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Biomedical subjects

E E Max

Publications and source records attributed to E E Max.

At least 37 records · Page 2Linked to original sources
PubMed1995

The role of BSAP in immunoglobulin isotype switching and B-cell proliferation.

A role for the transcription factor B cell-specific activator protein (BSAP) in switch recombination has been proposed because binding sites for this protein have been found near switch regions of several isotypes. We have attempted to assess BSAP's role by altering the expression of this protein in B cells switching in culture to IgG1. We found that a phosphorothioate oligonucleotide antisense to the BSAP translation initiation site was able, when incubated with B cells, to decrease BSAP activity in nuclear extracts, and that IgG1 expression was reduced in such cells compared to cells incubated with control oligonucleotides. However, it is not clear whether this apparent reduction in switch recombination was mediated by the known BSAP binding sites in the immunoglobulin heavy chain locus because the antisense experiments revealed an additional activity of this protein: it is a rate-limiting regulator of cell proliferation. Down-regulation of BSAP was associated with decreased proliferation, while increasing BSAP (by transfection with a BSAP expression plasmid) increased proliferation. Thus because switch recombination apparently requires cell division, the effect of BSAP down-regulation on switching might have resulted from decreased proliferation. The role of BSAP in B cell proliferation suggests that dysregulation of this protein could contribute to neoplastic transformation of B cells. Because of BSAP's many activities, experiments to elucidate the mechanisms of its effects on switching and proliferation will be challenging.

Animals↗
PubMed1994

Recombinant human IgA expressed in insect cells.

IgA serves as the first line of humoral defense at all mucosal surfaces and is present in large quantities in serum. To map the sites of interaction of immune effector molecules with the IgA constant region (C alpha), we have expressed soluble, chimeric human IgA in insect cells using recombinant baculoviruses. This antibody is correctly assembled into heavy chain/light chain heterodimers, N-glycosylated, and secreted by the insect cells; further, when coexpressed with a human J chain, the antibodies can assemble into dimers. The recombinant protein is authentic by a number of criteria, including antigen-binding, recognition by monoclonal antibodies, complement fixation via the alternative pathway, and specific binding to the monocyte IgA Fc receptor. We have also constructed viruses which encode structurally altered IgA heavy chains. Using one of these variant viruses, we have shown that glycosylation of the second domain of C alpha is required for interaction with the monocyte IgA Fc receptor. This system should prove useful in further characterization of the structure-function relationships in human C alpha.

Animals↗
PubMed1994

The B cell-specific transcription factor BSAP regulates B cell proliferation.

The B cell-specific activator protein (BSAP) is a DNA-binding transcription factor expressed in pro-B, pre-B, and mature B cells, but not in plasma cells. In this study, we explored the role of BSAP in B cell function by assessing how the content of this protein varies in cells driven by proliferative stimuli and, conversely, how artificial manipulation of BSAP activity affects cell proliferation. We found that BSAP activity of nuclear extracts increased when B cells were activated by mitogen (lipopolysaccharide [LPS]), antigen receptor-mediated signaling (surface immunoglobulin D [IgD] cross-linking) or T cell-dependent stimulation (CD40 cross-linking). We could suppress BSAP activity by exposure of B cells to phosphorothioate oligonucleotides antisense to the BSAP translation initiation start site, whereas control oligonucleotides were virtually inactive. Antisense-induced BSAP suppression was associated with a striking reduction in LPS-induced proliferation of splenic B cells and in the spontaneous proliferation of B lymphoma cells (CH12.LX), but the antisense oligonucleotide had virtually no effect on proliferation of two cell lines lacking BSAP: the T lymphoma line EL-4 and the plasma cell line MOPC-315. Overexpression of BSAP in splenic B cells or de novo expression in MOPC-315 plasma cells induced by transfection of a BSAP expression plasmid stimulated cell proliferation. Taken together, these results suggest that BSAP activity is a rate-limiting regulator of B cell proliferation. We also found that treatment with the antisense BSAP oligonucleotide downregulated Ig class switching induced by interleukin 4 plus LPS. This effect may be secondary to reduced proliferation or could be mediated through BSAP binding sites in the IgH locus.

Animals↗
PubMed1994

Complex alternative RNA splicing of epsilon-immunoglobulin transcripts produces mRNAs encoding four potential secreted protein isoforms.

