Persistence of partially functional double-stranded (ds) DNA binding B cells in mice transgenic for the IgM heavy chain of an anti-dsDNA antibody

Yih-Pai Chu, Devon Taylor1, Han-Guang Yan2, Betty Diamond and Linda Spatz2

Departments of Microbiology & Immunology and Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA
1 Department of Chemistry, and
2 Department of Microbiology and Immunology, Sophie Davis School of Biomedical Education, City College of New York, New York, NY 10031, USA

Correspondence to: L. Spatz


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
One mechanism by which anti-double stranded (ds) DNA B cells are regulated is anergy. Multiple phenotypes have been attributed to anergic B cells in various transgenic models. Differences in the nature of the antigen and in the avidity of antigen–antibody interactions may account for these variations in phenotype. In the present study we describe a population of dsDNA binding B cells that display many of the features of anergic B cells, but have characteristics which suggest they are partially functional as well. These B cells do not spontaneously secrete antibody nor can they be induced to secrete antibody following receptor cross-linking in vitro. Furthermore, they display an immature phenotype and have a shortened lifespan, characteristic of anergic B cells. However, they can be induced to secrete anti-dsDNA antibody following activation with T cell-derived factors as well as with lipopolysaccharide (LPS) and they can be recovered by somatic cell hybridization even in the absence of LPS stimulation prior to fusion. These results suggest that antigen receptor signaling can be uncoupled from signaling induced by T cell-derived factors or LPS and that this may be a mechanism for maintaining tolerance. This may have protective advantages because it may enable B cells to be down-regulated in response to autoantigen yet be available for recruitment in an inflammatory response.

Keywords: anergy, double-stranded DNA, systemic lupus erythematosus, tolerance


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Understanding the regulation of autoreactive B cells has been challenging, in part because the frequency of these B cells in the total lymphocyte population is small. Transgenic technology has diminished this problem by making it possible to generate larger numbers of autoreactive B cells. Thus, their fate can be followed, and the cell fate decisions that are made in non-autoimmune and autoimmune hosts can be elucidated.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of IgG anti-double stranded (ds) DNA antibodies. The presence of these antibodies correlates with disease activity, especially glomerulonephritis. To understand the immunological defects in SLE and the regulation of anti-dsDNA B cells, we previously generated non-autoimmune, NZW mice transgenic for the {gamma}2b heavy chain of the R4A anti-dsDNA antibody (1). The R4A anti-dsDNA antibody is encoded by an S107, V11 heavy chain gene and a V{kappa}1 light chain gene, and has been shown to deposit in glomeruli of SCID mice (2,3). We have previously demonstrated that tolerance induction is intact in NZW R4A-C{gamma}2b transgenic mice and have characterized three populations of anti-DNA B cells that are differentially regulated in these mice (1,4). There is an anergic population that secretes high-affinity anti-dsDNA antibodies after in vitro stimulation with LPS. These B cells predominantly use V{kappa}1 light chain genes which are somatically mutated. There is a second subset of B cells with high affinity for dsDNA, that is targeted to deletion but can be rescued in autoimmune, NZB/W F1 mice transgenic for R4A-C{gamma}2b and in mice transgenic for both R4A-C{gamma}2b and the proto-oncogene, bcl-2 (4,5). This population utilizes non-V{kappa}1 light chain genes. The third population is not tolerized, and produces antibodies that display low-affinity for dsDNA and utilizes a spectrum of light chain genes (6).

Other laboratories have studied the regulation of IgM anti-DNA B cells using transgenic mouse models and have observed that several mechanisms of tolerance, including anergy, deletion and receptor editing, contribute to the regulation of dsDNA binding B cells. While B cells specific for single-stranded (ss) DNA have been shown to be targeted to anergy only, those specific for dsDNA have been shown to be targeted to receptor editing, anergy or deletion depending upon the affinity of the anti-dsDNA antibody for the autoantigen (711).

We and others have demonstrated that IgG transgenic heavy chains can promote normal B cell development in mice (12,13). However, we have been concerned that the mechanisms of tolerance induction in NZW mice transgenic for R4A-C{gamma}2b may not be representative of tolerance mechanisms in non-transgenic animals since the expression in naive B cells of IgG heavy chains prior to IgM heavy chains is not physiological. This study was, therefore, undertaken to generate NZW mice transgenic for the R4A-Cµ heavy chain and to determine whether tolerance is maintained in these mice in a manner similar to that observed in R4A-C{gamma}2b mice. The R4A-Cµ heavy chain utilizes a VDJ region that is identical to that used by the R4A-C{gamma}2b transgene, previously described (1). The only difference between these transgenes is that the {gamma}2b constant region has been replaced by a µ constant region in the R4A-Cµ construct. In R4A-Cµ mice the transgenic µ heavy chain can pair with a variety of endogenous light chains to produce both dsDNA binding and non-dsDNA binding antibodies.

