JSI-124 inhibits IgE production in an IgE B cell line.

IgE is a key effector molecule in atopic diseases; however, the regulation mechanisms of IgE production in IgE B cells remain poorly understood. In the present study, we demonstrate that JSI-124 (cucurbitacin I), a selective STAT3 inhibitor, selectively inhibits production of IgE by a human IgE B cell line, CRL-8033 cells, while does not affect the IgG production by IgG B cell lines. In the aspect of molecular mechanism, we found that Igλ, but not Ighe, gene expression was suppressed by JSI-124. The above effects of JSI-124 were not mediated by affecting cellular proliferation or apoptosis. Furthermore, multiple B cell differentiation-related genes expression was not significantly affected by JSI-124. Taken together, we demonstrate a potential strategy of therapeutically suppressing IgE production without affecting IgG production in atopic patients.

Modulation of immunoglobulin gene conversion frequency and distribution by the histone deacetylase HDAC2 in chicken DT40.

Modifications of histones are reportedly associated with the regulation of immunoglobulin (Ig) gene diversification mechanisms, but the extent of their involvement in promoting sequence alterations at the Ig variable (V) regions still remains to be elucidated. We have previously demonstrated that Ig gene conversion in the B cell line DT40 is accompanied by the local hyperacetylation of histones, and that its frequency is highly increased in cells treated with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA). In this report, we describe the enhancing effects of the homozygous deletion of HDAC2 (HDAC2-/-) on Ig gene conversion. Remarkably, sequence analysis revealed that the distribution of the gene conversion tracts induced throughout the Ig V regions in HDAC2-/- was significantly different from the diversification patterns in TSA-treated wild-type cultures. Furthermore, we found that the effects of HDAC2-/- and of the treatment with TSA were additive as regards histone acetylation, Ig gene transcription, gene conversion frequency and distribution of gene conversion tracts. These results underscore the potential participation of HDAC-mediated histone acetylation in Ig diversification, but also suggest a specific role of HDAC2 to control the spatial targeting of Ig gene conversion.

Loss of PU.1 expression is associated with defective immunoglobulin transcription in Hodgkin and Reed-Sternberg cells of classical Hodgkin disease.

Immunoglobulin transcription is impaired in Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin disease (cHD). We recently demonstrated that defective immunoglobulin promoter transcription correlates with the down-regulation of the B-cell transcription factors Oct2 and BOB.1/OBF.1. These results prompted us to investigate whether immunoglobulin enhancer activity is also impaired in HRS cells and whether as yet unidentified factors could be necessary for immunoglobulin enhancer activity in HRS cells of cHD. Here we analyzed 30 cases of cHD for expression of the Ets family member PU.1 that is known to collaborate with multiple transcription factors and to regulate expression of immunoglobulin genes. We show that PU.1 is not expressed in primary and cultured HRS cells. Reintroduction of PU.1 and Oct2 in cultured HRS cells restored the activity of cotransduced immunoglobulin enhancer constructs. Our study identifies PU.1 deficiency as a recurrent defect in HRS cells that might contribute to their impairment of immunoglobulin transcription.

Chromatin remodeling at the Ig loci prior to V(D)J recombination.

Rearrangement of Ig H and L chain genes is highly regulated and takes place sequentially during B cell development. Several lines of evidence indicate that chromatin may modulate accessibility of the Ig loci for V(D)J recombination. In this study, we show that remodeling of V and J segment chromatin occurs before V(D)J recombination at the endogenous H and kappa L chain loci. In recombination-activating gene-deficient pro-B cells, there is a reorganization of nucleosomal structure over the H chain J(H) cluster and increased DNase I sensitivity of V(H) and J(H) segments. The pro-B/pre-B cell transition is marked by a decrease in the DNase I sensitivity of V(H) segments and a reciprocal increase in the nuclease sensitivity of Vkappa and Jkappa segments. In contrast, J(H) segments remain DNase I sensitive, and their nucleosomal organization is maintained in mu(+) recombination-activating gene-deficient pre-B cells. These results indicate that initiation of rearrangement is associated with changes in the chromatin structure of both V and J segments, whereas stopping recombination involves changes in only V segment chromatin. We further find an increase in histone H4 acetylation at both the H and kappa L chain loci at the pro-B cell stage. Although histone H4 acetylation appears to be an early change associated with B cell commitment, acetylation alone is not sufficient to promote subsequent modifications in Ig chromatin.