In RNA from human IgE-producing lymphocytes, we previously discovered two alternatively spliced epsilon-immunoglobulin mRNA isoforms that encode a novel secreted form of IgE and a membrane-bound species. Further analysis using epsilon-specific reverse transcriptase polymerase chain reactions has elucidated several additional alternatively spliced species of epsilon mRNA. One RNA isoform is generated by splicing the CH4 exon to a novel distal splice acceptor site, forming an epsilon RNA species (designated CH4-M2") that encodes a secreted epsilon protein 6 amino acids larger than the classic secreted epsilon protein. The other three novel epsilon RNAs are generated by splicing from within CH4 to a new exon structure (designated CH5) that is located between CH4 and the membrane exons. Since the three new mRNAs using CH5 share the same stop codon in CH5, they all encode the same novel protein, which is 10 amino acids shorter than the classic secreted epsilon heavy chain. The new alternatively spliced epsilon mRNAs reported here, in addition to the previously reported forms encoding membrane and larger secreted IgE, appear to reflect the normal splicing pattern in humans, as we have detected all these epsilon RNAs in all the human IgE-secreting cells and cell lines tested.

Alternative Splicing↗
PubMed1994

A processed J chain pseudogene on human chromosome 8 that is shared by several primate species.

Human DNA contains two sequences that hybridize to a human J chain gene probe: the J chain gene itself and a second previously uncharacterized sequence. By cloning and sequence analysis we now show this related sequence to be a processed pseudogene, which we have localized using somatic hybrids to chromosome 8 (distinct from the functional gene on chromosome 4) and mapped by linkage analysis to 8q13-q21. The pseudogene provides evidence of an additional DNA insertion event as it contains an AluI element embedded in sequence corresponding to the 3' untranslated region of the gene. The extent of sequence divergence between the pseudogene and the functional J chain gene suggests that the pseudogene was created roughly 40-50 million years ago; consistent with this estimate, Southern blots suggest that the pseudogene is shared by great apes as well as Old World monkeys.

Animals↗
PubMed1993

Interleukin 5 induces S mu-S gamma 1 DNA rearrangement in B cells activated with dextran-anti-IgD antibodies and interleukin 4: a three component model for Ig class switching.

The cellular signals required for induction of immunoglobulin (Ig) class switching are only partially understood. Two processes that are considered to be necessary for such induction are DNA synthesis and germline constant heavy (CH) gene transcription. We now show that an additional signal, as mediated by interleukin 5 (IL-5), is also required. To induce proliferation of resting B cells, but not Ig secretion, we utilized anti-IgD antibodies conjugated to dextran (alpha delta-dex). The addition of IL-4, a well-established switch factor for the IgG1 subclass, to alpha delta-dex-activated cell cultures failed to induce IgG1 secretion or mIgG1+ cells unless IL-5 was also present. While IL-4 stimulated an increase in germline gamma 1 RNA in alpha delta-dex-activated cells, this effect could neither be induced nor enhanced by IL-5. By contrast, IL-5 strongly enhanced steady-state levels of productive gamma 1 RNA induced by alpha delta-dex and IL-4, suggesting that IL-5 stimulated IgG1 switch rearrangement. To test this possibility we measured switch (S) mu-S gamma 1 DNA recombination events using a newly developed assay, digestion circularization polymerase chain reaction (DC-PCR). We demonstrated that IL-5 was necessary for induction of S mu-S gamma 1 DNA rearrangement in alpha delta-dex plus IL-4-activated cells but that it had little effect on rearrangement in the absence of IL-4. Our data strongly suggest, therefore, a three-component model for induction of Ig class switching. This model includes germline CH gene transcription, DNA synthesis, and a third component that is necessary for recombination.

Animals↗
PubMed1993

DNA rearrangement can account for in vitro switching to IgG1.