In this study, we observe that tolerance is maintained in the R4A-Cµ mice. Moderate- to high-affinity dsDNA binding B cells persist in the periphery of these mice and like their {gamma}2b counterparts they do not spontaneously secrete antibody. These B cells display characteristics of anergic B cell including reduced surface expression of the BCR, arrested development, shortened lifespan and inability to be activated by BCR cross-linking. However, unlike their {gamma}2b counterparts they can be easily rescued by hybridoma technology in the absence of prior stimulation with lipopolysaccharide, and can be activated to differentiate and secrete antibody in vitro by T cell-derived factors. Hence there appears to be a qualitative difference in the level of anergy induced in the R4A-Cµ and the R4A-C{gamma}2b B cells which may be a consequence of differences in expression levels of the transgenes or may be due to differences in heavy chain isotypes. Nevertheless, the observation that R4A-Cµ dsDNA binding B cells are responsive to T cell-derived factors but not BCR cross-linking suggests that these B cells are partially functional and that signaling pathways leading to their activation can be uncoupled.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Transgene construction and generation of transgenic mice
A genomic clone containing a 3.5-kb fragment consisting of the rearranged VDJ (variable–diversity–joining) region and the heavy chain enhancer region, from the anti-dsDNA producing hybridoma R4A, was ligated to a 9.9-kb fragment obtained from a BALB/c mouse, containing all exons of the µ heavy chain gene including the secreted and membrane exons. This construct was cloned into pBluescript. The entire 13.4-kb VDJ–Cµ fragment was cut out of pBluescript using the restriction enzyme NotI, and then purified by electroelution and microinjected into the male pronucleus of C57BL/6xCBA fertilized eggs (14). The R4A-Cµ transgene was bred onto the NZW background for at least 10 generations.

Southern and Northern blot analysis
Tail DNA was digested with SacI, transferred to nitrocellulose after electrophoresis and hybridized with a 570-bp BamHI–PstI probe containing part of the CH2 and CH3 domains of the µ constant region (15). Total RNA was isolated from the lung, kidney, heart, spleen and bone marrow of a transgenic NZW mouse and from the spleen of a non-transgenic littermate using Tri-Reagent (MRC, Cincinnati, OH). RNA was electrophoresed on a denaturing formaldehyde gel, transferred to nitrocellulose and hybridized to the Cµ probe described above or a probe specific for the S107 VH gene family (16).

ELISAs
To detect IgMa anti-dsDNA antibodies, Immulon-2, 96-well plates (Dynatech, Chantilly, VA) were coated with salmon sperm dsDNA (Calbiochem, La Jolla, CA) or calf thymus dsDNA diluted to 100 µg/ml in PBS and filtered through a 0.45 µm nitrocellulose filter (Millipore, Bedford, MA) to remove ssDNA. Plates were dried overnight at 37°C and blocked in PBS, 1.0% BSA, pH 7.2 for 2 h at room temperature. Wells were then incubated with serum samples diluted 1:100 in PBS, 0.1% BSA, culture supernatants from in vitro activation assays or hybridoma supernatants normalized to 5 µg/ml, for 2 h at 37°C. Plates were washed 6 times in PBS, 0.05% Tween 20 and then incubated with biotinylated rat anti-mouse IgMa antibody (biotin–anti-mouse IgMa; PharMingen, San Diego, CA) diluted 1:200 in PBS, 0.1% BSA for 1 h at 37°C. Plates were washed again and incubated with a 1:1000 dilution of alkaline phosphatase-conjugated streptavidin (streptavidin–AP) (Southern Biotechnology, Birmingham, AL) for 1 h at 37°C and then developed with p-nitrophenyl disodium phosphate as substrate (Sigma, St Louis, MO). Plates were read at 405 nm using a Titertek Multiscan ELISA reader.

Total IgMa antibodies in mouse sera and in culture supernatants were detected by ELISA. Briefly, 96-well polystyrene, Falcon plates (Becton Dickinson, Franklin Lakes, NJ) were coated at 1.0 µg/well with goat anti-mouse IgM antibody (Southern Biotechnology). Plates were blocked and washed as described above and then incubated with mouse sera or culture supernatants for 1 h at 37°C. Wells were washed again and incubated with biotin–anti-mouse IgMa followed by streptavidin–AP. Plates were developed with substrate solution and read at 405 nm on a Titertek Multiscan ELISA reader. IgMa antibodies in mouse sera were quantitated by ELISA, as previously described using biotin–anti-mouse IgMa antibody followed by streptavidin–AP and an allotype-matched standard (3).

In vitro LPS stimulation
Splenocytes isolated from five, 8- to 10-week-old NZW mice, transgenic for the R4A-Cµ heavy chain, were cultured in triplicate at 2x106cells /ml in RPMI 1640 medium (Sigma) supplemented with 10% FCS (Hyclone, Logan, UT), 2 mM L-glutamine (Sigma), 100 U/ml penicillin, 100 µg/ml streptomycin (Sigma), 100 µM non-essential amino acids (Sigma) and 50 µM ß-mercaptoethanol, in 24-well tissue culture plates with and without 10 µg/ml lipopolysaccharide (LPS) (Escherichia coli. serotype 055:B5; Sigma). After incubation for 72 h at 37°C, 5% CO2, cell supernatants were collected and assayed by ELISA for the presence of IgMa antibodies to dsDNA and for total IgMa antibodies.

