The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shankar, M.
Right arrow Articles by Webb, C. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shankar, M.
Right arrow Articles by Webb, C. F.
The Journal of Immunology, 2007, 178: 2996-3006.
Copyright © 2007 by The American Association of Immunologists, Inc.

Anti-Nuclear Antibody Production and Autoimmunity in Transgenic Mice That Overexpress the Transcription Factor Bright1

Malini Shankar*, Jamee C. Nixon*, Shannon Maier{dagger}, Jennifer Workman{dagger}, A. Darise Farris{dagger},{ddagger} and Carol F. Webb2,*,{ddagger},§

* Immunobiology and Cancer Program and {dagger} Arthritis and Immunology Program, Oklahoma Medical Research Foundation, {ddagger} Department of Microbiology and Immunobiology, and § Cell Biology Department, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The B cell-restricted transcription factor, B cell regulator of IgH transcription (Bright), up-regulates Ig H chain transcription 3- to 7-fold in activated B cells in vitro. Bright function is dependent upon both active Bruton’s tyrosine kinase and its substrate, the transcription factor, TFII-I. In mouse and human B lymphocytes, Bright transcription is down-regulated in mature B cells, and its expression is tightly regulated during B cell differentiation. To determine how Bright expression affects B cell development, transgenic mice were generated that express Bright constitutively in all B lineage cells. These mice exhibited increases in total B220+ B lymphocyte lineage cells in the bone marrow, but the relative percentages of the individual subpopulations were not altered. Splenic immature transitional B cells were significantly expanded both in total cell numbers and as increased percentages of cells relative to other B cell subpopulations. Serum Ig levels, particularly IgG isotypes, were increased slightly in the Bright-transgenic mice compared with littermate controls. However, immunization studies suggest that responses to all foreign Ags were not increased globally. Moreover, 4-wk-old Bright-transgenic mice produced anti-nuclear Abs. Older animals developed Ab deposits in the kidney glomeruli, but did not succumb to further autoimmune sequelae. These data indicate that enhanced Bright expression results in failure to maintain B cell tolerance and suggest a previously unappreciated role for Bright regulation in immature B cells. Bright is the first B cell-restricted transcription factor demonstrated to induce autoimmunity. Therefore, the Bright transgenics provide a novel model system for future analyses of B cell autoreactivity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Appropriate regulation of Ig synthesis by B lymphocytes is critical to maintain a balance between induction of strong humoral responses to pathogenic organisms and maintenance of tolerance to self Ags. Several autoimmune diseases, including systemic lupus erythematosus, are first characterized by high titers of anti-nuclear Abs (ANA)3 in serum. In such cases, inappropriate production and regulation of these Abs occur before presentation of other systemic disease responses and, therefore, represent an early indication of tolerance failure. In most cases, ANA production and autoimmune disease progression is severely influenced by the genetic background of the animals used (reviewed in Refs. 1, 2, 3), confirming the complex multigenic nature of most autoimmune diseases. The mechanisms that first lead to overproduction of ANAs and disease progression are unclear, but likely involve disruption of regulatory signals at multiple steps and may even originate from malfunctions in various cell types. Nonetheless, inappropriate Ab production is a defining and primary step in autoimmune disease.

Antigenic signals through the IgR cause B lymphocytes to differentiate and secrete Ab in the case of foreign Ags, or to become tolerized if the Ag is a "self" Ag. B cell tolerance can occur by anergy with survival of antiself cells that are nonfunctional, by receptor editing of L or H chains yielding IgRs that no longer bind self Ags, or by apoptosis and clonal deletion of the self-reactive B cells (4, 5, 6, 7, 8). Tolerant B cells are produced at two major checkpoints during B cell differentiation, the pre-B cell to immature B cell stage in the bone marrow, and in newly emigrating peripheral cells called transitional B cells (9, 10). More recent studies also suggest that tolerance is influenced at later stages in the germinal centers (11). Fetal and neonatal B cells up to day 7 after birth are also tolerant, and signals through the surface IgR more closely resemble those seen in immature adult B cells (12). The complex nature of most autoimmune diseases suggests that not only is tolerance controlled at multiple checkpoints, but also that defects in multiple gene products can lead to breaks in tolerance (13, 14, 15). In some lupus patients, it is now clear that production of ANAs and early breaks in B cell tolerance can precede disease progression by many years (reviewed in Ref. 2). Understanding the molecular events that can lead to production of autoantibodies, before complications due to secondary disease related events occur, is critical for early diagnosis and better disease treatment.

B cell regulator of IgH transcription (Bright) is a member of the A-T-rich interaction domain family of DNA-binding proteins and interacts with the matrix association regions flanking both sides of the intronic H-chain enhancer and 5' flanking sequences of several, but not all, VH genes (16, 17, 18, 19). Several Bright-binding sites were identified 5' of the basal promoter of the V1 S107 family gene, and these sites were required for IL-5 and Ag-induced µ H chain transcription by Bright in vitro (16, 20). More recent data indicate that Bright-enhanced transcription of a V1 reporter construct critically requires both the kinase activity of Bruton’s tyrosine kinase (Btk) and the Btk substrate, TFII-I (21, 22). These data suggest that Bright and Btk share common pathways in some cases, but may also have independent functions.

Bright is expressed in multiple embryonic tissues, but becomes B cell-restricted in the adult (23). Bright transcription in vivo is tightly regulated during B cell differentiation–so that its expression is high in bone marrow pre-B cell subpopulations and in germinal center-activated B cells, but most circulating and splenic mature B cells lack detectable Bright mRNA and protein (23, 24). Although Bright increases Ig transcription in vitro, it is not required for basal Ig production in mature B cells. Thus, the importance of Bright activity for Ig production and normal B cell development in vivo is unknown.

To determine whether appropriate regulation of Bright is important for B cell differentiation in vivo, transgenic mice constitutively expressing Bright in all B lineage cells were produced. Sera from nonimmunized Bright-transgenic mice contained slightly increased levels of total Ig compared with nontransgenic littermates. Expression of the V1 gene, previously shown to be regulated by Bright in vitro (21, 22), was also enhanced in transgenic spleen cells relative to littermate controls. Because the V1 gene is used predominantly in the antiself response against phosphorylcholine (PC) (25, 26, 27), Ag-specific responses reactive with this hapten were also examined. Although anti-PC responses were significantly enhanced in the transgenic mice, responses to other foreign Ags did not differ from littermate controls. Strikingly, sera from even very young Bright-transgenic mice contained ANAs. Moreover, these mice exhibit increases in B lymphopoiesis with significantly increased numbers of transitional type 1 immature B cells, a well-documented B cell tolerance checkpoint, in the spleen. These data suggest that inappropriate regulation of Bright alone during B cell development results in an early autoimmune phenotype.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Transgenic mice

A full-length cDNA for mouse Bright tagged at the C-terminal end with His-Myc sequences (28) was ligated to the SV40 poly(A) site. The native Bright Kozak sequence was modified to (GCCACCATGC) (29), the resulting DNA was ligated to a 6.3-kb fragment containing the human CD19 promoter (30), and was cloned into pUC19. Excised DNA was injected into FVB/N blastocysts by the Oklahoma Medical Research Foundation Transgenic Core Facility. All animal care and procedures were performed with prior institutional approval and within the review board-specified guidelines. Toe DNA from 10- to 11-day-old pups was assessed for the transgene with PCR primers from the Bright coding sequence (5'-GGAAGAGCAAGAGCTGGAAG-3') and the His-Myc tag (5'-CAGATCCTCTTCTGAGATGAG-3'). Seven positive founders were obtained and were assessed for transgene expression by retro-orbital bleeding and RT-PCR analyses of white blood cell RNA. Age-matched male mice, 8–15 wk of age unless otherwise indicated, were used for all assays.

Cell preparation and flow cytometry

Mice were euthanized, thymus lobes and spleens were harvested, and single-cell suspensions in RPMI 1640 with 7% FCS were produced using 70-µm strainers. Whole bone marrow cells were obtained from femurs by flushing with a 23-gauge needle containing PBS-3% FCS.