A DNase I hypersensitive site near the murine gamma1 switch region contributes to insertion site independence of transgenes and modulates the amount of transcripts induced by CD40 ligation.

Several cis-acting elements regulate the expression of germline transcripts of heavy chain constant region genes and their subsequent switch recombination. To study such elements in the murine gamma1 gene, we have utilized a transgenic approach. In this study we focused on a DNase I hypersensitive site (termed 'Site II') that lies about 2 kb 3' of the gamma1 promoter region and I exon, just 5' to the gamma1 switch region. We have reported that gamma1 transgenes with Site II display the characteristics of a locus control region (LCR) in that they are insertion site independent and copy number dependent. For the present study we prepared six lines of transgenic mice that have the promoter region and I exon, but lack Site II. Expression of RNA from gamma1 transgenes that lack Site II is not correlated with transgene copy number; expression is insertion site dependent. This result indicates that DNase hypersensitive Site II is an important part of the LCR-like elements in the murine gamma1 gene. RNA expression from the gamma1 transgenes that lack Site II is inducible by IL-4 and by CD40 ligation. However, the induction of transgenic RNA expression by CD40 ligation is greater than expected, suggesting that elements within Site II participate in negative regulation of the amount of germline transcripts after CD40 ligation.

In vitro induction of the expression of multiple IgA isotype genes in rabbit B cells by TGF-beta and IL-2.

The rabbit genome has 13 different Calpha genes that are expressed at different levels in mucosal tissues. To analyze the factors involved in the differential expression of these Calpha genes, we cloned and sequenced the promoters of the Ialpha regions that control the expression of sterile mRNA. We found that all Calpha genes, including Calpha3 and Calpha8, which are not expressed, and Calpha4, which is expressed at high levels, have similar nucleotide sequences in the Ialpha region, and all contain the recognition elements for TGF-beta in the promoter. B lymphocytes from popliteal lymph nodes or Peyer's patch activated in vitro could be induced by TGF-beta to express sterile IgA transcripts of all IgA isotypes, except Calpha2, Calpha3, and Calpha8. Many single B lymphocytes transcribed sterile mRNA of more than one IgA isotype, which demonstrates that transcription of sterile mRNA alone does not regulate the IgA isotype switch. The addition of IL-2 led to the expression of transcripts of mature IgA of all isotypes, except Calpha2, Calpha3, and Calpha8. The predominantly expressed isotype in these experiments was Calpha4. With the use of an IgA4-specific mAb we found that IgA4+ plasma cells are unevenly distributed throughout the small intestine such that many of the IgA+ plasma cells in the duodenum-jejunum produced IgA4, whereas in the lower part of the ileum IgA4-producing cells were almost absent. Because the microbial flora varies throughout the intestine, we suggest that the microbial flora creates different local environments and thus affects either isotype switching or homing of IgA-expressing cells.

Pentoxifylline inhibits Ig kappa gene transcription and rearrangements in pre-B cells.

Pentoxifylline (PF) has been used in a wide variety of clinical situations; however, the molecular consequences of this drug are not well characterized. In this paper we assayed the effects of PF in two models of pre-B differentiation. In 70Z pre-B cells, transcriptional induction of rearranged Ig kappa-chain gene in response to LPS was suppressed by PF, without affecting the induction of Rel family proteins. In contrast, kappa induction by IFN-gamma was not suppressed by PF, indicating that the drug inhibited certain activation pathways. We also found that LPS-induced activation of germline kappa transcription and V kappa to J kappa recombination were inhibited by PF in the pre-B cell line 38B9. These observations suggest that PF may adversely affect B lymphopoiesis during chronic administration.