In vitro activation and ELISpot
Splenocytes isolated from transgenic mice were enriched for B cells by depletion of T cells using anti-Thy-1.2 magnetic beads and MACS separation columns (Miltenyi Biotec, Auburn, CA). Enriched B cell preparations were diluted in RPMI 1640, 10% FCS, 50 µM ß-mercaptoethanol to a concentration of 2.0x106 cells /ml and incubated at 37°C, 5% CO2 in the presence of either 20 µg/ml of LPS, 10µg/ml F(ab')2 fragment of goat anti-mouse IgM (ICN, Cappel, Aurora, OH), 10 µg/ml of antibody to CD40 (PharMingen, San Diego, CA), 300 U of recombinant mouse IL-4 (rIL-4) (PharMingen), 10 µg/ml of anti-CD40 plus 300 U of rIL-4, 200 U of rIL-5 (PharMingen), 300 U of rIL-6 (PharMingen) or a cocktail of rIL-4, rIL-5 and r-IL-6. After 48 h in culture, cells were harvested and plated on Immulon-2 plates coated with 100 µg/ml of salmon sperm dsDNA or on Falcon plates coated with a 1:1000 dilution of anti-IgM antibody (Southern Biotechnology). After 6 h, plates were washed 5 times with 10 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.2 and then biotin–anti-mouse IgMa diluted 1:300 in PBS, 0.1% BSA was added to wells and plates were incubated overnight at 4°C. Plates were washed again and then incubated with a 1:1000 dilution of streptavidin–AP for 3 h at room temperature. 5-Bromo-4-chloro-3-indolyl phosphate (Sigma) was added as substrate and plates were allowed to develop at room temperature for 2–4 h. Antibody-secreting cells were counted under a dissecting microscope.

Flow cytometry
Single-cell suspensions were prepared from the bone marrow and spleen of transgenic mice and non-transgenic littermates, and were depleted of red blood cells by ammonium chloride lysis. Cells were resuspended at a concentration of 1.0x106 cells/0.1ml of PBS, 1% BSA and were treated with Fc receptor block (CD16/CD32) (PharMingen) for 5 min on ice. Cell suspensions were then surface stained by incubation with a combination of antibodies for 30 min at 4°C followed by 2 washes with PBS, 1% BSA. The following antibodies were used: CyChrome–anti-B220, FITC–anti-CD24 (PharMingen), FITC–anti-{kappa} (Southern Biotechnology), phycoerythrin (PE)–anti-IgMa and FITC–anti-IgMb (PharMingen). In some experiments biotin–anti-mouse IgMa or biotin–anti-mouse IgMb (PharMingen) followed by streptavidin–PE (Southern Biotechnology) were used. All samples were analyzed on a Coulter dual laser cytometer using Elite software. Gating was set for live lymphocytes based on forward and side scatter, and 50,000 events were collected for each sample. Analysis was performed using WinMDI, version 2.8 (Scripps Institute). For in vitro activation studies, splenocytes were enriched for B cells by depleting T cells by magnetic bead cell sorting using anti-thy1.2 microbeads (Miltenyi Biotec, Auburn, CA).

5-Bromo-2'-deoxyuridine (BrdU) labeling
BrdU labeling was according to Mandik-Nayak et al. (17). Briefly, NZW mice transgenic for R4A-Cµ or R4A-C{gamma}2b or wild-type control NZW mice were injected i.p. every 12 h for 8 days with 200 µl of 3 mg/ml BrdU (Sigma). Splenocytes were isolated on day 8 and surface stained with CyChrome–anti-B220 and either biotin–anti-IgMa or biotin–anti-IgMb followed by streptavidin–PE. Cells were then fixed and permeabilized in 1% paraformaldehyde plus 0.1% Tween 20, and DNA was denatured in 0.14M NaCl, 4.2 mM MgCl2 buffer containing 10 µM HCl and 100 U/ml of DNase I. Cells were then stained with FITC-labeled anti-BrdU (Becton Dickinson) in order to detect BrdU incorporation and analyzed by flow cytometry. Gates were set on B220+, IgMa or B220+, IgMb and percent FITC staining was displayed as a histogram.

Generation of hybridomas
Spleen cells derived from (six) 8- to 10-week-old transgenic R4A-Cµ, NZW mice were fused with NSO cells as described previously (12).

Analysis of V{kappa} gene expression
Hybridomas were screened for expression of V{kappa}1 genes by RNA dot-blot, as previously described (12). A probe specific for the mouse V{kappa}1 gene family was used. Sequencing the variable regions of V{kappa} light chain genes was according to Spatz et al. (4).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Generation of R4A-Cµ transgenic mice
The R4A-Cµ transgene was generated by ligating a 3.5-kb genomic fragment containing an unmutated, rearranged V11 gene and the heavy chain enhancer region, to a 9.9-kb genomic fragment containing both the secreted and membrane exons of Cµ. This 3.5-kb fragment containing the R4A VDJ region is identical to the one used to generate the R4A-C{gamma}2b construct as previously described (1). The Cµ fragment was derived from a BALB/c mouse so it bears the a allotype. The R4A-Cµ transgene was injected into C57BL/6xCBA F1 fertilized eggs and then backcrossed onto the NZW background for at least 10 generations. Southern blot analysis of tail DNA digested with SacI and hybridized with a probe specific for the CH2–CH3 regions of Cµ demonstrated integration of the transgene in a single site in the genome (Fig. 1AGo). Densitometry revealed the presence of five copies of the transgene in the genome.