Cell surface phenotype analyses were performed on 1.5 x 106 cells by flow cytometry using a FACSCalibur or LSRII (BD Biogenics). Cell sorting experiments were performed on a FACSARIA cell sorter (BD Biosciences). Abs purchased from BD Biosciences were: FITC-conjugated CD19 (1D3), CD21 (7G6), and CD4 (RM4-4); PE-conjugated CD8 (53-6.7), CD3 (145-2C11), CD43 (57), CD40 (1C10), CD69 (Hi.2F3), CD80 (1G10), CD86 (GL1), and CD23 (B3B4); allophycocyanin-conjugated CD45R/B220 (RA3-6B2), CD93/C1qRp (AA4.1); and PerCP-conjugated CD45R/B220(RA3-6B2). FITC-IgM, PE-IgD (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26), goat anti-mouse IgM- and 1-A9, MHC class II (KH116)-biotin, appropriate isotype controls and streptavidin conjugated-allophycocyanin were from BD Biosciences or Southern Biotechnology Associates. Cells were stained as previously described (23) and fixed in 0.2% paraformaldehyde overnight. Data were analyzed using CellQuest Pro software (BD Biosciences).

Western blots

Single-cell suspensions were resuspended in SDS-sample buffer and electrophoresed through 7.5% SDS-polyacrylamide gels under standard denaturing conditions. Proteins were transferred to nitrocellulose membranes and developed with polyclonal rabbit anti-Bright as previously described (31). Blots were developed with alkaline phosphatase substrate (Bio-Rad).

ELISA and ANA assays

Mice were anesthetized for retro-orbital bleeding and sera were collected. Costar 96-well U-bottom polyvinyl plates (Corning) were coated with 100 µl/well of 2 µg/ml goat anti-mouse Ig in borate saline (pH 8.4) and incubated overnight at 4°C, washed three times with PBS, blocked with 1% BSA (Sigma-Aldrich) in PBS for 1 h at 20°C, and four dilutions of duplicate serum samples were added overnight at 4°C. Wells were washed four times, and developed with isotype-specific alkaline phosphatase-labeled Abs from the Clonotyping System-AP kit (Southern Biotechnology Associates) and 4-nitrophenylphosphate disodium salt hexahydrate (Sigma- Aldrich). Reactions were stopped with 50 µl/well of 3 N NaOH and read on a Dynex MRX microtiter reader (Dynatech Laboratories). Standard curves were generated using serially diluted duplicates of known concentrations of each Ig isotype and Ig levels were determined using Excel software. For Ag-specific responses, plates were coated with nitrophenyl (NP)-BSA or PC-BSA and duplicate sera samples were serially diluted. The last dilution to give a reading above background was taken as the final dilution. Detection of anti-dsDNA was performed according to the manufacturer’s directions (kit 5100) using duplicate sera samples at multiple dilutions and the provided positive and negative controls (Alpha Diagnostics). Urine albumin concentrations were assessed with the Mouse Albumin ELISA Quantitation kit (Bethyl Laboratories) as per the manufacturer’s directions. A standard curve for albumin was calculated using a semilogarithmic scale in Graph Pad Prism version 4.0.

ANA assays were performed using the NOVA Lite HEp-2 kit (INOVA Diagnostics) according to the manufacturer’s directions at 1/40, 1/120, 1/360, 1/1080 dilutions in PBS. Slides were incubated with anti-mouse IgG-FITC conjugate and visualized with a Zeiss Axioplan 2i microscope. Pictures were taken with an AxioCam HRm camera (Carl Zeiss). Data were analyzed using AxioVision LE.

Immunizations

Mice were immunized i.p. with PC-keyhole limpet hemocyanin (KLH) or NP-KLH (0.5 µg/ml) in CFA (Sigma-Aldrich) and boosted on day 7 with the same dose of Ag. Serum was collected at days 0, 7, and 14 postimmunization.

RT-PCR analyses

Total RNA was extracted from sorted B cell subpopulations using TriReagent (MRC) according to manufacturer’s instructions. cDNA was generated in reactions containing RNA from ~7.5 x 103 cells as previously described (32). Levels of S107 family VH-specific IgM and GAPDH mRNA were assessed as described (22). Samples were electrophoresed through 1.0% agarose gels, transferred to GeneScreen Plus hybridization membrane (PerkinElmer Life Science Products), and hybridized with a [32P]V1 cDNA probe. Relative intensities of the PCR products were quantified using LumiAnalyst 3.0 software (Roche).

Cell culture assays

Splenic B cells were T cell depleted using anti-Thy-1 and guinea pig complement and isolated by centrifugation through a Ficoll gradient as previously described (31). Cells were plated at 1 x 106 cells/ml in RPMI 1640 (supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 5 x 10–5 M 2-ME, and 1 mM sodium pyruvate) alone or with 20 µg/ml LPS (Escherichia coli 0111:1B4 (Sigma-Aldrich)) or with CD40L-expressing SF9 or wild-type SF9 control cells at a ratio of 1 SF9 cell to 10 B cells (31). After 72 h, cells and supernatants were harvested for flow cytometry and ELISA. In some cases, wells were pulsed with 1 µCi [3H]thymidine for 6 h, harvested, and [3H] incorporation was measured.

Histological analyses

Spleens from euthanized animals were harvested and embedded in Tissue-Tek OCT (Electron Microscopy) and flash-frozen for sectioning on a Leica CM3050 cryomicrotome (Leica). Sections were rehydrated in PBS for 45 min and blocked with 2% BSA in PBS for 20 min at room temperature. Goat anti-mouse IgM-Alexa 546 (Molecular Probes) and rat anti-mouse metallophilic macrophages (MOMA-1; Serotec) with goat anti-rat IgG-Alexa350 were used for staining. Sections were sealed with Prolong Antifade (Molecular Probes) and viewed with a Zeiss LSM150 confocal microscope and Zeiss LSM Image Browser software (Carl Zeiss).

Mice were anesthetized i.p. with 1 ml of 20 mg/ml Avertin (2, 2, 2-tribromoethyl and tertiary amyl alcohol), prebled retro-orbitally and kidneys were extracted after perfusion with Dulbeco’s PBS followed by chilled 2% paraformaldehyde (EMS Biosciences). Kidneys were cut across the juxtamedullary gap and transversely and incubated in 2% paraformaldehyde overnight before tissue processing and paraffin embedding or cryopreservation. Sections were prepared on positively charged slides and blocked with 5% nonfat dairy milk for 45 min at 20°C. Sections were stained with an isotype control (donkey anti-goat IgG) or Cy3-conjugated AffiniPure Fab (donkey anti-mouse IgG; Jackson Immunochemical) for 45 min at room temperature. Slides were rinsed with PBS plus 0.05% Tween 20 for 5 min, twice with PBS, and 4', 6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) was added for nuclear staining for 2 min. For complement C3 staining, Alexa Fluor 546 rabbit anti-mouse IgG (H+L) (Molecular Probes) and goat anti-mouse complement C3-FITC (MP Biomedicals- Cappel) were used. Slides were visualized with a Zeiss Axioplan 2i microscope and visualized with an AxioCam HRm camera (Carl Zeiss) at x20 magnification. Glomerular and tubulointerstitial immunofluorescence staining was assessed by evaluating 10 average fields per animal and grading them on a scale from 1 to 4.

Urinalyses

Urine was collected from 1-year-old transgenic and control mice and analyzed for protein using Multistix10 SG Reagent Strips (Bayer). Clinitek Reagent Strips (Bayer) were used to detect albumin and creatinine.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Generation of transgenic mice overexpressing Bright

Transgenic mice were produced that expressed Bright from the human CD19 promoter previously used to express Btk in a B cell-specific fashion (30). A His-Myc tag was added to the C terminus of the protein causing the transgenic protein to be of slightly higher m.w., thereby distinguishing it from the endogenous Bright protein (Fig. 1a). The addition of the His-Myc tag did not alter DNA binding or functional activity of Bright (21, 28). Among seven founders produced, three did not breed well or maintain transgene expression in the progeny, and an additional line failed to exhibit robust protein expression. The remaining three lines were initially characterized. Two of those lines, the C line, which expressed the highest level of transgenic Bright, and the R line that expressed lower transgenic protein levels compared with endogenous Bright, were chosen for more detailed evaluation (Fig. 1b). Western blots of multiple tissues from both of these lines indicated that Bright expression did not occur broadly in tissues that do not contain B lymphocytes. Representative data are shown for line C (Fig. 1c) and similar data were obtained for line R.