Expression of epsilon germ-line gene transcripts and mRNA for the epsilon heavy chain of IgE in nasal B cells and the effects of topical corticosteroid.

We have studied the expression of the gene encoding the epsilon heavy chain of IgE in nasal B cells of hayfever patients. We developed probes to detect transcripts of the epsilon germ-line gene and the rearranged gene by in situ hybridization of biopsy sections from the nasal mucosa. We compared tissue from hayfever patients out of season with that of normal controls, and also of hayfever patients treated with topical corticosteroid (fluticasone propionate) or placebo for 6 weeks and then challenged with antigen. epsilon chain mRNA was expressed in an unexpectedly high proportion of nasal B cells of both hayfever patients and normal subjects. However, although similar numbers of B cells were found in both groups, the proportion of cells that express epsilon chain mRNA was several times higher in the hayfever patients. No transcripts of the epsilon germ-line gene were detected in either group before allergen challenge. When hayfever patients were administered antigen locally, early (10-30 min) and late (1-24 h) symptoms ensued. After 24 h, coincident with an increase in the number of cells expressing mRNA for IL-4 in the tissue, epsilon germ-line gene transcripts appeared in the nasal B cells. The induction by allergen of IL-4 mRNA and epsilon germ-line gene transcripts was suppressed by fluticasone propionate treatment. Our results suggest that local IgE synthesis and cytokine regulation of heavy chain switching to IgE occur in the nasal mucosa.

Analysis of the promoter elements necessary for IL-4 and anti-CD40 antibody induction of murine Fc epsilon RII (CD23): comparison with the germline epsilon promoter.

IL-4 and CD40 ligand stimulate transcription of CD23 (Fc epsilonRII) in B cells and are necessary for the expression of germline epsilon mRNA and production of IgE. Because in vivo studies have shown that the Fc epsilonRII is involved in the regulation of IgE, a study was initiated to compare how IL-4 and engagement of CD40 up-regulate the Fc epsilonRII and epsilon genes. Herein, we describe the preparation of a series of linker-scanning mutants that cover the IL-4 response region in the murine Fc epsilonRII promoter, and their function when transfected into M12.4.5 and M12.4.1 B lymphoma cell lines. Several discrete elements were found to be necessary for IL-4 induction of the Fc epsilonRII gene, some of which have homology with the binding sites of known transcription factors, including NF-IL-4 and NF-kappaB. In contrast, the response element for anti-CD40 (plus IL-4) mapped to a single discrete sequence, a NF-kappaB-like site. Aligning the Fc epsilonRII and germline epsilon promoters in the region that is highly conserved between the human and mouse homologues of both genes reveals a high degree of identity, particularly within discrete clusters. Comparing the function of linker-scanning mutants of the Fc epsilonRII promoter with a similar report for germline epsilon shows that both genes require at least two homologous and similarly located DNA elements in their promoters for a full IL-4 induction. Moreover, the similar response of Fc epsilonRII and epsilon promoter-driven chloramphenicol acetyl transferase plasmids to several cytokines and other agents suggests that the two proximal promoter regions are activated by a similar cassette of factors.

Early B-cell factor (EBF) down-regulates immunoglobulin heavy chain intron enhancer function in a plasmacytoma cell line.

The immunoglobulin heavy chain intron enhancer contains two potential binding sites for early B-cell factor (EBF). To investigate the functional properties of these, EBF was expressed in the EBF non-expressing S194 plasmacytoma cell line and found to down-regulate the activity of a co-transfected immunoglobulin heavy chain intron enhancer reporter construct. The expression of an unrelated reporter construct was unaltered. Dividing the immunoglobulin heavy chain intron enhancer into two subregions showed that the EBF mediated down-regulation of expression was mediated by at least two independent sites. These data indicate a role for EBF in the regulation of immunoglobulin gene expression.

