 21 sera by immunofixation electrophoresis. Further analysis with anti-delta- and anti-epsilon-specific antisera identified four delta heavy chains, all associated with a lambda light chain, and no epsilon heavy chains. For evaluation of the role of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in the diagnosis of IgD paraproteins, as a possible alternative or complement to immunofixation, IgD paraproteins were retrospectively analyzed by 2D-PAGE. Delta Heavy chains migrated to gel areas clearly distinguishable from other heavy chains alpha, gamma, or mu, and in a wide range of isoelectric points (pI: 5.4-8). In one serum, the monoclonal delta chain had a pI range comparable to that of albumin and was undetectable. However, all four delta chains were easily identified when analyzed from affinity-purified immunoglobulin fractions. These observations showed the following: 1) IgD paraproteins are not rare among apparently isolated monoclonal light chains detected by routine immunofixation, strongly confirming the need for further analysis with anti-delta antisera, before assumption of a light-chain disease. 2) 2D-PAGE analysis of affinity-purified immunoglobulin fractions allowed correct identification of IgD monoclonal gammopathies in all cases. 3) However, although 2D-PAGE analysis is now easy to perform, well standardized, and highly sensitive, this technique remains time-consuming and expensive, and does not appear suitable for routine practice as a first-line diagnostic procedure. 2D-PAGE should find its place as a complement to immunofixation and in the definitive demonstration, in selected ambiguous cases, of the clonal pattern of a suspected gammopathy at immunofixation.

Partial sequence of the equine immunoglobulin epsilon heavy chain cDNA

In order to isolate a part of the immunoglobulin E (IgE) heavy chain cDNA of the horse, primers have been designed based upon well conserved sequences in humans, sheep and rats. The PCR resulted in a 500 bp fragment which hybridised with a human IgE constant region probe. The fragment was cloned and sequenced and its derived protein sequence compared with the corresponding sequences in humans, sheep and mice. Most amino acids common to these three species are also shared by the horse.

Hydrodynamic studies of a complex between the Fc fragment of human IgE and a soluble fragment of the Fc epsilon RI alpha chain.

The interaction between immunoglobulin E (IgE) and its high-affinity receptor Fc epsilon RI is central to allergic disease. The binding site for Fc epsilon RI lies in the third constant region domain of the epsilon heavy chain of IgE (C epsilon 3). Identical epitopes on the two C epsilon 3 domains in the IgE-Fc are predicted to be on opposite sides of the structure, and therefore each could bind independently to a receptor molecule. Titrations, however, reveal that the IgE-Fc forms an equimolar complex with a soluble fragment of the Fc epsilon RI alpha chain (sFc epsilon RI alpha), and the molecular weight of the complex, as determined by sedimentation equilibrium, confirms this stoichiometry. The measured sedimentation coefficients of the two ligands are in good agreement with computed values for a compact IgE-Fc and an elongated sFc epsilon RI alpha structure. The calculated sedimentation coefficients for possible models of a 1:1 complex lead to exclusion of all highly extended geometries for the complex. Possible explanations for the paradoxical stoichiometry of the IgE-Fc/sFc epsilon RI alpha complex, in terms of the curved shape of IgE, a conformational change in IgE when the receptor binds, and steric interference between two molecules of Fc epsilon RI binding to identical sites, are discussed.

The non‐histone chromosomal protein HMG‐I(Y) contributes to repression of the immunoglobulin heavy chain germ‐line ε RNA promoter

The rate of germ-line RNA transcription correlates with the rate of immunoglobulin heavy chain isotype switching. A promoter element for the transcription of RNA from the germ-line mouse immunoglobulin epsilon heavy chain constant region gene is induced by interleukin(IL)-4 and lipopolysaccharide, and is bound at its transcription initiation sites by an IL-4-inducible nuclear protein, NF-BRE. To examine the function of the binding site for this IL-4-inducible complex, substitution mutations were introduced in the promoter. These binding site mutations increased promoter activity and decreased binding of NF-BRE. To investigate the paradox of an IL-4-inducible protein binding to a repressor site in an IL-4-inducible promoter, we determined that the non-histone chromosomal protein HMG-I(Y) binds at the transcription initiation sites of the germ-line epsilon promoter. Assays with antisera against HMG-I(Y) revealed monomeric HMG-I(Y) in nuclear extracts. Cotransfection of an expression construct directing the synthesis of anti-sense HMG-I(Y) RNA also increased promoter activity, consistent with a repressor function of HMG-I(Y). Thus, the data are most consistent with a model in which HMG-I(Y) participates in repression of promoter activity. The effects of IL-4 may include derepression at this site.