Figure 1
View larger version (25K):
[in this window]
[in a new window]

  		FIGURE 1. Production of mice constitutively expressing Bright in all B lymphocytes. a, Diagram of transgenic construct with CD19 promoter for B cell-specific expression. b, A Western blot was prepared using 10 µg of total spleen cell extract from two independent transgenic lines (R and C) and nontransgenic littermate controls (LM) and was developed with anti-Bright Abs. The His-myc-tagged transgenic protein has a higher m.w. than endogenous Bright. c, Western blots of whole tissues from the transgenic C line and a littermate control were developed for the presence of transgenic (T) or endogenous (E) Bright. A positive control containing both proteins is shown in lane 1 (Control). d, B cell subpopulations were isolated from littermate and C-transgenic mice by flow cytometry using anti-B220, CD43, and IgM; 50,000 cells per lane were analyzed for Bright protein expression. Levels of transgenic and endogenous Bright protein in individual samples were quantified and are indicated as the ratio of transgenic to endogenous Bright (T/E). Similar results were found with the R line. e, Splenic B cells were sorted into transitional 1, 2, and 3 (T1, T2, T3), marginal zone (MZ) and follicular (FO) subpopulations and were analyzed for Bright and actin protein expression. Signals were normalized to actin levels and relative Bright transgene expression is shown. All data are representative of at least three experiments.

 
To determine whether Bright transgene expression was maintained throughout B cell differentiation, consistent with expression of the CD19 promoter, individual B cell subpopulations were sorted and evaluated for transgenic and endogenous Bright expression by Western blotting. Western blots of sorted bone marrow B cell subpopulations indicated that the Bright transgene is expressed at the early pro-B cell stage when the CD19 promoter begins to be expressed and is maintained throughout late B cell differentiation in the recirculating B cells that have lost endogenous Bright expression (Fig. 1d). Comparison of transgenic vs endogenous Bright within the C transgenics suggests that the transgenic protein is much more abundant than endogenous Bright, particularly in more mature B cells where endogenous Bright levels were previously shown to decrease (23). Similarly, fractionation of splenic B cell subsets (Fig. 1e) shows that transgene expression is maintained in splenic B cell subpopulations. Endogenous Bright was not detectable in immature transitional T2, T3, and follicular cells, and expression of transgenic Bright was also particularly low in the follicular B cells. Expression of both endogenous Bright and transgenic Bright was highest in the transitional T1 and marginal zone B cell populations. Relative levels of transgene expression among the different B cell subpopulations was quantified after normalization to actin levels and showed a >35-fold difference in transgenic Bright expression (Fig. 1e). Immature B cells were previously shown to express increased levels of CD19 relative to mature B cells (33), so some of the variation in transgene expression among the different subpopulations may be the result of increased use of the CD19 promoter in those cell types. These data demonstrate that transgenic Bright protein is consistently expressed at higher levels than the endogenous protein throughout B cell differentiation in the transgenic mice.

Expansion of early B lymphocyte subpopulations in transgenic Bright mice

Flow cytometric analyses of the bone marrow subpopulations (Fig. 2a) revealed that total numbers of B220+ B lymphocyte lineage cells in the transgenic mice were significantly increased by ~1.8-fold in the C line (Table I). However, this increase was not the result of expansion of individual subpopulations, but rather reflected a general increase in B lineage cells. Although the R line that expressed lower levels of Bright consistently exhibited an ~1.4-fold increase in B lineage cells compared with littermate control values, those increases were not statistically significant by Student’s t test. These data suggest that overexpression of Bright in bone marrow B lineage cells leads to expansion of those cells.


Figure 2
View larger version (52K):
[in this window]
[in a new window]

  		FIGURE 2. Bone marrow and splenic B cell subpopulations in Bright-transgenic mice. a, Bone marrow from representative Bright C-transgenic (C TG) and littermate control (LM) male mice were stained with anti-CD43, anti-IgM, and anti-B220. Pro-B (Pro), pre-B (Pre), recirculating (RC), and immature (Imm) B cells were identified by FACS analyses as shown by the boxed areas. b, Spleen cells from a littermate control (LM) and Bright C-transgenic (C TG) mice were stained with anti-CD93 and B220, and the B220+ cells were gated into CD93+ (R2) and CD93– (R3) cells for analyses of IgM and CD23 levels. Transitional type 1 (T1), 2 (T2), and 3 (T3) subpopulations and marginal zone (MZ) and follicular (FO) B cells were identified. Only T1 cells (LM-2.2% vs TG-4.6%) and MZ cells (LM-1.9% vs TG-7.6%) were significantly increased in the transgenics. Data are representative of 14 littermates and 16 transgenic mice.

 

View this table:
[in this window]
[in a new window]

  		Table I. B cell subpopulations are increased in C-transgenic bone marrowa

 
Thymic T cell numbers did not differ significantly between littermate controls and transgenic lines, as expected due to the CD19-specific expression of the transgene. All mice examined appeared to be generally healthy and had normal sized thymi consistent with absence of stress and illness. In addition, total numbers of CD3-positive T lymphocytes in the thymus (data not shown) of the transgenic R and C lines did not differ significantly from numbers observed in the littermate controls.

Examination of splenic lymphocytes by flow cytometry indicated slightly increased numbers of total lymphocytes, including T cells, in the C line relative to the R line and littermate controls (Table II). Individual B cell subsets in the spleen were skewed such that early immature B cells were consistently increased in the Bright-transgenic mice (Fig. 2b). Marginal zone B cells (identified as IgMhigh, CD93–, CD23–/low, B220+) (34) were significantly increased in the C line by as much as 4-fold, but the R line that expresses lower levels of the transgene did not exhibit increased marginal zone B cells. In contrast, the transitional type 1 (T1) immature B cell subpopulation was increased significantly in both the R and C lines using several different staining protocols (34, 35, 36), both when assessed as total cell numbers and when analyzed as percentages relative to other B cell subpopulations. IgMhigh, IgD–/low, CD21–/low T1 cell numbers (36) were 3.3 x 106 ± 0.7 in littermate controls, compared with 8.5 x 106 ± 1.5 (p = 0.011) for the C line and 5.5 x 106 ± 0.6 (p = 0.034) for the R line. Increased percentages of T1 cells relative to T2 cells were easily observed by flow cytometry (Fig. 2b). Transitional type 2 and 3 (T2 and T3) immature cell numbers were not significantly different from littermate controls in either transgenic line. Furthermore, limited analyses of a third transgenic line (A) showed 2.4-fold increases in T1 cell numbers and 1.5-fold increases in marginal zone B cells, while other B cell subsets including T2 cells were not different from the littermate controls (data not shown). These data indicate that Bright overexpression results in expanded numbers of splenic T1 immature B cells in three independent lines and suggest that marginal zone B cell numbers are also increased in some lines relative to littermate controls.


View this table:
[in this window]
[in a new window]

  		Table II. B cell subpopulations are altered in transgenic spleensa

 
Splenic architecture is not grossly perturbed in Bright-transgenic mice

Spleen sections from unimmunized transgenic and littermate controls were analyzed for marginal zone B cells and follicle formation by staining with anti-IgM and anti-MOMA (37). Fig. 3 shows typical splenic architecture in both transgenic and littermate controls. Consistent with the increased numbers of marginal zone B cells identified by FACS analyses in Table II, the C-transgenic mice appeared to have slightly larger marginal zone areas (red) relative to normal control sections. However, transgenic mice did not exhibit any gross abnormalities in spleen histology.


Figure 3
View larger version (103K):
[in this window]
[in a new window]

  		FIGURE 3. Enhanced marginal zone in Bright-transgenic mice. Spleen sections from unimmunized male 12-wk-old mice were sectioned and stained for IgM (red) and MOMA (green) to identify B cells and metallophilic macrophages, respectively. Two views of follicles are shown centered (left panels) and off-center (right panels) for a representative littermate control (LM) and a Bright C-transgenic line (C TG). Magnification was x25.