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Fig. 1. (A) Map of the R4ACµ heavy chain transgene. A 3.5-kb EcoRI fragment containing the rearranged VDJ and heavy chain enhancer (EH) regions was ligated to a 9.9-kb fragment containing the µ constant region including the membrane exons (mµ). White boxes depict exons. Dark boxes depict introns. CH(1–4), constant region exons; E, EcoRI; B, BamHI; N, NotI; S, SacI; P, PstI, sµ, secretion coding exon; Mµ, membrane coding exons. (B) Southern blot of tail DNA digested with SacI and hybridized to a Cµ probe. Lane 1, DNA from a non-transgenic mouse; lane 2, DNA from a transgenic littermate (the transgene is indicated by a band at 9.7 kb). (C) Tissue-specific expression of the R4A-Cµ transgene. RNA isolated from different organs from a R4A-Cµ transgenic (Tg) mouse (lanes 1–4) was hybridized with a probe specific for the S107 VH gene family (top panel) and a probe specific for the Cµ constant region (bottom panel). RNA from the spleen of a non-transgenic littermate (NTg) was used as control (lane 5).

 
RNA expression
Expression of the R4A-Cµ transgene was demonstrated by Northern blot analysis of tissue RNA hybridized with a probe specific for the S107 VH gene family. Elevated levels of expression of the transgene were observed in the spleen of a transgenic mouse (top panel, Fig. 1CGo). Expression of endogenous S107 in the non-transgenic spleen was too low to be detected at this exposure. Minimal levels of expression of the transgene were observed in the lung, kidney and heart of the transgenic mouse. A probe specific for the µ constant region hybridized to splenic RNA from the transgenic mouse and its non-transgenic littermate indicating that equivalent concentrations of RNA from both mice were loaded onto the gel (bottom panel, Fig. 1CGo).

Assays for serum Ig
Serum from 10, 7- to 12-week-old transgenic NZW mice and 10 non-transgenic NZW littermates were assayed for IgMa anti-dsDNA binding by ELISA (Fig. 2AGo). Since the transgene is of the a allotype and endogenous IgM in the NZW mouse strain is of the b allotype, transgenic IgM could be distinguished from endogenous IgM using an anti-IgM antibody specific for the a allotype. A purified monoclonal IgMa anti-dsDNA antibody (4D4) that utilizes the R4A heavy chain was used at a concentration of 1 µg/ml, as a positive control for dsDNA binding.



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Fig. 2. Measurement of R4A-Cµ anti-dsDNA antibody in the sera of transgenic mice and their non-transgenic littermates. (A) Sera from 10 transgenic mice (Tg) and 10 non-transgenic littermates (NTg) were diluted 1/100, and then screened by ELISA for binding of IgMa antibody to salmon sperm dsDNA-coated plates. (B) The concentration of total IgMa antibody in the sera of Tg and NTg mice was quantitated by ELISA. Mice ranged in age from 7 to 12 weeks. Mean values are indicated by a bar. Purified IgMa anti-dsDNA antibody designated 4D4, obtained from a hybridoma line generated from R4A-Cµ mice, was diluted to 1 µg/ml and used as a positive control.

 
Serum concentrations of total IgMa were determined by a quantitative ELISA (Fig. 2BGo). Despite the observation that transgenic mice contain between 3 and 18 µg/ml of total IgMa antibody in their sera, none of this antibody was observed to bind dsDNA. As mentioned previously, the transgenic heavy chain can pair with some endogenous light chains to produce non-DNA binding antibodies which can account for the serum concentration of IgMa antibody.

Characterization of transgenic B cells by flow cytometry
Bone marrow and splenic B cells from transgenic mice and non-transgenic littermates were examined by flow cytometry. By staining bone marrow and splenic cells with CyChrome–anti-B220 only or in combination with PE–anti-IgMa and FITC–anti-IgMb we determined the frequency of total B cells, B cells expressing the transgene (IgMa) and B cells expressing endogenous IgM (IgMb). Analysis of B220+ gated B cells revealed that the majority of transgene-expressing B cells in the bone marrow and spleen express IgMa only and not endogenous IgMb. This demonstrates that the transgene maintains intact allelic exclusion (Fig. 3AGo). The splenic B cells that fail to surface stain with PE–anti-IgMa or FITC–anti-IgMb express IgG heavy chains and none of the IgG B cells co-express IgMa (data not shown).




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Fig. 3. FACS analysis of transgenic and non-transgenic mice. (A) Heavy chain allelic exclusion; bone marrow and splenic B cells from Tg and NTg mice were tripled stained with CyChrome–anti-B220, PE–anti-IgMa and FITC–anti-IgMb. Gates were set on B220+ cells, and percent IgMa, IgMb and IgMab B cells are displayed by dot-plot. (B) Developmental status of transgenic B cells. Bone marrow cells from Tg and NTg mice were triple stained with CyChrome–anti-B220, FITC–anti-CD24 and either biotin–anti-IgMa or biotin–anti-IgMb followed by streptavidin–PE. Gates were set on B220+ B cells. Percentages of CD24hi and CD24lo B cells are indicated. This experiment is representative of four others.

 
Table 1Go summarizes the results obtained from six transgenic mice and six non-transgenic littermates. While the percent of B220 B cells in the bone marrow of transgenic mice and non-transgenic littermates are similar, the percent of B220 B cells is ~2-fold less in the spleens of transgenic mice. Likewise, there is <1.5-fold difference in the absolute number of B cells in the bone marrow of transgenic mice and non-transgenic littermates, while there is almost a 5-fold difference in the absolute number of B cells in the spleen of transgenic mice and non-transgenic littermates. While we cannot rule out the possibility that the diminished B cell number in the spleens of transgenic mice is simply due to the presence of the rearranged transgene and unrelated to B cell specificity, a feature occasionally observed in transgenic mice, results suggest that B cell deletion may be occurring at some time in transit from the bone marrow to the spleen in transgenic mice, presumably because of autoreactivity. In addition, we observe that the relative increase in the number of B cells expressing endogenous IgM (IgMb) from the bone marrow to the spleen in transgenic mice (32-fold) is greater than the increase of B cells expressing the transgene (4-fold) (Table 1Go). This may be due to a selective expansion of B cells expressing endogenous IgM or to anergy and/or deletion of autoreactive, transgenic B cells. Alternatively, receptor revision in immature B cells in the periphery may be promoting the disappearance of B cells bearing the transgenic heavy chain.


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Table 1. Frequency of bone marrow and splenic B cells in transgenic mice and non-transgenic littermates
 
Since other studies have demonstrated that tolerized B cells are arrested in an immature stage of development, we next examined whether more B cells expressing the transgene have an immature phenotype than B cells expressing endogenous IgM (18). Immature B cells express surface IgM, have a CD24hi phenotype and can be detected using an antibody to CD24 (19). We stained bone marrow cells from 2-month-old transgenic mice and non-transgenic littermates with CyChrome–anti-B220, FITC–anti-CD24 and biotin-anti-IgMa or biotin-anti-IgMb followed by streptavidin–PE. A higher percent of bone marrow B cells expressing the transgene is in an immature stage of development than B cells expressing endogenous IgM (Fig. 3BGo). The ratio of CD24hi to CD24lo is 4 for B cells expressing IgMa and 0.9 for B cells expressing IgMb. In the spleen of transgenic mice, however, we did not consistently observe an increased frequency of CD24hi transgene-expressing B cells (data not shown). We speculate this reflects maturation of non-DNA binding IgMa B cells. The observation that at least some IgMa B cells can acquire the mature phenotype indicates that the R4A-Cµ transgene is capable of signaling B cell maturation. The development of a reagent that can specifically track the dsDNA binding subset of transgenic B cells is necessary to accurately determine the fate of autoreactive B cells and this is currently underway.

Density of receptor
Studies by Goodnow et al. demonstrated that in mice transgenic for soluble hen egg lysozyme (HEL) and antibody to HEL, tolerant HEL binding B cells exhibited an altered phenotype characterized by a reduction in membrane IgM (20). To determine whether a subset of B cells in our transgenic mice also exhibited this receptor down-modulation, we stained splenocytes from transgenic and non-transgenic littermates with CyChrome–anti-B220, FITC–anti-{kappa} and either biotin–anti-mouse IgMa or biotin–anti-mouse IgMb respectively, followed by streptavidin–PE. Cells were gated on B220+, IgMa cells (transgenic mice) or B220+, IgMb cells (non-transgenic mice), and mean fluorescent intensity of {kappa} staining was compared for IgMa and IgMb B cells (Fig. 4Go). A subset of transgene-expressing B cells displayed reduced expression of IgMa relative to IgMb, while another subset displayed a level of expression of IgMa comparable to that of IgMb. We speculate that the transgenic B cells expressing a reduced level of IgM are dsDNA binders while those expressing levels of IgM similar to endogenous B cells are non-dsDNA binders.



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Fig. 4. Reduced expression of surface IgMa. Splenocytes from R4A-Cµ transgenic mice and non-transgenic littermates were triple stained with PE–anti-IgMa or PE–anti-IgMb respectively, and CyChrome–anti-B220 and FITC–anti-{kappa}. A gate was set on either B220+, IgMa or B220+, IgMb cells for splenocytes from R4A-Cµ or non-transgenic mice respectively, and {kappa} staining was analyzed by histograms. Results are representative of five experiments.

 
In vitro activation studies
We previously demonstrated that anergic B cells in the spleens of R4A-C{gamma}2b transgenic mice can be activated to secrete anti-dsDNA antibody following in vitro stimulation with LPS. In the present study, we were interested in determining whether an anergic population of anti-dsDNA B cells also exists in the R4A-Cµ mice. We stimulated splenocytes from five R4A-Cµ mice in vitro with 10 µg/ml of LPS and assayed culture supernatants for the presence of anti-dsDNA antibody. Spleen cells cultured with LPS displayed elevated levels of IgMa anti-dsDNA antibodies relative to unstimulated control cells (Fig. 5AGo). Likewise, total IgMa antibody secretion was enhanced by LPS stimulation (Fig. 5BGo). The number of B cells secreting anti-dsDNA antibodies also increased following LPS stimulation as detected by ELISpot (Fig. 6Go).



