IgM and IgA anti-erythrocyte autoantibodies induce anemia in a mouse model through multivalency-dependent hemagglutination but not through complement activation.

By generating IgM and IgA switch variants of the 34-3C IgG2a anti-red blood cell (RBC) autoantibody, we evaluated the pathogenic activity of these 2 isotypes in view of the Fc-associated effector functions (ie, complement activation and polyvalency-dependent agglutination). We found that polymeric forms of 34-3C IgM and IgA anti-RBC autoantibody were as pathogenic as IgG2a, which was the most pathogenic among 4 different IgG subclasses, whereas their monomeric variants completely lacked pathogenic effects. Histological examination showed that 34-3C IgM and IgA autoantibodies caused anemia as a result of multivalency-dependent hemaggultination and subsequent sequestration of RBC in the spleen, in contrast to Fc receptor- and complement receptor-mediated erythrophagocytosis by Kupffer cells with IgG isotypes. In addition, the development of anemia induced by IgM and IgA isotypes of 34-3C antibody and by 2 additional IgM anti-RBC monoclonal autoantibodies was not inhibited at all in C3-deficient mice, indicating the lack of involvement of complement activation in the pathogenesis of IgM- and IgA-induced anemia. Our data demonstrate a remarkably high pathogenic potential of polymeric forms of IgM and IgA anti-RBC autoantibodies due to their ability to induce hemagglutination but completely independent of complement activation.

A weakened transcriptional enhancer yields variegated gene expression.

Identical genes in the same cellular environment are sometimes expressed differently. In some cases, including the immunoglobulin heavy chain (IgH) locus, this type of differential gene expression has been related to the absence of a transcriptional enhancer. To gain additional information on the role of the IgH enhancer, we examined expression driven by enhancers that were merely weakened, rather than fully deleted, using both mutations and insulators to impair enhancer activity. For this purpose we used a LoxP/Cre system to place a reporter gene at the same genomic site of a stable cell line. Whereas expression of the reporter gene was uniformly high in the presence of the normal, uninsulated enhancer and undetectable in its absence, weakened enhancers yielded variegated expression of the reporter gene; i.e., the average level of expression of the same gene differed in different clones, and expression varied significantly among cells within individual clones. These results indicate that the weakened enhancer allows the reporter gene to exist in at least two states. Subtle aspects of the variegation suggest that the IgH enhancer decreases the average duration (half-life) of the silent state. This analysis has also tested the conventional wisdom that enhancer activity is independent of distance and orientation. Thus, our analysis of mutant (truncated) forms of the IgH enhancer revealed that the 250 bp core enhancer was active in its normal position, approximately 1.4 kb 3' of the promoter, but inactive approximately 6 kb 3', indicating that the activity of the core enhancer was distance-dependent. A longer segment--the core enhancer plus approximately 1 kb of 3' flanking material, including the 3' matrix attachment region--was active, and the activity of this longer segment was orientation-dependent. Our data suggest that this 3' flank includes binding sites for at least two activators.

A system for precise analysis of transcription-regulating elements of immunoglobulin genes.

BACKGROUND: Precise analysis of expression-regulating elements, such as enhancers and insulators, requires that they be tested under reproducible, isogenic conditions. The commonly used methods of transfecting DNA into cell lines and selecting for drug resistance lack the requisite precision, as they yield cell lines in which varying numbers of gene copies have inserted at varying and undefined sites. By contrast, recombination-mediated cassette exchange (RMCE), by which a site-specific recombinase is used to place a single copy of a transgene at a constant chromosomal site of a cell line, offers the necessary precision. Although RMCE is generally applicable, many regulatory elements of interest are tissue-specific in their function and so require cell lines in the appropriate ontogenetic state. RESULTS: As reported here, we have used RMCE in a mouse B hybridoma cell line to establish a system with several additional advantages. To avoid the non-physiological features of prokaryotic DNA, this system uses the immunoglobulin mu heavy chain (IgH) gene from the hybridoma as the reporter. Expression can be measured simply by bulk culture assays (ELISA, Northern blot) and single cell assays (flow cytometry). Expression of the IgH reporter gene varies only 1.5 fold among independent transfectants, and expression is greatly (> 50 fold) increased by inclusion of the IgH intronic enhancer. CONCLUSION: This system is suitable for precise analysis of the regulatory elements of the immunoglobulin loci.

Complex regulation of somatic hypermutation by cis-acting sequences in the endogenous IgH gene in hybridoma cells.