During immune responses, B lymphocytes may switch from the expression of immunoglobulin M (IgM) to the expression of another isotype (e.g., IgG, IgE, IgA). In stable hybridomas and myelomas expressing a "switched" (S) isotype, DNA deletions between S mu and a "downstream" S region (S region recombination) have been found. In primary B cells, studies of the molecular basis of switching have been limited by the ability to sensitively quantitate the amount of DNA deletion; such studies would be of interest because other nondeletional mechanisms (trans-splicing, alternative processing of a long transcript) have been proposed to account for isotype switching in certain circumstances. We have applied the digestion-circularization polymerase chain reaction (DC-PCR) technique to measure the amount of S region recombination that occurs in the course of class switching in primary B lymphocytes. Resting B cells were cultured in lipopolysaccharide (LPS) and interleukin 4 (IL-4) to stimulate switching to IgG1. These cells begin to express membrane IgG1 at day 2.5 of culture and reach maximum expression by day 4.5. DNA was prepared from cultured cells and analyzed for S mu-S gamma 1 rearrangement by DC-PCR. Chimeric switch regions, indicating S mu-S gamma 1 recombination, were detected in amounts that, in most cases, correlated with surface expression. Furthermore, when cells were sorted on the basis of surface IgG1 expression, a mean of at least one S mu-S gamma 1 rearrangement per cell was seen in five out of seven experiments. In general, the IgG1+ cells obtained at 4.5 and 5.5 d of culture had close to 2 S mu-S gamma 1 rearrangements per cell. In IgG1- cells, S mu-S gamma 1 rearrangements were detectable, but at frequencies substantially lower that in IgG1+ cells. Thus, these results indicate that DNA deletion accompanies class switching in normal B cells stimulated with LPS and IL-4.

Animals↗
PubMed1993

Variable IgH chain enhancer activity in Burkitt's lymphomas suggests an additional, direct mechanism of c-myc deregulation.

The deregulation of the c-myc gene in small non-cleaved cell lymphomas (SNCL) with 8;14 translocations is thought to be due to the juxtaposition of this gene with the IgH chain locus, but exactly how the Ig locus contributes to the deregulation is unclear. One widely considered hypothesis is that Ig gene enhancers, when moved near c-myc, might stimulate inappropriate transcription of this gene. To evaluate this hypothesis, we have tested the ability of the two known H chain enhancers, the JH-C mu intronic enhancer and the 3' alpha enhancer, to support transcription from c-myc promoters, by using transient transfection assays in a panel of 32 SNCL cell lines with 8;14 translocations. The activity of the JH-C mu intronic enhancer varied widely among the cell lines tested and correlated with the presence of nuclear factors binding to the E4/octamer regions of the enhancer. The 3' alpha enhancer was much less active than the intronic enhancer in all of our cell lines and seems unlikely to account for the c-myc deregulation (although the enhancer we tested was from the rat, because the human homologue is unidentified at present). A marked difference was also seen in the ability of individual cell lines to support transcription from the unenhanced c-myc constructs. Cell lines supporting the lowest enhancer activity tended to support the highest level of transcription from unenhanced c-myc promoters. The differences in transcription from c-myc promoters were confirmed by stably transfecting constructs containing c-myc promoters in SNCL cell lines. Our data strongly suggest that the importance of Ig enhancers for c-myc deregulation varies markedly in different cell lines and that, for many, trans-acting regulators of the c-myc promoters are as important or more important, than Ig enhancers in the deregulation of the c-myc gene.

Base Sequence↗
PubMed1992

Quantitation of immunoglobulin mu-gamma 1 heavy chain switch region recombination by a digestion-circularization polymerase chain reaction method.

B lymphocytes expressing surface IgM with or without IgD may switch to the expression of other isotypes (IgG, IgA, or IgE) in the course of immune responses. Analyses of genomic DNA from cloned myelomas and hybridomas have shown that the isotype switch is accompanied by a rearrangement characterized by deletion of DNA between the switch (S) region of the mu gene and that associated with the new isotype, resulting in the formation of a composite S region. Measurement of this deletional rearrangement has been difficult in populations of normal B cells but would be useful for investigating the mechanism of the rearrangement and determining whether deletional rearrangement is responsible for all instances of class switching. We have developed a sensitive assay for deletional rearrangement that we designate the digestion-circularization polymerase chain reaction (PCR). In this assay, genomic DNA is digested with a restriction enzyme that recognizes sites that flank the recombined composite S region. The digested DNA is then ligated at low concentrations to favor the formation of circles. The ligation joins the 5' and 3' ends of each restriction fragment, making it possible to amplify by PCR across the ligated restriction site by using appropriate primers. From DNA that has undergone deletional rearrangement, a single-sized PCR product is produced and can be quantitated. We demonstrate here that the digestion-circularization PCR assay can detect S mu-S gamma 1 rearrangements in B cells cultured with lipopolysaccharide and interleukin 4. The assay is sensitive enough to quantitate switched cells constituting only 1-2% of the population.

Animals↗
PubMed1992

Ig mu-epsilon isotype switch in IL-4-treated human B lymphoblastoid cells. Evidence for a sequential switch.