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Fig. 5. In vitro LPS stimulation of IgMa dsDNA binding B cells. Splenocytes from five (#1–#5) 8- to 10-week-old R4A-Cµ transgenic mice were cultured for 3 days with (open bars) and without (cross-hatched bars) 10 µg/ml LPS. Supernatants were tested by ELISA for (A) IgMa anti-dsDNA antibody secretion and (B) total IgMa antibody secretion. Triplicate samples from each mouse were analyzed.

 


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Fig. 6. In vitro activation of IgMa dsDNA binding B cells. Splenocytes from R4A-Cµ transgenic mice that were enriched for B cells by negative depletion of T cells were incubated in vitro at a concentration of 2.0x106 cells/ml for 48 h at 37°C in medium alone, or with either 20 µg/ml of LPS, 10 µg/ml of antibody to CD40 plus 10 µg/ml of F(ab')2 anti-IgM or 10 µg/ml of anti-CD40 plus 300 units of rIL-4. DNA binding B cells and total IgMa B cells were detected by ELISpot on microtiter plates coated with dsDNA (A) or antibody to IgM (B) respectively, followed by incubation with biotin–anti-IgMa and streptavidin–AP. A hybridoma cell line, 4D4, producing IgMa anti-dsDNA antibody was used as a positive control. Results are representative of four experiments. (C) B cell-enriched splenocytes were cultured with rIL-4, rIL-5, rIL-6 or a cocktail of all three cytokines and dsDNA binding B cells were detected by ELIspot. Results are representative of three experiments.

 
Anergic B cells have been defined as functionally inactivated B cells that fail to secrete antibody in response to cross-linking of their surface Ig with antigen or antibody to Ig (21,22). To determine whether the IgM DNA binding B cells are anergic or resting B lymphocytes, we attempted to activate them with anti-IgM F(ab')2 in the presence of an antibody to CD40 which mimics the action of CD40 ligand (CD40L) thereby mediating T cell help (2224). While the combination of these signals induced total IgMa antibody secretion, as assayed by ELISpot (Fig. 6BGo), it failed to induce the production of anti-dsDNA antibody (Fig. 6AGo), suggesting that the transgenic, dsDNA binding B cells are anergic.

It has been reported in some transgenic mouse models that anergic B cells can be induced to differentiate and secrete antibody in response to CD40L plus IL-4 while others have reported that B cells can proliferate but not secrete antibody under the same conditions (22,2527). We wished to determine whether anti-CD40 plus IL-4 could induce antibody secretion by dsDNA binding B cells from our transgenic mice. We, therefore, cultured T cell-depleted splenocytes for 48 h in the presence of antibody to CD40 and rIL-4. We observed by ELISpot a significant increase in the number of transgenic B cells secreting anti-dsDNA antibody, comparable to that observed with LPS (Fig. 6Go). Based on these results we also sought to determine the affect of incubating splenic B cells with a cocktail of T cell cytokines. We observed that a combination of rIL-4, rIL-5 and rIL-6 could also activate IgMa dsDNA binding B cells to differentiate and secrete anti-dsDNA antibody (Fig. 6CGo).

Lifespan measurement
Anergic B cells have been shown to have a shortened lifespan (17,28). We assessed the lifespan of B cells expressing the R4A-Cµ transgene relative to B cells expressing endogenous IgM. B cells from transgenic mice and non-transgenic littermates were continuously labeled with BrdU for 8 days, and the incorporation of BrdU was measured by flow cytometry. Populations that are rapidly turning over and have a reduced lifespan are replaced more rapidly by labeled cells from the bone marrow, and this is reflected as a higher percentage of cells that have incorporated BrdU. In three out of three experiments we observed that IgMa B cells have a slightly higher incorporation of BrdU relative to non-transgenic B cells, suggesting a reduced lifespan (Table 2Go).


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Table 2. BrdU incorporation in splenic B cells
 
Generation of transgene-expressing IgMa anti-DNA B cell hybridomas
We previously demonstrated that hybridomas secreting high-affinity anti-DNA antibody could be obtained from R4A-C{gamma}2b transgenic mice only after stimulation of spleen cells with LPS prior to fusion. We were unable to generate high-affinity R4A-C{gamma}2b anti-dsDNA hybridomas from unstimulated B cells. To analyze tolerance mechanisms in the R4A-Cµ transgenic mice, we generated hybridomas from LPS stimulated spleen cells. Approximately 750 wells from three fusions with LPS-stimulated splenocytes showed growth of hybridoma cells; 37 displayed IgMa dsDNA binding activity (5%) and 16 were cloned for further analysis.