To create high-affinity antibodies, B cells target a high rate of somatic hypermutation (SHM) to the Ig variable-region genes that encode the antigen-binding site. This mutational process requires transcription and is triggered by activation-induced cytidine deaminase (AID), which converts deoxycytidine to deoxyuridine. Mistargeting of AID to non-Ig genes is thought to result in the malignant transformation of B cells, but the mechanism responsible for targeting SHM to certain DNA regions and not to others is largely unknown. Cis-acting elements have been proposed to play a role in directing the hypermutation machinery, but the motifs required for targeting SHM have been difficult to identify because many of the candidate elements, such as promoters or enhancers, are also required for transcription of Ig genes. Here we describe a system in cultured hybridoma cells in which transcription of the endogenous heavy-chain Ig gene continues in the absence of the core intronic enhancer (Emu) and its flanking matrix attachment regions (MARs). When AID is expressed in these cells, SHM occurred at the WT frequency even when Emu and the MARs were absent together. Interestingly, SHM occurred at less than the WT frequency when Emu or the MARs were individually absent. Our results suggest that these intronic regulatory elements can exert a complex influence on SHM that is separable from their role in regulating transcription.

The epigenetic stability of the locus control region-deficient IgH locus in mouse hybridoma cells is a clonally varying, heritable feature.

Cis-acting elements such as enhancers and locus control regions (LCRs) prevent silencing of gene expression. We have shown previously that targeted deletion of an LCR in the immunoglobulin heavy-chain (IgH) locus creates conditions in which the immunoglobulin micro heavy chain gene can exist in either of two epigenetically inherited states, one in which micro expression is positive and one in which micro expression is negative, and that the positive and negative states are maintained by a cis-acting mechanism. As described here, the stability of these states, i.e., the propensity of a cell to switch from one state to the other, varied among subclones and was an inherited, clonal feature. A similar variation in stability was seen for IgH loci that both lacked and retained the matrix attachment regions associated with the LCR. Our analysis of cell hybrids formed by fusing cells in which the micro expression had different stabilities indicated that stability was also determined by a cis-acting feature of the IgH locus. Our results thus show that a single-copy gene in the same chromosomal location and in the presence of the same transcription factors can exist in many different states of expression.

Use of a simple, general targeting vector for replacing the DNA of the heavy chain constant region in mouse hybridoma cells.

It is often necessary to modify the constant region of the immunoglobulin (Ig) heavy chain in order to produce Ig with optimal properties. In the case of Ig production by mouse hybridoma cells, it is possible to modify the Ig heavy chain (IgH) locus by gene targeting to achieve the desired changes. DNA segments from the JH-S micro region and from the region 3' of Calpha are normally present in the functional IgH gene of all hybridomas, regardless of the heavy chain class which is expressed. Consequently, these DNA segments could in principle serve as 5' and 3' homology regions to create a "universal" targeting vector for replacing the constant region exons in the IgH locus of any hybridoma cell. The practicality of this vector design has been uncertain. That is, the extent of the chromosomal DNA which would be replaced by a universal targeting vector would be as little as 5 kb (in a cell producing the alpha heavy chain) and as much as 180 kb (in a micro -producing cell), and it has been uncertain whether it would be practical to generate such long chromosomal deletions by gene targeting. Using a vector of this design, we found (a) that correctly targeted recombinant cells lacking the 180 kb DNA segment occurred at a low but usable frequency, (b) that these recombinants expressed the modified IgH locus at the same rate as the original hybridoma and (c) that IgH expression in these cell lines was stable. Our results thus indicate that this vector design is suitable for modifying IgH loci expressing any heavy chain, provided that an efficient selection or screening for targeted recombinants is available.

Novel genetic markers in the 5'-flanking region of ANKH are associated with ankylosing spondylitis.