Inducing the loss of immunoglobulin lambda light chain production and the rearrangement of a previously excluded allele in human plasma B cell lines with concanavalin A.

We investigated the expression of differential lambda light chains in human B cell lines secreting immunoglobulin (Ig). When these cell lines were cultured with concanavalin A for a long period of time, a subpopulation of some but not all of these cell lines was induced to express new lambda light chains replacing the original lambda chain (light chain shifting). Production of the new lambda chain, which replaces the original lambda chain, results from a VJ rearrangement at a previously excluded allele and a dramatic reduction of the original lambda chain transcript, although no difference was found in the level of heavy chain transcript. Recombination activating genes RAG-1 and RAG-2, which are normally expressed during specific early stages of lymphocyte development, were expressed in not only the light chain shifting-inducible lines but also in the non-inducible cells. Treatment of these Ig secreting cell lines with dibutyryl cAMP, which is known to enhance expression of the RAG genes, could not induce the creation of new lambda light chain-producing cells from the inducible lines, suggesting that the expression of the two RAG genes is not sufficient for inducing new lambda light chain production. Concanavalin A induced a gradual but significant production lost of the original lambda chain in a subpopulation of the light chain shifting-inducible cells but not in the non-inducible cells. Association of new lambda light chain production with loss of original lambda chain raises the possibility that, when the RAG genes are expressed, concanavalin A may act on a novel intracellular mechanism controlling lambda light chain allelic exclusion in these plasma cell lines.

Staphylococcal enterotoxin D functions as a human B cell superantigen by rescuing VH4-expressing B cells from apoptosis.

Staphylococcal enterotoxins are potent superantigens, in that they activate T cells bearing specific V beta-chain gene segments. In this study, we analyzed the capacity of staphylococcal enterotoxin D (SED) to function as a B cell superantigen. SED induced T cell-dependent polyclonal proliferation and differentiation of B cells. In the absence of T cells, SED induced survival of B cells uniquely expressing VH4 containing IgM. The mechanism of survival of VH4-expressing B cells appeared to relate to the countering of apoptosis initiated by the engagement of HLA-DR by SED. Analysis of the VH4 gene products expressed by SED-stimulated B cells revealed the usage of six of the known functional VH4 genes with a variety of different CDR3 regions, employing different DH and JH gene segments. Moreover, the sequence analysis identified a possible site for SED binding of VH4 that includes the solvent-exposed surfaces of 3' CDR2/FR3 and/or FR1. Thus, SED appears to function as a unique B cell superantigen by inducing survival of VH4-expressing B cells.

Regulation of 3' IgH enhancers by a common set of factors, including kappa B-binding proteins.

Targeted disruption of the p50 subunit of NF-kappa B resulted in isotype class switch defects resembling those observed in mice in which the downstream IgH enhancer 3'alpha E(hs1,2) was deleted. We postulated that kappa B binding proteins may regulate class switching by interacting with 3'alpha E(hs1,2) or with other IgH 3' enhancers with which 3'alpha E(hs1,2) synergizes. kappa B binding sites were identified in 3'alpha E(hs1,2) and 3' alpha-hs4, the distal 3' IgH enhancer. A kappa B binding site within 3'alpha E(hs1,2) contributes to at least half the activity of the enhancer in plasma cells, while the same kappa B binding site participates in the complex repression of the enhancer in B cells. In the case of 3'alpha-hs4, a kappa B binding complex activates the enhancer in pre-B, B cells and plasma cells. Additional binding sites within 3'alpha-hs4 for factors known to regulate 3'alpha E(hs1,2), including Oct-1 and BSAP, were identified, and their contribution to 3'alpha-hs4 regulation during B cell development was assessed. Oct-1 positively regulates the enhancer in pre-B and B cells, while BSAP is a repressor in pre-B cells and an activator at the B cell stage. These studies identify kappa B binding proteins as key modulators of 3'alpha E(hs1,2) and 3'alpha-hs4, and suggest coregulation of the two enhancers by a common set of factors.