IgE is produced by B lymphocytes that have undergone a deletional rearrangement of their Ig H chain gene locus, a rearrangement that joins the switch region of the mu gene, S mu, with the corresponding region of the epsilon gene, S epsilon. To examine the resulting composite S mu-S epsilon junctions of human lymphoid cells, we have used a polymerase chain reaction strategy to clone the switch regions of the human myeloma U266 and of two IgE-producing human cell lines generated by treatment of lymphocytes with EBV plus IL-4. The switch junction of one of the EBV lines is a complex rearrangement in which a fragment of S gamma is interposed between S mu and S epsilon. This finding suggested that the switch to epsilon in this human lymphoid cell was preceded by a S mu-S gamma recombination. To determine whether this sequential switch rearrangement represented a unique event or occurred with some regularity in human B cells switching to IgE production, DNA samples from bulk cultures of lymphocytes treated with IL-4 were subjected to polymerase chain reaction amplification of their S mu-S epsilon junctions. When the resulting fragments were examined by Southern blotting, a substantial fraction hybridized to an S gamma probe. This finding suggests that sequential recombination involving S gamma is not rare in the switch to epsilon production in humans. Our polymerase chain reaction strategy should be useful in studying isotype switching at the DNA level.

B-Lymphocytes↗
PubMed1992

Two unusual forms of human immunoglobulin E encoded by alternative RNA splicing of epsilon heavy chain membrane exons.

We present evidence for RNA transcripts encoding two forms of human epsilon immunoglobulin (Ig) heavy chain that differ significantly from those of other isotypes. We previously demonstrated three human epsilon mRNA species, instead of the two, corresponding to membrane and secreted proteins, seen with other heavy chain transcripts. In human genomic DNA downstream of the C epsilon gene, we identified sequences homologous to the two putative murine exons M1 (encoding a hydrophobic, presumably transmembrane region) and M2 (encoding hydrophilic residues). To determine the structures of epsilon transcripts containing these sequences, we amplified epsilon-related RNAs with the reverse transcriptase polymerase chain reaction. RNA was examined from fresh human B cells stimulated to IgE production by interleukin 4 plus anti-CD40, as well as from the human IgE-producing line AF10. Instead of the single CH4-M1-M2 splice product predicted for murine membrane IgE, we found two other RNA species. One form has the structure CH4-M1'-M2, in which M1' includes the human sequence homologous to the murine M1 as well as a unique segment of 52 codons further upstream in the genomic sequence; this RNA species apparently encodes the IgE expressed on the membrane of IgE-producing lymphocytes. The other RNA has the structure CH4-M2', in which M2' is spliced in an alternative reading frame that includes an additional 109 codons downstream of the termination codon of the CH4-M1'-M2 form. Because the CH4-M2' mRNA form does not encode a hydrophobic segment, its translated product should be secreted. A secreted epsilon protein of approximately the size predicted for this form was identified by Western blotting. This novel IgE protein could play a significant and distinctive role in allergic disorders.

Amino Acid Sequence↗
PubMed1992

Characterization of the human immunoglobulin kappa gene 3' enhancer: functional importance of three motifs that demonstrate B-cell-specific in vivo footprints.

Using a combination of in vivo footprinting and site-directed mutagenesis, we have functionally characterized an enhancer located 12 kb downstream of the human immunoglobulin kappa constant-region gene. The core enhancer region is highly homologous to the murine 3' kappa enhancer. However, in addition to two regulatory elements homologous to the functional motifs of the murine enhancer, we find a third positive regulatory element in the human enhancer. This element is associated with an 11/12-bp direct repeat (DR) that is well conserved in the murine locus but was not recognized as functionally important in the murine enhancer. Mutation of any of the three motifs of the human enhancer decreases its activity to 3 to 20% of the wild-type level, indicating cooperative interaction between these elements. The DR motif does not resemble any known enhancer element and does not appear to function as a transcriptional activator on its own when present in multiple copies. Interestingly, nuclear extracts from both B- and T-cell lines contain factors binding to DR in vitro, but in vivo footprinting shows no evidence of protein-DNA binding in the T-cell line. This finding suggests that an additional regulatory mechanism, such as the effect of chromatin configuration on accessibility, may be involved in the B-cell-restricted activity of the human 3' kappa enhancer.

B-Lymphocytes↗
PubMed1991

Inhibition of human IgE production via Fc epsilon R-II stimulation results from a decrease in the mRNA for secreted but not membrane epsilon H chains.