OBJECTIVE: To use a candidate gene approach for the identification of genetic markers that are significantly linked to and associated with ankylosing spondylitis (AS). METHODS: We searched for novel polymorphisms in the ANKH gene (human homolog of the murine progressive ankylosis gene) and genotyped 2 polymorphic sites, one in the 5'-noncoding region and the other in the promoter region of ANKH, using DNA from affected (n = 273) and unaffected (n = 112) individuals from 124 AS families. We used these ANKH and other nearby polymorphisms to perform linkage and family-based association analyses. RESULTS: We identified 2 novel polymorphic sites: one in the 5'-noncoding region of ANKH involving 1-2 copies of an 8-bp repeat (denoted as ANKH-OR), and the other in the promoter region involving different copy numbers of a triplet repeat (denoted as ANKH-TR). ANKH-OR and ANKH-TR were in complete linkage disequilibrium. Five markers (D5S1953, ANKH-TR, ANKH-OR, D5S1954, and D5S1963) were used for both the linkage and association analyses. Multipoint linkage analysis of 124 AS families showed a modest level of significance (nonparametric linkage score 2.15; P = 0.015) at the ANKH region. The contribution of ANKH to AS susceptibility (lambda(s)) was 1.9. A family-based association study on the same AS families revealed that both ANKH-OR allele 1 and ANKH-TR allele 7 were significantly associated with disease, assuming an additive model (for ANKH-OR allele 1, P = 0.03; for ANKH-TR allele 7, P = 0.04). CONCLUSION: Our results indicate that ANKH-OR and ANKH-TR are novel genetic markers that are significantly associated with AS.

Positive and negative transcriptional states of a variegating immunoglobulin heavy chain (IgH) locus are maintained by a cis-acting epigenetic mechanism.

Analyses of transgene expression have defined essential components of a locus control region (LCR) in the J(H)-C(mu) intron of the IgH locus. Targeted deletion of this LCR from the endogenous IgH locus of hybridoma cells results in variegated expression, i.e., cells can exist in two epigenetically inherited states in which the Ig(mu) H chain gene is either active or silent; the active or silent state is typically transmitted to progeny cells through many cell divisions. In principle, cells in the two states might differ either in their content of specific transcription factors or in a cis-acting feature of the IgH locus. To distinguish between these mechanisms, we generated LCR-deficient, recombinant cell lines in which the Ig(mu) H chain genes were distinguished by a silent mutation and fused cells in which the mu gene was active with cells in which mu was silent. Our analysis showed that both parental active and silent transcriptional states were preserved in the hybrid cell, i.e., that two alleles of the same gene in the same nucleus can exist in two different states of expression through many cell divisions. These results indicate that the expression of the LCR-deficient IgH locus is not fully determined by the cellular complement of transcription factors, but is also subject to a cis-acting, self-propagating, epigenetic mark. The methylation inhibitor, 5-azacytidine, reactivated IgH in cells in which this gene was silent, suggesting that methylation is part of the epigenetic mark that distinguishes silent from active transcriptional states.

Differential activation of human and guinea pig complement by pentameric and hexameric IgM.

Human and mouse IgM can be polymerized as a hexamer in addition to a pentamer. Our previous work with mouse IgM measured activation of guinea pig complement by highly enriched preparations of hexamer and pentamer and showed that hexamer is >100-fold more active than pentamer. In this report pentamer and hexamer were compared for their capacity to activate complement in a homogeneic system, i.e. chimeric mouse V/human Cmu IgM pentamer and hexamer were assayed separately for their capacity to activate human (and guinea pig) complement. In both the homogeneic and the xenogeneic systems hexamer was more active than pentamer, but the magnitude of the difference between hexamer and pentamer depended on the complement source. Whereas chimeric hexamer activated guinea pig complement >100-fold more efficiently than did chimeric pentamer, this hexamer was only 4-13-fold more active than pentamer when assayed with human complement. Similarly, mouse hexamer, which was >100-fold more active than mouse pentamer with guinea pig complement, was only approximately 2-fold more active than mouse pentamer with human complement. Mouse hexameric and pentameric IgM were each approximately 20-fold more active with human complement than were the corresponding chimeric isoforms of IgM.

Activation-induced cytidine deaminase turns on somatic hypermutation in hybridomas.

The production of high-affinity protective antibodies requires somatic hypermutation (SHM) of the antibody variable (V)-region genes. SHM is characterized by a high frequency of point mutations that occur only during the centroblast stage of B-cell differentiation. Activation-induced cytidine deaminase (AID), which is expressed specifically in germinal-centre centroblasts, is required for this process, but its exact role is unknown. Here we show that AID is required for SHM in the centroblast-like Ramos cells, and that expression of AID is sufficient to induce SHM in hybridoma cells, which represent a later stage of B-cell differentiation that does not normally undergo SHM. In one hybridoma, mutations were exclusively in G*C base pairs that were mostly within RGYW or WRCY motifs, suggesting that AID has primary responsibility for mutations at these nucleotides. The activation of SHM in hybridomas indicates that AID does not require other centroblast-specific cofactors to induce SHM, suggesting either that it functions alone or that the factors it requires are expressed at other stages of B-cell differentiation.