Surprisingly, we were also able to generate hybridomas from unstimulated, naive splenocytes from R4A-Cµ transgenic mice. Approximately 560 wells from six fusions showed growth of hybridomas. Seventeen of these clones (3%) produced high-affinity IgMa anti-dsDNA antibody. Ten of them were cloned for further analysis. These results indicate that the high-affinity anti-dsDNA B cells present in the R4A-Cµ transgenic mice can form viable hybridomas even without prior LPS activation. Thus, they differ from R4A-C{gamma}2b anti-DNA B cells which can only be immortalized as hybridomas following activation with LPS. By ELISA we demonstrated that anti-dsDNA antibodies produced by hybridomas obtained from either unstimulated or LPS-stimulated splenocytes show a similar binding to dsDNA (Fig. 7Go). We also measured affinities of anti-dsDNA antibodies obtained from both LPS-stimulated and unstimulated R4A-Cµ B cells by inhibition ELISA, and observed that all antibodies have similar apparent affinities for dsDNA ranging from 107 to 108.



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Fig. 7. DNA binding activity in hybridoma supernatants. Normalized supernatants (5 µg/ml) from hybridomas from LPS-stimulated spleen cells (dotted bars) and naive spleen cells (open bars) were measured for dsDNA binding activity by ELISA. µ{kappa} is an IgM antibody without specificity for dsDNA. 2D2 is an IgM anti-DNA antibody of known pathogenic potential.

 
Analysis of V{kappa} gene usage in LPS-stimulated and naive hybridomas
In NZW, R4A-C{gamma}2b mice, we previously observed that the anergic population of transgenic B cells predominantly utilizes a mutated V{kappa}1 light chain gene, while the subset that is targeted to deletion utilizes non-V{kappa}1 genes (4). In the present study we observed by RNA dot-blot that the light chains of all of the R4A-Cµ anti-dsDNA antibodies produced by hybridomas generated following LPS activation utilize V{kappa}1 genes. Similarly, the light chains from seven of nine anti-dsDNA antibodies produced by hybridomas generated from unstimulated B cells utilize V{kappa}1 genes. We sequenced the light chains from 11 antibodies obtained from LPS-stimulated splenocytes and nine antibodies obtained from naive splenocytes to confirm these results.

Ten of 11 antibodies produced by LPS-activated B cells utilize the V{kappa}1-A gene and one utilizes the V{kappa}1-C gene. Only two V{kappa}1-A genes are mutated; one contains a somatic mutation in FR3, resulting in the substitution of a phenylalanine for a serine and one contains a substitution at the VJ joining region resulting in the replacement of a leucine with a proline. This latter change may not be the result of mutation in the periphery, but a consequence of rearrangement of the V J regions in the bone marrow. The V{kappa}1-C gene is unmutated. Seven of nine light chains sequenced from the antibodies obtained from unstimulated anti-dsDNA B cells utilize the V{kappa}1A light chain gene. One of the V{kappa}1A genes contains a substitution in the VJ joining region which results in the replacement of a tryptophan with a proline. Two of the light chains utilize non-V{kappa}1 genes (V{kappa}21-E and V{kappa}OX-1), neither of which is mutated. All sequences have been reported to GenBank.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have observed that dsDNA binding B cells are present, but are anergic, in the periphery of R4A-Cµ mice. They fail to be activated to differentiate and secrete antibody in vitro following cross-linking of their BCR with anti-IgM F(ab')2. However, they can be activated to secrete antibody by LPS, anti-CD40 as a surrogate for T cell help plus IL-4 or a combination of the cytokines IL-4, IL-5 and IL-6. We have also observed that some B cells in the transgenic mice display reduced levels of surface IgMa, have a lag in development as evidenced by CD24hi expression and have a shortened lifespan. Since this phenotype is consistent with that of anergic B cells we believe that these represent the dsDNA binding B cells. We have been able to rescue R4A IgMa dsDNA binding B cells as hybridomas by somatic cell hybridization. Anti-dsDNA antibodies obtained from these hybridomas predominantly utilize unmutated V{kappa}1 light chain genes. This suggests that tolerance induction is occurring in a naive B cell population. We have previously demonstrated that the R4A heavy chain can associate with several V{kappa} families to produce antibodies with specificity for DNA; however, those B cells producing antibodies that utilize non-V{kappa}1 light chains are targeted to deletion in vivo. Failure to obtain anti-dsDNA-producing hybridomas that utilize non-V{kappa}1 light chains from the R4A-Cµ mice confirms that these B cells are targeted to deletion in these mice.

It is intriguing that high-affinity, IgMa V{kappa}1 dsDNA binding B cells can be rescued as hybridomas from unstimulated splenocytes since earlier studies demonstrated that high-affinity, IgG2b, V{kappa}1 dsDNA binding B cells could only be rescued if B cells were activated with LPS prior to fusion (4,12). It is also interesting that IgM dsDNA binding B cells can be activated to secrete antibody following in vitro stimulation with anti-CD40 plus IL-4 since recent observations revealed that IgG2b dsDNA binding B cells from R4A-C{gamma}2b mice cannot be activated to secrete antibody under these conditions (data not shown). The IgM dsDNA binding B cells may be partially functional relative to their IgG2b counterparts and in a weaker stage of anergy, making them more conducive to fusion and more responsive to T cell-derived factors. While qualitative differences in anergy in dsDNA bindng R4A-Cµ and R4A-{gamma}2b B cells may be due to differences in the expression level of the transgenes, we speculate that differences in heavy chain isotype may play a role as well and {gamma} heavy chains may transduce qualitatively different signals leading to tolerance induction than µ heavy chains.