 
Bright-transgenic mice exhibit slight increases in serum Ig levels

Because Bright up-regulates Ig H chain transcription in in vitro model systems (20, 21), we asked whether serum Ab levels were increased in the transgenic mice. Sera were obtained from 8- to 12-wk-old mice and analyzed by ELISA for IgM and IgG isotypes (Fig. 4). IgM levels were abnormally high only in the C line, consistent with the expansion of IgM-secreting marginal zone B cells observed in that line (Table II). Marginal zone B cells account for approximately half of serum IgM levels (37, 38). IgG2a and IgG2b levels were statistically increased ~2-fold relative to levels from the littermate controls in both the R and C lines. In contrast, circulating IgG3 levels were slightly decreased in the C-transgenic line relative to the littermate controls, but not in the R line. The reasons for this apparent decrease are unclear, but xid mice also exhibit reduced levels of IgG3 (39). IgG1 levels could not be accurately assessed in the FVB/N mice because anti-isotype Abs failed to react with the sera from this strain in a titratable fashion, albeit standards and sera from C57BL/6 mice titered normally. Together, these data indicate that overexpression of Bright results in slight increases in serum Ig levels.


Figure 4
View larger version (24K):
[in this window]
[in a new window]

  		FIGURE 4. Transgenic serum Ig levels are increased relative to control mice. Serum samples from 12 littermate, 12 C, and 10 R male transgenic mice (8–12 wk of age) were measured by ELISA for IgM and IgG Abs and averaged from duplicate samples at three dilutions. Concentrations were determined using purified Ab controls of each specific isotype to produce standard curves. Error bars are indicated. Asterisks indicate values significantly different from those of the littermate controls. Student’s t test p values are as follows: IgM: C 0.00067, R 0.45; IgG2a: C 0.000001, R 0.036; IgG2b: C 0.00000004, R 0.0013; and IgG3: C 0.02, R 0.75.

 
Transgenic B cells do not display a hyperactivated phenotype

To determine whether the increased Ig production resulted from hyperactivation, or increased susceptibility to activation, of B cells in the transgenic mice, splenic B cells were stimulated for 3 days in culture with medium alone, LPS, CD40L-expressing SF9 insect cells, or SF9 cells alone. Quadruplicate samples from six transgenic mice and six nontransgenic littermates were evaluated for [3H]thymidine incorporation after 6 h. None of the unstimulated transgenic cultures exhibited significantly increased [3H]thymidine incorporation relative to the control B cell cultures, suggesting that the transgenic B cells were not constitutively activated. No statistically relevant differences were observed between the transgenic and wild-type B cells in their ability to up-regulate activation and costimulatory markers (MHC class II, CD69, CD40, CD80, or CD86) after treatment with LPS or CD40L (data not shown). Furthermore, supernatants taken from the CD40L- and LPS-activated cells contained similar levels of secreted Ig in both control and transgenic cultures suggesting that the increased levels of serum Abs observed in the transgenic mice were not due to increased numbers of previously activated, or more easily activated, B cells. Therefore, the transgenic B cells do not appear to be hyperactivated compared with wild-type controls.

Anti-PC Abs are overproduced in transgenic mice overexpressing Bright

Although many mouse H chain promoters do not contain obvious Bright consensus sites (18, 40), the S107 family V1 gene that is used predominantly in responses to PC (25, 26, 27) is regulated by Bright and contains Bright-binding sites in its 5' flanking sequence (16). Therefore, we predicted that responses to PC-KLH might be increased in the transgenic mice. Responses to Ag were examined by immunizing six transgenic and six littermate controls with the T-dependent Ags NP-KLH and PC-KLH. Mice were prebled and immunized on day 0, boosted, and bled at day 7 and bled again at day 14 after the initial immunization. Hapten-specific anti-IgM and anti-IgG responses were measured using either NP-BSA or PC-BSA-coated ELISA plates and results were reported as the last positive dilution where both of the duplicate samples were above background (Fig. 5). No significant differences were observed in either preimmune or primary 7-day immune IgG or IgM responses between the littermate and transgenic mice for either hapten. However, the transgenic mice consistently produced more anti-PC IgM Ab (average end dilution of 1/18,300) at day 14 than did the control littermates (endpoint dilution of only 1/1,070) (Fig. 5a). Anti-PC IgG Abs were low in both transgenic and littermate mice. Others have shown that the anti-PC response is almost entirely germline and of the IgM isotype (25). Anti-NP IgM Abs were slightly lower in the transgenics (1/2,200) than in the littermates (1/13,325), but NP-specific IgG at day 14 averaged 1/17,000 in the Bright C line transgenics and 1/13,000 in the littermates, a statistically insignificant difference. These results are consistent with previous data indicating that Bright enhances expression of PC-specific Abs in vitro and suggest that the increase in humoral Abs in the Bright-transgenic mice does not reflect a global increase in all Ig production.


Figure 5
View larger version (28K):
[in this window]
[in a new window]

  		FIGURE 5. Ag-specific immune responses to PC are increased in Bright-transgenic C mice. Sera were collected from preimmune and either PC-KLH (a and c) or NP-KLH (b and d) immunized 8- to 12-wk-old control and transgenic mice 7 and 14 days postimmunization (first and second bleeds) and were serially diluted in duplicate and assessed by ELISA for NP-specific and PC-specific IgM (a and b) and IgG (c and d) Abs using hapten-BSA-coated plates. The last dilution in both duplicates above background was taken as the endpoint. Data show average values from seven C-transgenic mice and six littermates. SE bars are shown. *, Significance. e, S107 V1 mRNA was amplified by RT-PCR from FACS purified T1 and follicular (FO) littermate control and wild-type (WT) Bright-transgenic B cells from unimmunized mice. Data for one littermate (LM) and two C-transgenic (Tg1 and Tg2) mice are shown. Relative RNA levels per sample were standardized by amplification of GAPDH and are listed numerically below each sample.

 
To further analyze the repertoire of the transgenic B cells for increases in anti-PC responses, relative levels of the S107 family V1 H chain mRNA were examined in FACS-purified B cells from both littermate controls and Bright transgenics. The S107 family V1 gene is the predominant H chain used against PC (25, 41). T1 and follicular B cells were isolated as shown in Fig. 2 and RNA was subjected to semiquantitative RT-PCR analyses for the presence of V1-hybridizing cDNA (Fig. 5e). T1 cells from the transgenic mice contained ~50-fold more V1 H chain mRNA than did the littermate controls. Follicular B cells in the Bright-transgenic mice also expressed significantly more (20-fold) V1 mRNA than the littermate controls. It is not clear whether the increase in V1 mRNA reflects an increase in the numbers of cells expressing V1 or in the levels of Ig produced per V1-positive cell. However, these data suggest that expression of Ig from the S107 family is more abundant in the transgenic mice relative to littermate controls even at immature stages of B cell differentiation.

Bright-transgenic mice produce ANAs

PC-specific Abs have previously been associated with autoimmune syndromes and atherosclerotic plaques (42). We therefore sought to determine whether the Bright-transgenic mice produced other Abs that were self-reactive. Sera from 8 C- and 12 R-transgenic mice and 17 littermate controls were analyzed for reactivity with nuclear Ags by staining to HEP-2 cells. Representative positive staining is shown (Fig. 6a). One hundred percent of the C-transgenic mice examined showed ANA staining of HEP-2 cells at serum dilutions of 1/360. Only samples that resulted in obvious staining at this dilution were considered positive. None of the littermates analyzed were positive for ANA production. ANAs were already evident in the C mice analyzed at 4 wk of age. Furthermore, the C-transgenic line has now been backcrossed for six generations onto the nonautoimmune C57BL/6 background, and ANA production was retained in three of three young C transgenics on that background. Production of ANAs in the R Bright-transgenic line appeared only in older male mice (4–6 mo of age) and was present in only three of the 13 R mice examined. Thus, the R Bright-transgenic mice that expressed lower levels of transgenic Bright exhibited an intermediate phenotype with respect to ANA production compared with the C-transgenic mice that were all positive for ANA production from early ages.


Figure 6
View larger version (39K):
[in this window]
[in a new window]

  		FIGURE 6. Bright-transgenic mice produce anti-nuclear Ag Abs. a, HEP-2 cells were incubated with varying dilutions of sera from littermate or transgenic R and C Bright mice and were developed with anti-mouse IgG FITC. Data shown are representative of typical positive sera samples for each transgenic line at a 1/360 serum dilution. b, A summary of the data for all mice analyzed is shown graphically. The highest Ab dilution that yielded positive staining as shown in the middle and right panels of a is indicated for male littermate controls (LM) and R- and C-transgenic (TG) mice. Each point represents sera from an individual animal.