We have previously shown, using IgE-anti-IgE immune complexes or mAb directed against CD23, that ongoing production of secreted IgE from the human plasma cell line AF-10 can be inhibited via the low affinity FcR for IgE (CD23). Changes occurring in the various forms of epsilon messenger RNA (epsilon mRNA) during this suppression were investigated. mRNA levels of lambda L chain were also assessed as well as beta-actin controls. Changes in membrane IgE and secreted IgE and lambda protein were simultaneously measured. Using a genomic probe corresponding to the human epsilon C region domains, three sets of epsilon-mRNA bands were identified (2.1, 3.0, and 3.8 kb). Only the largest of these (3.8 kb) contained the full epsilon membrane sequence and coded for true membrane epsilon protein. The 2.1-kb species of epsilon-mRNA contained no membrane sequence and coded for classical secreted epsilon protein. The intermediate epsilon-mRNA species (3.0 kb) was shown to contain membrane sequence but did not contain the full epsilon membrane sequence. The product of this mRNA would, in fact, function as a secreted protein. When IgE production by AF-10 cells was suppressed via Fc epsilon R-II, there was a 50% fall in the steady state levels of both forms of mRNA (2.1 and 3.0 kb) that code for secreted epsilon protein. Similarly, there was a fall in lambda-mRNA corresponding to the observed decrease in free lambda secretion. In marked contrast, levels of both membrane IgE protein and mRNA coding for membrane epsilon were unaltered on the suppressed AF-10 cells. These data suggest that inhibition of IgE production via Fc epsilon R-II is related to a fall in mRNA for secreted proteins (epsilon and lambda) and probably reflects a post-transcriptional mechanism effecting mRNA for secreted vs membrane protein mRNA.

Antigen-Antibody Complex↗
PubMed1991

Generation and cloning of stable human IgE-secreting cells that have rearranged the C epsilon gene.

Although the secretion of Ig isotypes other than IgM is generally accompanied by a DNA rearrangement that deletes C mu (and the other IgCH genes located between VDJ and the expressed CH gene), a system has recently been described that generates a high frequency of IgE-secreting cells that have failed to delete IgCH genes or to rearrange their C epsilon genes. These cells, derived from EBV-transformed human PBMC, secrete IgM and IgD as well as IgE. To determine whether the absence of C epsilon rearrangement and CH gene deletion is a general phenomenon for human IgE-secreting cells, we have characterized IgE-secreting cells that are generated by culturing purified human B cells with EBV plus IL-4 in the presence of irradiated human PBMC. In contrast to the earlier observation, we have not been able to detect any cells that demonstrate cytoplasmic staining for IgE and concurrently stain for a second Ig isotype. Stable IgE-secreting cell lines and clones produced by this method have rearranged one of their C epsilon genes and have deleted both C mu genes. These observations demonstrate that the generation of human IgE-secreting cells can involve the same gene rearrangement and deletional mechanisms that lead to the generation of cells that secrete other isotypes.

B-Lymphocytes↗
PubMed1989

Inheritance of idiotype expression unlinked to the H chain allotype locus. Novel features of genetic control in the Ia.7 system.

We have observed a pattern of inherited idiotype expression in three mouse strains that is unexpected from the genetics of the strains: a dominant idiotype that was expressed at high levels in two parental strains was expressed only at low levels in a heavy chain allotype congenic strain derived from them. In the C3H.SW strain, the antibody response to the class II MHC Ag I-E is of limited diversity, with dominant expression of an idiotype and the V kappa 21 L chain. The C57BL/10 strain expresses the same idiotype at high levels, whereas the CWB/12 strain, which was derived by replacing the Ig H chain Igh-Cj allele of C3H.SW with the Igh-Cb allele derived from C57B1/10, has been found to express little of this dominant idiotype. CWB/12 responds, with titers equal to those of the parental strains, to the I-E epitope responsible for dominant idiotype expression, and it expresses normal V kappa 21 levels; thus deficiencies in epitope-specific responsiveness or in V kappa 21 expression cannot explain the low Id expression in CWB/12. Furthermore, Southern blot analysis of three VH families gave no evidence of recombination within the the VH locus of CWB/12, which was Igh-Vb throughout. Black-cross analysis demonstrated that expression of the dominant idiotype segregated independently of Ig allotype, and was therefore due to genes unlinked to the H chain gene locus. To our knowledge, this pattern of Id expression is unprecedented, and indicates the need for caution in the interpretation of studies using allotype congenic strains. It also demonstrates a role for genes outside the Igh locus in the control of Id expression.

Animals↗