Although anergy is defined as functional unresponsiveness to antigen, the characteristics of anergic B cells seem to vary in different transgenic models. Goodnow et al. first demonstrated that in mice transgenic for soluble HEL and antibody to HEL, anergic HEL binding B cells display reduced levels of surface Ig known as receptor down-modulation and have a shortened lifespan but are not arrested in development (28,29). They also showed that anergic HEL B cells can be induced to proliferate and differentiate into antibody-secreting cells, although at suboptimal levels following in vitro activation with LPS or CD40L plus IL-4 and IL-5 as well as with an antisera to CD40 plus IL-4 (22,25,30).

Erikson and colleagues observed differences in the characteristics of anergic ssDNA and dsDNA binding B cells (17,18,26,27). They observed that anergic ssDNA binding B cells cannot be induced to secrete antibody following BCR cross-linking; however, they can be induced to proliferate following LPS stimulation or cross-linkage with CD40L plus IL-4. Furthermore, they observed that these B cells have a normal lifespan and are not arrested in development. In contrast, they observed that dsDNA binding B cells have a shortened lifespan and are arrested in development, and display an antigen-experienced phenotype. In addition, they do not proliferate in response to LPS or BCR cross-linking and do not differentiate into antibody secreting cells in response to CD40L plus IL-4, although they do proliferate in response to these T cell factors (17,26,27).

Our studies demonstrate a phenotype that differs from that described above for anergic ssDNA and dsDNA binding B cells. While the dsDNA binding B cells in our transgenic mice show evidence of receptor down-modulation, arrested development, shortened lifespan and inability to be activated to secrete antibody following BCR cross-linking, they can be activated to secrete antibody following incubation with LPS or T cell-derived factors. These results are significant because the vulnerability of some `anergic' B cells to non-antigenic stimuli may provide a pathway to a breakdown in tolerance.

Differences in the avidity of interaction between autoantibodies and their autoantigen which reflects receptor affinity, surface density, and concentration of the autoantigen and differences in fine specificity, and the nature of the autoantigen may account for the range of phenotypes observed in anergic B cells. Just as the extent of receptor cross-linking determines whether autoreactive B cells are anergized, deleted or ignored, the strength and/or nature of antigen–antibody interactions may determine qualitative differences in anergy (31,32).

The observation that R4A-Cµ dsDNA binding B cells are partially functional and can respond by antibody secretion to T cell-derived factors and LPS but not BCR cross-linking suggests an uncoupling of signaling cascades. Signaling pathways transduced via the BCR, CD40 ligation or LPS activates the transcription factor, NF-{kappa}B (33 ,34). It is not clear whether these signaling pathways are distinct or have common intermediates upstream of NF-{kappa}B. Protein kinase C (PKC) has been shown to be necessary for BCR but not CD40 or LPS-induced activation of NF-{kappa}B while phosphoinositide 3' kinase (PI-3 kinase) has been shown to be necessary for both BCR- and LPS-induced activation of NF-{kappa}B (3335). Depletion or inhibition of a molecule unique to one pathway may have little affect on the other pathways. This could explain why a block in BCR signaling could be overcome by signaling through CD40 ligation or LPS. Inhibition of a factor common to all three pathways would prevent signaling following BCR cross-linking as well as CD40 ligation and LPS. Therefore, qualitative differences in anergy may be the consequence of blocking one or more signaling pathways. A paradigm for the uncoupling of signaling pathways comes from recent studies in B and T cells. Okkenhaug et al. demonstrated that a point mutation in the co-stimulatory molecule CD28 uncouples signals required for T cell proliferation and survival (36). Bone and Williams demonstrated that an inhibitor of PKC uncouples BCR signaling from signaling through LPS by blocking a PI-3 kinase-dependent pathway to NF-{kappa}B activation that is distinct for BCR signaling (35). Future studies will be aimed at determining mechanisms by which BCR uncoupling may be occurring in anergic R4A-Cµ dsDNA binding B cells.

In summary, this study describes a novel phenotype for dsDNA binding B cells; one in which B cells can be activated to differentiate and secrete antibody in response to T cell-derived factors or LPS, but are unresponsive to signaling through the BCR. The persistence of these `partially functional' B cells due to the uncoupling of the BCR signaling pathway from other signaling pathways may offer the immune system a protective advantage by enabling B cells cross-reactive to both self and foreign antigen to be down-regulated in response to autoantigen while still being available for recruitment in an inflammatory response.


    Acknowledgments
 
This work was supported by NIH grant AI-40163 and an SLE Research grant awarded to L. S., and by NIH grants AI31229 and AR32371 awarded to B. D. The NIH-RCMI grant G12RR-A103060 also aided this work.


    Abbreviations
 
AP alkaline phosphatase
BrdU 5-bromo-2'-deoxyuridine
CD40L CD40 ligand
ds double stranded
HEL hen egg lysozyme
LPS lipopolysaccharide
PE phycoerythrin
PI-3 kinase phosphoinositide 3' kinase
PKC protein kinase C
SLE systemic lupus erythematosus
ss single stranded

    Notes
 
The first two authors contributed equally to this work

Transmitting editor: J. V. Ravetch

Received 6 July 2001, accepted 3 October 2001.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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