 
Sera from mice that produced ANAs were analyzed by ELISA for activity with dsDNA. Compared with four littermate controls that produced neither ANA nor anti-dsDNA Ab, 3 of 3 sera from the R-transgenic line reacted with dsDNA and only 2 of the 11 C sera tested were reactive with dsDNA (data not shown). Thus, the nature of the strong ANA response in the C line is likely to be polyclonal.

Overexpression of Bright in old transgenic mice results in kidney sequelae

Autoimmunity often leads to immune complex nephritis, with deposition of IgG Abs in the kidney with age. Therefore, 1-year-old male mice were perfused with saline to remove circulating Abs, and kidney sections were prepared and stained with anti-mouse IgG. Fig. 7a shows representative kidney sections from a C transgenic and an age- and gender-matched littermate control stained with the nuclear stain DAPI (blue). Anti-mouse IgG is detected as red. Although the 8 littermates tested showed low reactivity with the anti-mouse Ig reagent, 12 of the 17 Bright-transgenic mice (two-thirds) exhibited higher levels of mouse IgG deposition on the kidney basement membranes and/or in the glomeruli (Fig. 7b). In contrast, complement C3 staining was not remarkable in any of the mice examined (data not shown). These data suggest that overexpression of the Bright transgene does not result in pathologic immune complexes and kidney malfunction.


Figure 7
View larger version (61K):
[in this window]
[in a new window]

  		FIGURE 7. Kidney glomeruli exhibit Ig deposits in 1-year-old male C-transgenic mice. a, Cryosections of kidney from perfused C Bright transgenics and littermates (LM) were costained with DAPI (blue, left panels) to show nuclei and anti-mouse IgG Fab' Abs (red, middle panels). Arrows indicate glomeruli with Ig deposits in the transgenic sections. The panel on the far right is an overlay of the two stains. Sections shown are representative of a very positive stain (4+). b, Graphic representation of the IgG deposits observed in 8 littermate, 17 C-transgenic, and 6 R-transgenic mice ranging in age from 7 to 9 mo. Scoring was as follows: 1+ background staining; 2+ low staining; 3+ moderate staining; and 4+ bright staining. Each point indicates an individual animal assessed.

 
Nevertheless, to determine whether IgG deposition was associated with renal impairment in these mice, we assessed urine protein excretion in the transgenic mice. Although the R Bright-transgenic mice exhibited no differences in urine protein levels compared with the littermate controls (Fig. 8), 8 of the 12 C Bright-transgenic mice (67%) showed total protein levels in the high range. Only 1 littermate control of 16 examined exhibited high range proteinuria. A more in-depth analysis of albumin and creatinine levels in the urine by HPLC (data not shown) failed to show substantial increases in the majority of the mice. These data are consistent with the failure to find appreciable complement deposition in the kidney and suggest that renal function is not impaired in the Bright-transgenic mice. Indeed, at least out to 1 year of age, mortality of the mice was not affected.


Figure 8
View larger version (10K):
[in this window]
[in a new window]

  		FIGURE 8. One-year-old C Bright-transgenic mice exhibited proteinuria. Urine protein levels were measured using Multistix 10 SG reagent strips. Samples were read as indicated. Each point represents an individual animal.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Overexpression of the transcription factor, Bright/A-T-rich interaction domain 3a, in B lineage cells resulted in increased numbers of B lineage cells in both the bone marrow and periphery, increased levels of serum Ig, and production of ANAs. These effects appeared to occur in a dose-dependent fashion in three independent transgenic lines, but we cannot exclude the possibility that some of the variation in phenotype observed among the three lines were due to integration of the transgenes into other loci. Bright was originally identified as a protein that up-regulated Ig H chain transcription, so the effects on serum Ab production were not unexpected. However, selective overproduction of a subset of Abs associated with autoimmune syndromes was observed. Intriguingly, immature transitional T1 cells in both lines, and marginal zone B cells in the line with highest transgene expression, were statistically increased relative to other splenic B lymphocytes in the Bright-transgenic mice. These data suggest that inappropriate Bright regulation at the immature T1 stage may contribute to the breaches in B cell tolerance observed in the transgenic mice. Marginal zone B cells have also been implicated as a subset containing enriched numbers of autoreactive B cells (43) and the increased numbers observed in Bright-transgenic mice may contribute to the autoimmune phenotype observed. These data suggest a previously unappreciated role for Bright in immature B cells and are the first to suggest the importance of a B cell-restricted transcription factor in tolerance maintenance.

Endogenous Bright expression in the B cell lineage is tightly regulated such that transcription in the bone marrow begins at the pro-B to pre-B cell stages, peaks in immature B cells, and then is turned off in recirculating IgM+ mature B cells (23). Previous experiments indicated that bulk splenocytes also show very low levels of Bright expression, with the highest transcription observed in the PNA+ germinal center subpopulation (23). The Bright-transgenic mice express the transgene throughout B cell differentiation (Fig. 1) and at much higher levels than the endogenous proteins. Individual B cell subpopulations vary in the levels of transgene expression. This may be partially explained by the fact that expression is controlled by the CD19 promoter. CD19 expression varies during B differentiation with particularly high expression in immature B cells (33). Alternatively, protein stability may vary among the different B cell subsets. It may not be surprising that transgenic Bright had no apparent effect on follicular, T2, or T3 cells because endogenous Bright is not expressed in those subpopulations and additional cofactors and gene targets for Bright activity may be inactive in those cells. In contrast, the splenic B cell populations with the highest Bright transgene expression exhibited the greatest increase in cell numbers suggesting a potential correlation between Bright overexpression and expansion of these cells. Increased numbers of T1 and marginal zone B cells have been correlated with autoimmune disease in other mouse and human systems where the expansion is proposed to be the result of survival of self-reactive cells that would normally undergo apoptosis (44, 45, 46, 47, 48). Additional experiments will be required to determine the role Bright may play in proliferation and survival of T1 and marginal zone B cells.

Finding increased numbers of T1 cells in the transgenic mice was unexpected, but correlates with others’ studies suggesting an important role for Btk in these cells. Xid mice with a point mutation in Btk exhibit blocks in B cell differentiation at the immature B cell stage, although the block is incomplete. Several groups have demonstrated that B cells from mice deficient in Btk do not proliferate normally at the T1 immature stage and that T2 transitional stage xid B cells fail to respond to signals through the surface IgR (9, 49). Our data, using an in vitro model system, suggested that Btk is critically required for Bright-mediated up-regulation of transcription of at least some H chains (21, 22). We hypothesize that Bright and Btk function coordinately in transitional B cells to mediate further differentiation through this important tolerance checkpoint. Whether Bright activity is mediated through direct effects on Ig H chain expression or through other gene targets remains to be seen.

Although serum Igs were increased in the Bright-transgenic compared with control mice, immunization with NP-KLH resulted in normal levels of anti-hapten Abs in vivo. This indicated that increased Bright expression did not uniformly increase all Ab production. Furthermore, stimulation of splenic B cells failed to show increased secretion of Ab in response to LPS or CD40L stimulation, suggesting that transgenic B cells were not simply hyperactive relative to littermate controls. However, ANAs and immune responses to PC were significantly enhanced by overexpression of Bright (Figs. 5 and 6). The increased anti-PC responses observed in these mice were not unexpected because the S107 family V1 H chain gene predominantly responsible for immune responses to PC (25, 26, 27) is regulated by Bright in vitro (21). Consistent with our data, xid mice that lacked functional Btk and were defective in Bright DNA-binding activity (31) exhibited the opposite phenotype showing preferential loss of anti-PC responses relative to other types of Ags (50). Together, our data are consistent with a model whereby Bright is more critically required for expression of the V1 S107 H chain gene that predominates the anti-PC response than for other IgH genes (51). Anti-PC Abs can also react with self Ags in atherosclerotic plaques, and overexpression of these Abs in knockin mice leads to down-regulation of antiself cells (42, 52, 53, 54). Thus, we speculate that Bright may preferentially affect a subset of IgH genes. Some of these may normally be self-reactive resulting in the ANA production observed in the Bright-transgenic mice. Experiments to formally examine the VH genes used in these mice for repertoire skewing are in progress.