Regulation of antibody class switching to IgE: characterization of an IL-4-responsive region in the immunoglobulin heavy-chain germline epsilon promoter.

A large body of data indicate that antibody class switching is directed by cytokines by inducing or repressing transcription from unrearranged, or germline, CH genes. IL-4 induces transcription of the germline C epsilon genes in activated B cells, and subsequently cells in this population will undergo switch recombination to IgE. Furthermore, the data suggest that transcription of germline C epsilon genes is required for class switching. In this paper we define DNA elements required for induction of transcription of the germline C epsilon genes by IL-4. To do this, segments of DNA from the 5' flank of the initiation sites for germline epsilon RNA were ligated to a luciferase reporter gene and transfected into two mouse B-cell lines, one of which can be induced to switch to IgE. By analysis of a series of 5' deletion constructs and linker-scanning mutations, we demonstrate that a 46-bp segment (residing at -126/-79 relative to the first RNA initiation site) contains an IL-4 responsive region. This segment binds three transcription factors: the recently described NF-IL4, one or more members of the C/EBP family of transcription factors, and NF-kappa B/p50. Mutation of any of the binding sites for these three factors abolishes or reduces IL-4 inducibility of the epsilon promoter. A 27-bp segment within this IL-4 response region containing binding sites for NF-IL4 and a C/EBP factor is sufficient to transfer IL-4 inducibility to a minimal c-fos promoter.

A DNA end-binding factor involved in double-strand break repair and V(D)J recombination.

We have identified a nuclear factor that binds to double-stranded DNA ends, independently of the structure of the ends. It had equivalent affinities for DNA ends created by sonication or by restriction enzymes leaving 5', 3', or blunt ends but had no detectable affinity for single-stranded DNA ends. Since X rays induce DNA double-strand breaks, extracts from several complementation groups of X-ray-sensitive mammalian cells were tested for this DNA end-binding (DEB) activity. DEB activity was deficient in three independently derived cell lines from complementation group 5. Furthermore, when the cell lines reverted to X-ray resistance, expression of the DEB factor was restored to normal levels. Previous studies had shown that group 5 cells are defective for both double-strand break repair and V(D)J recombination. The residual V(D)J recombination activity in these cells produces abnormally large deletions at the sites of DNA joining (F. Pergola, M. Z. Zdzienicka, and M. R. Lieber, Mol. Cell. Biol. 13:3464-3471, 1993, and G. Taccioli, G. Rathbun, E. Oltz, T. Stamato, P. Jeggo, and F. Alt, Science 260:207-210, 1993), consistent with deficiency of a factor that protects DNA ends from degradation. Therefore, DEB factor may be involved in a biochemical pathway common to both double-strand break repair and V(D)J recombination.

Alteration of cell cycle kinetics and immunoglobulin gene transcription as the result of multiple agonist stimulation of murine B cells.

Several laboratories have established that anti-IgM can inhibit polyclonal B cell activation by LPS or LPS/DxS. The use of intact anti-IgM results in an inhibition of both proliferation and differentiation, whereas F(ab')2 fragments inhibit only differentiation. Since signal transduction by both alpha-Ig's (intact and F(ab')2 fragments) is known to be mediated by PIP2 hydrolysis, we have investigated the effects of A23187 and PMA on LPS/DxS activation of splenic B cells. These agents mimic the second messengers generated as the results of PIP2 hydrolysis. As with intact alpha-IgM, either agent in conjunction with LPS/DxS resulted in an inhibition of proliferation as assessed by [3H]thymidine uptake. However, when proliferation was assessed by acridine orange (AO) staining and flow cytometric analysis, cells were observed to have entered cell cycle. This disparity between AO staining and proliferation was resolved by using BrDu/Hoechst quenching analysis and revealed a delay in cell cycle transit time as the result of multiple agent stimulation. Since both anti-IgM's result in the inhibition of differentiation, we also investigated the effects of these agents on differentiation normally observed with LPS/DxS alone activation of B cells. A23187 and PMA, either alone or in combination, were observed to result in a decrease in mRNA-encoding mu immunoglobulin of the 2.4-kb mRNA for secreted IgM.

Natural occurrence of Nuc in the sera of autoimmune-prone MRL/lpr mice.

We previously established a clone of cells termed KML1-7 which produces a soluble factor that boosts anti-DNA antibody production both in vitro and in vivo across the H-2 barrier. By using the purified protein, termed nucleobindin (Nuc), we cloned cDNA and produced recombinant(r) Nuc in E.coli. Although the purified rNuc showed biological activities such as anti-DNA antibody boosting and DNA binding, there was no evidence that Nuc is really associated with autoimmune status in lupus-prone MRL/lpr mice. Here we report that identification of Nuc was successful from the sera of MRL/lpr mice, but not from those of the substrain MRL/n mice, which show no apparent autoimmune syndrome at the same age of MRL/lpr mice, by means of immunochemical as well as N-terminal amino-acid sequencing methods.

Promoter specificity of basal transcription factors.

Regulation of expression of protein-encoding genes in eukaryotes is frequently mediated by sequence-specific transcription factors that control the activities of the basal factors and RNA polymerase II. Basal factors have been considered to be essential for all polymerase II promoters. Studies of the basal factor requirements for transcription from the immunoglobulin heavy chain gene (IgH) core promoter and the adenovirus major late gene core promoter (MLP) suggest that this paradigm is too simple. Basal transcription from the IgH promoter was reconstituted by TFIID, TFIIB, TFIIF, and polymerase, whereas basal transcription from the MLP is highly dependent upon TFIIE in addition to the above factors. Two novel protein activities, referred to as 700 kd and 90 kd, further stimulated the basal reaction from the MLP. Thus, these data indicate that not all basal factors are in fact general.

Deletional switch recombination occurs in interleukin-4-induced isotype switching to IgE expression by human B cells.

There is controversy as to whether deletional rearrangement occurs between the IgM and IgE switch regions (S mu and S epsilon, respectively) during switching to the IgE isotype. We have addressed the issue by stimulating normal human B cells, sorted for lack of expression of surface IgE, to produce IgE by infection with Epstein-Barr virus (EBV) in the presence of interleukin 4 (IL-4). Genomic DNA was amplified for S mu/S epsilon switch junction fragments by utilizing the nested-primer polymerase chain reaction. Switch junction fragments were amplified from B cells infected with EBV in the presence of IL-4 but not from B cells infected with EBV alone. The DNA sequence of these "switch fragments" revealed direct joining of S mu to S epsilon in each case. The recombination sites within S mu were clustered within 900 base pairs at the 5' end of the switch region, suggesting that there are "hot spots" for recombination within S mu. The S epsilon recombination sites were scattered throughout the S epsilon region. These findings indicate that IL-4-induced isotype switching to IgE production in human B cells is accompanied by DNA rearrangements with joining of S mu to S epsilon.

Phorbol ester treatment down-regulates immunoglobulin RNA steady-state levels in B type chronic lymphocytic leukemia and non-Hodgkin's lymphoma cells.

The human B-lymphoma cell lines BJAB and Daudi, as well as the human pre-B cell line KM3, were found to down-regulate steady-state immunoglobulin RNA levels 2- to 4-fold after stimulation with phorbol 12-myristate-13-acetate (PMA) for 24 hr. No down-regulation of the transcriptional rate of a kappa promoter could be observed in any of these cell lines upon transient expression transfection. The observed down-regulation of steady-state immunoglobulin RNA affected both the secretory and the membrane form of the mu transcript equally. When freshly isolated chronic lymphocytic leukemia (CLL) cells where tested for their response to PMA, three of four isolates responded by down-regulating their steady-state immunoglobulin RNA levels.