The mechanisms by which ANAs are produced in Bright-transgenic mice are unclear; however, the B cell-restricted expression of the transgene suggests that the effect is intrinsic to B cells. The immature B cell subpopulations expanded in the bone marrow and spleens of wild-type Bright-transgenic mice are the checkpoints in normal mice where autoreactive B cells are either deleted, become anergic, or undergo receptor editing effectively removing self-reactive Ig (9, 10). Because the ANA specificity is variable in these mice (only some produce anti-dsDNA Abs) it has not been possible to directly correlate the ANA production with either the T1 or marginal zone B cell populations. However, we hypothesize that ANA production in the Bright-transgenic mice is the direct result of breaks in tolerance at the T1 checkpoint. Accumulation of immature B cells in Bright-transgenic mice could be the result of failure of immature B cells to undergo apoptosis, increased survival and migration from the bone marrow, increased proliferation of these cells, and/or inappropriate responses to signaling through the surface IgR. None of these are mutually exclusive. The underlying mechanism by which overexpression of Bright leads to this phenotype may be the direct result of up-regulation of IgH transcription and surface Ig density at a specific stage of B cell differentiation. Several studies indicate that BCR density is critical for signaling cell survival vs apoptosis (55, 56). However, flow cytometry data do not show appreciable increases in IgM levels in transgenic B cells relative to control B cell populations, nor were surface IgG levels appreciably different (data not shown). Alternatively, Bright could act through the regulation of other non-Ig gene targets. Although no additional targets of Bright have been experimentally identified, microarray data indicate several antiapoptotic genes may be regulated by Bright (our unpublished results).

Transgenic mice expressing wild-type Bright were originally produced as controls for other transgenic mice that express a dominant-negative (DN) form of Bright (28). Because Bright associates with Btk, overexpression of Bright might have sequestered Btk such that it could not function properly leading to an additional Btk-deficient phenotype. However, the phenotype of the wild-type Bright-transgenic mice differs from Btk-deficient mice and from DN Bright-transgenic mice using the identical CD19 promoter for transgene expression. Intriguingly, the DN Bright-transgenic mice down-regulate transgene expression at the T1 to T2 transition, consistent with an important function for Bright in immature B cells (our unpublished results). In contrast, wild-type Bright-transgenic mice, with B cells that maintained Bright expression continuously throughout B cell development, developed ANAs early in life, and exhibited an autoimmune phenotype similar to that observed in Sle1 congenic mice (13). Sle1 congenic mice produce ANAs, but do not develop disease without expression of additional lupus-associated genes (15, 57). Together, these data strengthen the hypothesis that Bright plays an important role in maintaining B cell tolerance at early stages before production of autoreactive Abs.

Bright-transgenic mice represent a new category of autoimmune mice. Although overexpression of cytokines such as BAFF (B cell activating factor belonging to the TNF family) can result in autoimmunity (58, 59), in most autoimmune models that result in autoantibody production, cell surface receptor or intracellular signaling molecules, such as CD22, CD19, SHP-1 and Lyn, have been shown to play important roles (6, 60, 61, 62). The Bright-transgenic mice overexpress a downstream target of these signaling processes and represent the first transgenic model where an intrinsic B cell transcription factor capable of regulating endogenous IgH genes directly results in ANA production. Therefore, determining which checkpoints are overcome in these mice may lead to a better understanding of the initial events that are disrupted in autoimmune disease.

More recently, new data has suggested that B cells do not merely act as the end producer of Abs in autoimmune disease, but they may also be important regulators of earlier events involving T cells. Using a diabetes mouse model, Tian et al. (63) presented data that suggested B cells, rather than other APC types, are responsible for the spreading of T cell responses during type I diabetes. In another study, activated B cells from NZB mice that expressed high levels of CD86 induced hyperresponsiveness in CD4+ T cells from normal BALB/c mice (64). In this same study, B cells from tolerized mice induced T cell anergy in vitro (64). Others showed that B cells controlled T cell help and autoimmunity in lupus-prone NZB/W mice (65). We showed previously that Bright is expressed in activated mature B cells in response to a number of stimuli (31). Therefore, it is interesting to hypothesize that overexpression of Bright in the transgenic mice may result in B cells preferentially capable of stimulating T cell-mediated autoimmune responses. Additional experiments to characterize T cell-mediated functions in the Bright transgenics are in progress.

Although the data presented here expand the potential roles for Bright in the mouse, very little is known regarding the function of Bright in human B lymphocytes. Notably, while Bright activity is not detectable in peripheral blood B cells from normal individuals, peripheral blood from lupus patients is an abundant source of Bright protein and was used as a source of RNA for cloning human Bright (24). Experiments using peripheral blood cells from lupus patients also show an expansion of cells that resemble T1 transitional B cells (46, 66). Furthermore, preliminary data suggest that EBV, sometimes causally linked with lupus, can induce Bright expression in human cells (67). ANA production in lupus and rheumatoid arthritis patients has been shown to occur years before disease onset (47, 68, 69). Understanding more about how Bright regulation is related to ANA production may lead to new insights into human autoimmune disease.


   Acknowledgments
 
We thank Dr. U. Hochgeschwender, Dr. B. Gordon, P. Long, and R. Stephens for generation and maintenance of transgenic animals, K. Humphrey for manuscript preparation, and Drs. R. Perlmutter and A. Feeney for helpful comments.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
C. F. Webb has a patent related to the data presented.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant AI044215 (to C.F.W.). Back

2 Address correspondence and reprint requests to Dr. Carol F. Webb, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104. E-mail address: carol-webb{at}omrf.ouhsc.edu Back

3 Abbreviations used in this paper: ANA, anti-nuclear Ab; Bright, B cell regulator of IgH transcription; Btk, Bruton’s tyrosine kinase; PC, phosphorylcholine; KLH, keyhole limpet hemocyanin; DAPI, 4', 6-diamidino-2-phenylindole; DN, dominant negative; NP, nitrophenyl. Back

Received for publication June 8, 2006. Accepted for publication December 21, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Theofilopoulos, A. N., F. J. Dixon. 1985. Murine models of systemic lupus erythematosus. Adv. Immunol. 37: 269-390. [Medline]
  2. Anolik, J., I. Sanz. 2004. B cells in human and murine systemic lupus erythematosus. Curr. Opin. Rheumatol. 16: 505-512. [Medline]
  3. Fields, M. L., B. D. Hondowicz, G. N. Wharton, B. S. Adair, M. H. Metzgar, S. T. Alexander, A. J. Caton, J. Erikson. 2005. The regulation and activation of lupus-associated B cells. Immunol. Rev. 204: 165-183. [Medline]
  4. Nemazee, D.. 2000. Receptor selection in B and T lymphocytes. Annu. Rev. Immunol. 18: 19-51. [Medline]
  5. Melamed, D., D. Nemazee. 1997. Self-antigen does not accelerate immature B cell apoptosis, but stimulates receptor editing as a consequence of developmental arrest. Proc. Natl. Acad. Sci. USA 94: 9267-9272. [Abstract/Free Full Text]
  6. Goodnow, C. C., A. Basten. 1989. Self-tolerance in B lymphocytes. Semin. Immunol. 1: 125-135. [Medline]
  7. Heltemes-Harris, L., X. Liu, T. Manser. 2004. Progressive surface B cell antigen receptor down-regulation accompanies efficient development of antinuclear antigen B cells to mature, follicular phenotype. J. Immunol. 172: 823-833. [Abstract/Free Full Text]
  8. Norvell, A., M. L. Birkeland, J. Carman, A. L. Sillman, R. Wechsler-Reya, J. G. Monroe. 1996. Use of isolated immature-stage B cells to understand negative selection and tolerance induction at the molecular level. Immunol. Res. 15: 191-207. [Medline]
  9. Su, T. T., D. J. Rawlings. 2002. Transitional B lymphocyte subsets operate as distinct checkpoints in murine splenic B cell development. J. Immunol. 168: 2101-2110. [Abstract/Free Full Text]
  10. Hartley, S. B., M. P. Cooke, D. A. Fulcher, A. W. Harris, S. Cory, A. Basten, C. C. Goodnow. 1993. Elimination of self-reactive B lymphocytes proceeds in two stages: arrested development and cell death. Cell 72: 325-335. [Medline]
  11. William, J., C. Euler, N. Primarolo, M. J. Shlomchik. 2006. B cell tolerance checkpoints that restrict pathways of antigen-driven differentiation. J. Immunol. 176: 2142-2151. [Abstract/Free Full Text]
  12. Metcalf, E. S., N. R. Klinman. 1976. In vitro tolerance induction of neonatal murine B cells. J. Exp. Med. 143: 1327-1340. [Abstract/Free Full Text]
  13. Subramanian, S., Y. S. Yim, K. Liu, K. Tus, X. J. Zhou, E. K. Wakeland. 2005. Epistatic suppression of systemic lupus erythematosus: fine mapping of Sles1 to less than 1 mb. J. Immunol. 175: 1062-1072. [Abstract/Free Full Text]
  14. Chen, Y., D. Perry, S. A. Boackle, E. S. Sobel, H. Molina, B. P. Croker, L. Morel. 2005. Several genes contribute to the production of autoreactive B and T cells in the murine lupus susceptibility locus. Sle1c. J. Immunol. 175: 1080-1089.
  15. Wakui, M., L. Morel, E. J. Butfiloski, C. Kim, E. S. Sobel. 2005. Genetic dissection of systemic lupus erythematosus pathogenesis: partial functional complementation between Sle1 and Sle3/5 demonstrates requirement for intracellular coexpression for full phenotypic expression of lupus. J. Immunol. 175: 1337-1345. [Abstract/Free Full Text]
  16. Webb, C. F., C. Das, S. Eaton, K. Calame, P. W. Tucker. 1991. Novel protein-DNA interactions associated with increased immunoglobulin transcription in response to antigen plus interleukin-5. Mol. Cell. Biol. 11: 5197-5205. [Abstract/Free Full Text]
  17. Webb, C. F., C. Das, K. L. Eneff, P. W. Tucker. 1991. Identification of a matrix-associated region 5' of an immunoglobulin heavy chain variable region gene. Mol. Cell. Biol. 11: 5206-5211. [Abstract/Free Full Text]
  18. Goebel, P., A. Montalbano, N. Ayers, E. Kompfner, L. Dickinson, C. F. Webb, A. J. Feeney. 2002. High frequency of matrix attachment regions and cut-like protein x/CCAAT-displacement protein and B cell regulator of IgH transcription binding sites flanking Ig V region genes. J. Immunol. 169: 2477-2487. [Abstract/Free Full Text]
  19. Herrscher, R. F., M. H. Kaplan, D. L. Lelsz, C. Das, R. Scheuermann, P. W. Tucker. 1995. The immunoglobulin heavy-chain matrix-associating regions are bound by Bright: a B cell-specific trans-activator that describes a new DNA-binding protein family. Genes Dev. 9: 3067-3082. [Abstract/Free Full Text]
  20. Webb, C. F., C. Das, R. L. Coffman, P. W. Tucker. 1989. Induction of immunoglobulin m mRNA in a B cell transfectant stimulated with interleukin-5 and a T-dependent antigen. J. Immunol. 143: 3934-3939. [Abstract]
  21. Rajaiya, J., M. Hatfield, J. C. Nixon, D. J. Rawlings, C. F. Webb. 2005. Bruton’s tyrosine kinase regulates immunoglobulin promoter activation in association with the transcription factor Bright. Mol. Cell Biol. 25: 2073-2084. [Abstract/Free Full Text]
  22. Rajaiya, J., J. C. Nixon, N. Ayers, Z. P. Desgranges, A. L. Roy, C. F. Webb. 2006. Induction of immunoglobulin heavy chain transcription through the transcription factor Bright requires TFII-I. Mol. Cell. Biol. 26: 4758-4768. [Abstract/Free Full Text]
  23. Webb, C. F., E. A. Smith, K. L. Medina, K. L. Buchanan, G. Smithson, S. Dou. 1998. Expression of Bright at two distinct stages of B lymphocyte development. J. Immunol. 160: 4747-4754. [Abstract/Free Full Text]
  24. Nixon, J. C., J. B. Rajaiya, N. Ayers, S. Evetts, C. F. Webb. 2004. The transcription factor, Bright, is not expressed in all human B lymphocyte subpopulations. Cell. Immunol. 228: 42-53. [Medline]
  25. Claflin, J. L., J. Berry. 1988. Genetics of the phosphocholine-specific antibody response to Streptococcus pneumoniae germ-line but not mutated T15 antibodies are dominantly selected. J. Immunol. 144: 4012-4019.
  26. Claflin, J. L., M. Cubberley. 1980. Clonal nature of the immune response to phosphocholine VII. Evidence throughout inbred mice for molecular similarities among antibodies bearing the T15 idiotype. J. Immunol. 125: 551-558. [Medline]
  27. Feeney, A. J., S. H. Clarke, D. E. Mosier. 1988. Specific H chain junctional diversity may be required for non T15 antibodies to bind phosphorylcholine. J. Immunol. 141: 1267-1272. [Abstract]
  28. Nixon, J. C., J. Rajaiya, C. F. Webb. 2004. Mutations in the DNA-binding domain of the transcription factor Bright act as dominant negative proteins and interfere with immunoglobulin transactivation. J. Biol. Chem. 279: 52465-52472. [Abstract/Free Full Text]
  29. Kozak, M.. 1999. Initiation of translation in prokaryotes and eukaryotes. Gene 234: 187-208. [Medline]
  30. Maas, A., G. M. Dingjan, F. Grosveld, R. W. Hendriks. 1999. Early arrest in B cell development in transgenic mice that express the E41K Bruton’s tyrosine kinase mutant under the control of the CD19 promoter region. J. Immunol. 162: 6526-6533. [Abstract/Free Full Text]
  31. Webb, C. F., Y. Yamashita, N. Ayers, S. Evetts, Y. Paulin, M. E. Conley, E. A. Smith. 2000. The transcription factor Bright associates with Bruton’s tyrosine kinase, the defective protein in immunodeficiency disease. J. Immunol. 165: 6956-6965. [Abstract/Free Full Text]
  32. Webb, C. F., S. Dou, K. L. Buchanan, R. Resta, G. Smithson, E. A. Smith. 1997. Reassessment of germline heavy chain transcripts from two murine VH families. Mol. Immunol. 34: 743-750. [Medline]
  33. Sato, S., N. Ono, D. A. Steeber, D. S. Pisetsky, T. F. Tedder. 1996. CD19 regulates B lymphocyte signaling thresholds critical for the development of B-1 lineage cells and autoimmunity. J. Immunol. 157: 4371-4378. [Abstract]
  34. Allman, D., R. C. Lindsley, W. DeMuth, K. Rudd, S. A. Shinton, R. R. Hardy. 2001. Resolution of three nonproliferative immature splenic B cell subsets reveals multiple selection points during peripheral B cell maturation. J. Immunol. 167: 6834-6840. [Abstract/Free Full Text]
  35. Rolink, A. G., J. Andersson, F. Melchers. 2004. Molecular mechanisms guiding late stages of B-cell development. Immunol. Rev. 197: 41-50. [Medline]
  36. Loder, F., B. Mutschler, R. J. Ray, C. J. Paige, P. Sideras, R. Torres, M. C. Lamers, R. Carsetti. 1999. B cell development in the spleen takes place in discrete steps and is determined by the quality of B cell receptor-derived signals. J. Exp. Med. 190: 75-89. [Abstract/Free Full Text]
  37. Pillai, S., A. Cariappa, S. T. Moran. 2005. Marginal zone B cells. Annu. Rev. Immunol. 23: 161-196. [Medline]
  38. Hayakawa, K., R. R. Hardy, D. R. Parks, L. A. Herzenberg. 1983. The "LY-1 B" cell subpopulation in normal immunodefective, and autoimmune mice. J. Exp. Med. 157: 202-218. [Abstract/Free Full Text]
  39. Khan, W. N., F. W. Alt, R. M. Gerstein, B. A. Malynn, I. Larsson, G. Rathbun, L. Davidson, S. Muller, A. B. Kantor, L. A. Herzenberg, et al 1995. Defective B cell development and function in Btk-deficient mice. Immunity 3: 283-299. [Medline]
  40. Johnston, C. M., A. L. Wood, D. J. Bolland, A. E. Corcoran. 2006. Complete sequence assembly and characterization of the C57BL/6 mouse Ig heavy chain V region. J. Immunol. 176: 4221-4234. [Abstract/Free Full Text]
  41. Lotscher, M., H. Amstutz, C. H. Heusser, K. Blaser. 1990. Fine specificity and heavy chain V gene usage of antibodies to the phosphorylcholine hapten. Mol. Immunol. 27: 369-378. [Medline]
  42. Shaw, P. X., C. S. Goodyear, M. K. Chang, J. L. Witztum, G. J. Silverman. 2003. The autoreactivity of anti-phosphorylcholine antibodies for atherosclerosis-associated neo-antigens and apoptotic cells. J. Immunol. 170: 6151-6157. [Abstract/Free Full Text]
  43. Li, Y., H. Li, M. Weigert. 2002. Autoreactive B cells in the marginal zone that express dual receptors. J. Exp. Med. 195: 181-188. [Abstract/Free Full Text]
  44. Yurasov, S., J. Hammersen, T. Tiller, M. Tsuiji, H. Wardemann. 2005. B-cell tolerance checkpoints in healthy humans and patients with systemic lupus erythematosus. Ann. NY Acad. Sci. 1062: 165-174. [Medline]
  45. Atencio, S., H. Amano, S. Izui, B. L. Kotzin. 2004. Separation of the New Zealand Black genetic contribution to lupus from New Zealand Black determined expansions of marginal zone B and B1a cells. J. Immunol. 172: 4159-4166. [Abstract/Free Full Text]
  46. Yurasov, S., H. Wardemann, J. Hammersen, M. Tsuiji, E. Meffre, V. Pascual, M. C. Nussenzweig. 2005. Defective B cell tolerance checkpoints in systemic lupus erythematosus. J. Exp. Med. 201: 703-711. [Abstract/Free Full Text]
  47. Samuels, J., Y. S. Ng, C. Coupillaud, D. Paget, E. Meffre. 2005. Impaired early B cell tolerance in patients with rheumatoid arthritis. J. Exp. Med. 201: 1659-1667. [Abstract/Free Full Text]
  48. Quinn, W. J., III, N. Noorchashm, J. E. Crowley, A. J. Reed, H. Noorchashm, A. Naji, M. P. Cancro. 2006. Cutting edge: impaired transitional B cell production and selection in the nonobese diabetic mouse. J. Immunol. 176: 7159-7164. [Abstract/Free Full Text]
  49. Petro, J. B., R. M. Gerstein, J. Lowe, R. S. Carter, N. Shinners, W. N. Khan. 2002. Transitional type 1 and 2 B lymphocyte subsets are differentially responsive to antigen receptor signaling. J. Biol. Chem. 277: 48009-48019. [Abstract/Free Full Text]
  50. Brown, M., M. Stenzel-poore, M. Rittenberg. 1985. Immunologic memory to phosphocholine VII. Lack of T15 V1 gene utilization in Xid anti-PC hybridomas. J. Immunol. 135: 3558-3563. [Abstract]
  51. Webb, C. F.. 2001. The transcription factor. Bright, and immunoglobulin heavy chain expression. Immunol. Res. 24: 149-161. [Medline]
  52. Kenny, J. J., E. G. Derby, J. A. Yoder, S. A. Hill, R. T. Fischer, P. W. Tucker, J. L. Claflin, D. L. Longo. 2000. Positive and negative selection of antigen-specific B cells in transgenic mice expressing variant forms of the VH1 (T15) heavy chain. Int. Immunol. 12: 873-885. [Abstract/Free Full Text]
  53. Kenny, J. J., L. J. Rezanka, A. Lustig, R. T. Fischer, J. Yoder, S. Marshall, D. L. Longo. 2000. Autoreactive B cells escape clonal deletion by expressing multiple antigen receptors. J. Immunol. 164: 4111-4119. [Abstract/Free Full Text]
  54. Hu, L., L. J. Rezanka, Q. S. Mi, A. Lustig, D. D. Taub, D. L. Longo, J. J. Kenny. 2002. T15-idiotype-negative B cells dominate the phosphocholine binding cells in the preimmune repertoire of t15i knockin mice. J. Immunol. 168: 1273-1280. [Abstract/Free Full Text]
  55. Gauld, S. B., R. J. Benschop, K. T. Merrell, J. C. Cambier. 2005. Maintenance of B cell anergy requires constant antigen receptor occupancy and signaling. Nat. Immunol. 6: 1160-1167. [Medline]
  56. Kovesdi, D., K. Paszty, A. Enyedi, E. Kiss, J. Matko, K. Ludanyi, E. Rajnavolgyi, G. Sarmay. 2004. Antigen receptor-mediated signaling pathways in transitional immature B cells. Cell Signal. 16: 881-889. [Medline]
  57. Carter, R. H., H. Zhao, X. Liu, M. Pelletier, W. Chatham, R. Kimberly, T. Zhou. 2005. Expression and occupancy of BAFF-R on B cells in systemic lupus erythematosus. Arthritis Rheum. 52: 3943-3954. [Medline]
  58. Khare, S. D., I. Sarosi, X. Z. Xia, S. McCabe, K. Miner, I. Solovyev, N. Hawkins, M. Kelley, D. Chang, G. Van, et al 2000. Severe B cell hyperplasia and autoimmune disease in TALL-1 transgenic mice. Proc. Natl. Acad. Sci. USA 97: 3370-3375. [Abstract/Free Full Text]
  59. Rolink, A. G., F. Melchers. 2002. BAFFIed B cells survive and thrive: roles of BAFF in B-cell development. Curr. Opin. Immunol. 14: 266-275. [Medline]
  60. Hibbs, M. L., K. W. Harder, J. Armes, N. Kountouri, C. Quilici, F. Casagranda, A. R. Dunn, D. M. Tarlinton. 2002. Sustained activation of Lyn tyrosine kinase in vivo leads to autoimmunity. J. Exp. Med. 196: 1593-1604. [Abstract/Free Full Text]
  61. O’Keefe, T. L., G. T. Williams, S. L. Davies, M. S. Neuberger. 1996. Hyperresponsive B cells in CD22-deficient mice. Science 274: 798-801. [Abstract/Free Full Text]
  62. Taylor, D. K., E. Ito, M. Thorn, K. Sundar, T. Tedder, L. A. Spatz. 2006. Loss of tolerance of anti-dsDNA B cells in mice overexpressing CD19. Mol. Immunol. 43: 1776-1790. [Medline]
  63. Tian, J., D. Zekzer, Y. Lu, H. Dang, D. L. Kaufman. 2006. B cells are crucial for determinant spreading of T cell autoimmunity among beta cell antigens in diabetes-prone nonobese diabetic mice. J. Immunol. 176: 2654-2661. [Abstract/Free Full Text]
  64. Wu, H. Y., A. Monsonego, H. L. Weiner. 2006. The mechanism of nasal tolerance in lupus prone mice is T-cell anergy induced by immature B cells that lack B7 expression. J. Autoimmun. 26: 116-126. [Medline]
  65. Langnickel, D., P. Enghard, C. Klein, R. Undeutsch, B. Hocher, R. Manz, G. R. Burmester, G. Riemekasten. 2006. Induction of pathogenic anti-dsDNA antibodies is controlled on the level of B cells in a non-lupus prone mouse strain. J. Clin. Immunol. 26: 86-95. [Medline]
  66. Wehr, C., H. Eibel, M. Masilamani, H. Illges, M. Schlesier, H. H. Peter, K. Warnatz. 2004. A new CD21low B cell population in the peripheral blood of patients with SLE. Clin. Immunol. 113: 161-171. [Medline]
  67. McClain, M. T., L. D. Heinlen, G. J. Dennis, J. Roebuck, J. B. Harley, J. A. James. 2005. Early events in lupus humoral autoimmunity suggest initiation through molecular mimicry. Nat. Med. 11: 85-89. [Medline]
  68. Jackson, S. M., J. D. Capra. 2005. IgH V-region sequence does not predict the survival fate of human germinal center B cells. J. Immunol. 174: 2805-2813. [Abstract/Free Full Text]
  69. Samuels, J., Y. S. Ng, C. Coupillaud, D. Paget, E. Meffre. 2005. Human B cell tolerance and its failure in rheumatoid arthritis. Ann. NY Acad. Sci. 1062: 116-126. [Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
J. C. Nixon, S. Ferrell, C. Miner, A. L. Oldham, U. Hochgeschwender, and C. F. Webb
Transgenic Mice Expressing Dominant-Negative Bright Exhibit Defects in B1 B Cells
J. Immunol., November 15, 2008; 181(10): 6913 - 6922.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shankar, M.
Right arrow Articles by Webb, C. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shankar, M.
Right arrow Articles by Webb, C. F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS