Skip to main content
NCBI home page
Immunology logoLink to Immunology
. 2007 Feb;120(2):281–289. doi: 10.1111/j.1365-2567.2006.02500.x

Unexpected development of autoimmunity in BAFF-R-mutant MRL-lpr mice

Zhong L Ju 1,2, Gui Y Shi 1, Jin X Zuo 1, Jing W Zhang 1,2, Sun Jian 1,2
PMCID: PMC2265857  PMID: 17073941

Abstract

BAFF-R is the predominant receptor that mediates B-cell activating factor (BAFF)-dependent B-cell signalling and plays a critical role in late-stage B-cell maturation and survival. BAFF has been implicated in the development of autoimmunity and systemic lupus erythematosus (SLE). To define the role of BAFF-R in autoimmunity and SLE, we crossed A/WySnJ mice with MRL-lpr mice and generated BAFF-R-mutant MRL-lpr mice. The BAFF-R mutation markedly impaired the development of immature, mature and marginal zone B cells in the spleens of MRL-lpr mice. Unexpectedly, the BAFF-R mutation in MRL-lpr mice did not result in decreased autoantibody production, hypergammaglobulinaemia or immune complex-mediated glomerulonephritis. Rather, the ability of BAFF-R-mutant lpr splenic B cells to produce immunoglobulins in vitro was not decreased, although germinal centre formation, antibody response and B-cell proliferation were impaired. Further studies found increased numbers of B cells in the bone marrow of BAFF-R-mutant MRL-lpr mice compared to the BAFF-R-intact lupus mice. ELISPOT analysis revealed that BAFF-R-mutant MRL-lpr mice had more antibody-secreting cells in their bone marrow than the control mice. Thus, these findings could explain the development of autoimmunity and hypergammaglobulinaemia observed in BAFF-R-mutant MRL-lpr mice.

Keywords: autoimmunity, BAFF-R, B cells, MRL-lpr mice, systemic lupus erythematosus

Introduction

Previous studies indicated that B-cell-activating factor, BAFF (also called TALL-1, THANK, BlyS and zTNF4), which belongs to the tumour necrosis factor (TNF) family, is critical for late stage B-cell development and survival.1–5 BAFF is produced predominantly by myeloid cells and is expressed as a cell surface-bound molecule and as a proteolytically cleaved soluble molecule. BAFF knockout mice have a severe block in B-cell development at the transition from type 1 (transitional B1, T1) to type 2 (transitional B2, T2) immature B cells in the spleen. In contrast, the development of immature B cells in bone marrow, their transit to the periphery, and the development of B1 cells did not appear to be affected in BAFF knockout mice. T-cell-dependent and -independent responses to 4-hydroxy-3-nitrophenylacetyl (NP)–keyhole limpet haemocyanin (KLH) and 2,4,6-trinitrophenyl (TNP)–Ficoll, respectively, were severely decreased in these mice.6,7 Transgenic mice over-expressing BAFF exhibited B-cell hyperplasia, produced autoantibodies and developed phenotypes similar to those of systemic lupus erythematosus (SLE) and Sjögren syndrome.5,8–10 The serum level of circulating BAFF was increased in NZBW/F1 and MRL-lpr mice,5 which were used as spontaneous lupus models. Moreover, increased BAFF protein expression has been observed in a subgroup of SLE, rheumatoid arthritis and Sjögren syndrome patients.10,11

Currently, three receptors from the TNF receptor family that bind to BAFF have been identified: transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), B-cell maturation antigen (BCMA) and BAFF-R (also known as BR3/Bcmd), all of which are expressed on B cells.5,12–15 BAFF-R is known to bind specifically to BAFF with the highest affinity, whereas TACI and BMCA bind to another member of the TNF family, A Proliferation-Inducing Ligand (APRIL).16 TACI deficiency in mice results in the accumulation of peripheral B cells and increased B-cell responses to stimulation with lipopolysaccharide (LPS) or anti-CD40.17,18 Loss of the BCMA receptor did not affect the generation of mature peripheral B cells or short-lived plasma cells, nor did it alter humoral immune responses.19 However, BCMA is essential for the survival of long-lived bone marrow plasma cells.20 Our current understanding of BAFF-R function comes mainly from analysis of A/WySnJ mice that have a natural mutation in exon 3 of the BAFF-R gene, which encodes the intracellular signalling domain of the receptor.21–24 A transposon insertion of 4·7 kilobases in these mice replaces the last eight amino acids of the BAFF-R C terminus with 21 amino acids encoded by the insertion. The mutant protein is expressed on the B-cell surface and binds to BAFF; however, the profiles are qualitatively and quantitatively different from wild-type BAFF-R.13 Recently, BAFF-R null mice were generated.25,26 Similar to BAFF knockout mice, BAFF-R null mice show defective splenic B-cell maturation, reduced marginal zone (MZ) B-cell numbers and impaired T-cell-dependent responses. These results indicate that BAFF-R plays an important role in BAFF signalling. When the essential role of BAFF-R in mediating BAFF signalling was first described, it was speculated that BAFF-R might be critical for the development of autoimmunity. To test this idea, we crossed A/WySnJ mice with MRL-lpr mice to create BAFF-R-mutant MRL-lpr mice.

Materials and methods

Mice

A/WySnJ mice and MRL-lpr mice (The Jackson Laboratory, Bar Harbor, ME) were bred to produce F1 offspring heterozygous for lpr and BAFF-R. These mice were intercrossed to generate mice with a homozygous mutation for both Fas and BAFF-R. BAFF-Rmut/mut Faslpr/lpr mice were backcrossed to MRL-lpr mice seven to 10 times. All mice were bred and housed under specific pathogen-free conditions. The genotypes of mice were identified by polymerase chain reaction (PCR) of mouse-tail DNA.

Wild-type Fas DNA was amplified using two primers: FasIn2F, 5′-CTC CAG ACT CTC TTG CTT TAC-3′; and FasIn2R, 5′-GAC AAG AGA TTA GCC TCC AGG-3′ that generated a PCR product of 424 base pairs (bp). The mutant Fas DNA was amplified using the primers FasIn2F and FasIn2mR (5′-GAC ACC AGT TAT GAA GGA AGG-3′). This primer pair amplified a 380-bp DNA fragment that included the 280-bp insertion. The wild-type BAFF-R gene was amplified using BAFFR-E3F (5′-GGA AAA TGT CTT TGT ACC CTC-3′) and BAFFR-E3R (5′-CTA TTG CTC TGG GCC AGC TGT-3′), producing a product of 172 bp. The mutant BAFF-R gene was amplified with BAFFR-E3F and BAFFR-E3mR (5′-CTT AGA ACA CAG GAT GTC AGC-3′). The 204-bp PCR product contained a 140-bp fragment insertion. The cycling conditions for all PCRs were as follows: 94° for 10 min, 55° for 1 min, 72° for 2 min, total 35 cycles.

Flow cytometry analysis

The following fluorescently conjugated or biotinylated antibodies were used to detect cell surface markers on cells from spleen and bone marrow: CD19-fluorescein isothiocyanate (FITC), B220-FITC, B220-allophycocyanin (APC), immunoglobulin M (IgM)-biotin, IgD-phycoerythrin (PE), CD21-FITC and CD23-PE. Biotinylated antibody was detected with Streptavidin-PE or -FITC. Stained cells were analysed using a FACSCalibur (Becton Dickinson, Mountain View, CA). All reagents were purchased from Southern Biotechnology Associates (Birmingham, AL), except B220-APC (eBioScience, San Diego, CA) and CD21-FITC (BD PharMingen, San Diego, CA).

Detection of immunoglobulins and autoantibodies by enzyme-linked immunosorbent assay (ELISA)

Plates were coated with either anti-mouse immunoglobulin, or NP25-bovine serum albumin (BSA) (Biosearch Technologies, Novato, CA) or salmon sperm DNA (Sigma, St Louis, MO). Diluted serum or supernatant samples then were added. Immunoglobulin standards were used to quantify the concentration of immunoglobulins. Horseradish peroxidase-conjugated anti-IgM, anti-IgG, anti-IgG1, anti-IgG2a, anti-IgG2b, anti-IgG3, or IgA were used as the secondary antibodies. All reagents were purchased from Southern Biotechnology Associates except where indicated.

B-cell proliferation and immunoglobulin production in vitro

Splenic B cells were separated via negative selection using a mouse B-cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). B cells (purity: CD19+ cells > 95%; 2 × 105/ml) were cultured for 72 hr in triplicate with 20 μg/ml LPS (Sigma-Aldrich), 10 μg/ml anti-CD40 antibody (Southern Biotechnology Associates), 5 μg/ml F(ab′)2 of anti-IgM antibody (Jackson ImmunoResearch Laboratories, Baltimore, PA), and 10 μg/ml interleukin-4 (IL-4; PeproTech, London, UK). The cultures were pulsed with 1 μCi methyl-[3H]thymidine for the last 12 hr of culture. B-cell proliferation was determined by isotope incorporation measured by liquid scintillation counting. For immunoglobulin production in vitro, B cells (1 × 106/ml) were cultured with LPS, anti-CD40 antibody, anti-IgM antibody and IL-4. The amounts of the stimuli used were the same as those used in the proliferation experiment described above. Culture supernatants were collected at 7 days and immunoglobulin isotypes were assayed by ELISA.

Immunohistochemistry and renal pathology

The spleen and kidneys of mice were snap-frozen in optimal cutting temperature (OCT) compound (Sakura Finetek USA, Inc. Torrance, CA). Frozen sections were fixed in chilled acetone for 5 min. Splenic sections were stained with B220-FITC (BD PharMingen), IgM-rhodamine (Jackson ImmunoResearch Laboratories), or MOMA-1-FITC (Serotec Ltd, Oxford, UK) and peanut agglutinin (PNA)-biotin (Vector Laboratories, Burlingame, CA). Biotinylated PNA was detected using streptavidin-FITC. Renal samples were stained with either anti-IgG-FITC (Sino-American Biotech, Shanghai, China) or with anti-C3-FITC (Accurate Chemical & Scientific, New York, NY). For histological examination, kidneys were embedded in paraffin, and sections were stained with periodic acid-Schiff. Approximately 50 μl urine was collected from individual mice. Urine samples were kept at 4° and tested within 2 hr of collection. Urinary albumin was measured using the BioRad protein assay kit according to the manufacturer's instructions (Bio-Rad, Richmond, CA).

Detection of antibody-secreting cells by ELISPOT assay

MultiscreenHTS-IP filter plates (Millipore, Billerica, MA) were coated with 20 μg/ml anti-mouse immunoglobulin antibody (Southern Biotechnology Associates). After blocking, a five-fold serial dilution of a cell suspension (starting with 1 × 106 cells/ml in phosphate-buffered saline + 3% fetal calf serum) was added and incubated for 4 hr at 37°. Antibody-secreting cells were detected with biotinylated anti-mouse IgG antibody (Southern Biotechnology Associates), and then developed with Streptavidin-horseradish peroxidase (Southern Biotechnology Associates) and 3-amino-9-ethylcarbazole (AEC) substrate (Sigma). Spots were counted using a Bioreader 4000 (Biosys, Karben, Germany).

Statistical analysis

Statistical significance was evaluated using the Student's t-test. A P-value < 0·05 was considered statistically significant.

Results

Generation of BAFF-R-mutant MRL-lpr mice

To investigate the role of BAFF-R in autoimmunity and SLE in MRL-lpr mice, we generated BAFF-R-mutant MRL-lpr mice by crossing A/WySnJ mice with MRL-lpr mice. Mice with the following genotypes were used in these experiments: BAFF-Rwt/wt Faslpr/lpr, BAFF-Rwt/mut Faslpr/lpr and BAFF-Rmut/mut Faslpr/lpr. Identification of these genotypes is shown in Fig. 1.

Figure 1.

Figure 1

Open in a new tab

Identification of BAFF-R-mutant MRL-lpr mice. Representative PCR results from mice with the following genotypes are shown: BAFF-Rwt/wt Faslpr/lpr, BAFF-Rwt/mut Faslpr/lpr and BAFF-Rmut/mutFaslpr/lpr. (a) PCR detection of the wild-type and mutant Fas genes. MRL-lpr and C57BL/6 mice were used as controls. (b) PCR detection of the wild-type and mutant BAFF-R genes. A/WySnJ and A/J mice were used as controls.

BAFF-R-mutant MRL-lpr mice produced autoantibodies and developed hypergammaglobulinaemia and immune complex-mediated glomerulonephritis

Since BAFF-R plays an essential role in B-cell maturation and function by mediating BAFF signalling,13,21–24,27 we anticipated that the BAFF-R mutation might prevent or impair the development of lupus-like phenotypes in MRL-lpr mice. Therefore, we analysed the production of autoantibodies in the MRL-lpr mice with the BAFF-R mutation. In contrast to our expectation, the mice produced high titres of IgG1 and IgG2a anti-dsDNA antibodies, and the levels of the anti-dsDNA antibodies were comparable between the BAFF-R-mutant and BAFF-R-intact lupus mice (Fig. 2a). Next, we measured the serum level of immunoglobulins. Consistent with the level of autoantibody production, BAFF-R-mutant MRL-lpr mice exhibited hypergammaglobulinaemia and levels of serum immunoglobulins that were comparable to the BAFF-R-intact lupus mice (Fig. 2b). In addition, we examined the immune complex-mediated glomerulonephritis. As shown in Fig. 2(c), BAFF-R-mutant MRL-lpr mice had deposits of IgG and C3 in their glomeruli similar to those found in the MRL-lpr mice without the BAFF-R mutation. Histological examination of the kidneys demonstrated severe glomerulonephritic changes consisting of hypercellularity, lobularity, dilated capsules and crescent formation or enlarged glomeruli (Fig. 2c). Moreover, measurement of urinary protein showed that BAFF-R-mutant MRL-lpr mice developed proteinuria, which was also observed in the BAFF-R-intact lupus mice (Fig. 2d).

Figure 2.

Figure 2

Open in a new tab

BAFF-R-mutant MPL-lpr mice produced anti-dsDNA autoantibodies, and developed hypergammaglobulinaemia and immune complex-mediated glomerulonephritis. (a) Serum levels of IgG1 or IgG2a anti-dsDNA antibodies. (b) Serum levels of immunoglobulins and immunoglobulin subclasses. A/J and A/WySnJ mice were used as controls. (c) Immune complex-mediated glomerulonephritis. Deposits of IgG and C3 in the glomeruli were detected as indicated by immunofluorescence staining of frozen sections with FITC-conjugated anti-mouse IgG antibody and anti-mouse C3 antibody, respectively. Paraffin-embedded sections were stained with periodic acid-Schiff. Glomerulonephritic changes, including hypercellularity, lobularity, dilated capsules, crescent formation or enlarged glomeruli, were observed in the BAFF-R-mutant lupus mice. C57BL/6 mice were used as negative controls. Representative sections are shown. (d) Proteinuria: urinary albumin was measured as indicated in the Materials and methods. C57BL/6 mice were used as controls. Data shown are the mean ± SD in each group. Six mice at the age of 3–4 months in each genotype were analysed in each experiment.

BAFF-R mutation impaired splenic B-cell development in MRL-lpr mice

Our results above demonstrated that the BAFF-R-mutant MRL-lpr mice developed autoantibodies, hypergammaglobulinaemia and immune complex-mediated glomerulonephritis. To understand the mechanism for the unexpected phenotypes, we first investigated the effect of BAFF-R on splenic B-cell development in MRL-lpr mice. As shown in Fig. 3(a) and Table 1, the number of CD19+ B cells was reduced in the MRL-lpr mice with the BAFF-R heterozygous mutation. The number of CD19+ cells was markedly reduced in the BAFF-R homozygous mutant MRL-lpr mice. Consistent with this result, CD19+ IgM+ immature B cells were also reduced, especially in BAFF-R homozygous mutant MRL-lpr mice, which strongly affected further differentiation into CD19+ IgD+ mature cells. Moreover, analysis using IgM and IgD double staining revealed that the BAFF-R-mutant lupus mice had severely decreased numbers of mature IgMlow IgDhigh B cells (Fig. 3b). Similarly, the numbers of T2 (IgMhigh IgDhigh) and MZ B/T1 (IgMhigh IgDint) subsets were also diminished (Fig. 3b). Cell surface staining for B220, CD21 and CD23 showed a severe loss of follicular B cells (CD21low CD23high) and markedly reduced MZ B cells (CD21high CD23low/negative) in these mice (Fig. 3c, Table 1). The results were confirmed by immunohistochemical staining with anti-MOMA-1 and/or anti-IgM antibodies (Fig. 4). BAFF-R-intact MRL-lpr mice exhibited an IgM+ B-cell zone surrounded by a ring of MZ B cells. However, the IgM+ B cells and MZ B cells were markedly decreased in the MRL-lpr mice with the BAFF-R mutation, especially in mice with the homozygous mutation.

Figure 3.

Figure 3

Open in a new tab

Impairment of splenic B-cell maturation in BAFF-R-mutant MRL-lpr mice. Splenic cells from each genotype were double-stained with antibodies as indicated and analysed by flow cytometry. The percentage of positive cells for single or double antibody staining is given. Data are representative of six mice for each genotype.

Table 1.

Number of splenic B-cell populations in BAFF-R mutant and intact MRL-lpr mice1

B cells (CD19+) Immature B cells (CD19+ IgM+) Mature B cells (CD19+ IgD+) Follicle B cells (CD21low CD23high) MZ B cells (CD21high CDlow/neg)
BAFF-Rwt/wt Faslpr/lpr 87·3 ± 9·6 84·5 ± 8·0 84·1 ± 9·2 60·8 ± 8·2 17·3 ± 2·7
BAFF-Rwt/mut Faslpr/lpr 63·7 ± 7·0 62·0 ± 6·8 59·7 ± 7·6 31·8 ± 6·8 16·4 ± 2·1
BAFF-Rmut/mut Faslpr/lpr 18·3 ± 5·3 17·5 ± 5·9 15·0 ± 6·5 5·7 ± 2·3 6·2 ± 2·3
Open in a new tab
1

Values shown indicate the mean cell number (× 106) and SD for six mice in each group.

Figure 4.

Figure 4

Open in a new tab

Immunohistochemical staining of B-cell follicles and GCs in BAFF-R-mutant MRL-lpr mice. B-cell follicles were stained with anti-IgM (red) antibody. MZ B cells were revealed by counterstaining of anti-MOMA-1 (green) and anti-IgM (red). For GC staining, mice were challenged with 100 μg NP-KLH for 10 days and splenic sections were stained with PNA (green) and anti-IgM (red) to reveal GC and follicle, respectively. C57BL/6 mice were used as negative controls. Representative samples from each genotype are shown.

It was reported that BAFF regulates CD21 and CD23 expression independently of its B cell survival function.28 To evaluate whether the mutated BAFF-R in MRL-lpr mice retained some ability to mediate BAFF signalling, we assayed the expression of CD21 and CD23. BAFF-R-mutant MRL-lpr mice exhibited a severe loss of CD21+ B cells compared to the BAFF-R-intact lupus mice (Fig. 3d,e). Analysis of CD23 expression on B220+ cells showed markedly reduced numbers of CD23high CD21+ cells (Fig. 3e). These results indicated that the BAFF-R mutation could not have mediated the function of BAFF in the regulation of CD21 and CD23 expression in the lupus mice.

Taken together, these results demonstrated that BAFF-R is critical for late-stage B-cell maturation in autoimmune MRL-lpr mice and indicated that the haplo-insufficiency of BAFF-R could result in defective B-cell maturation in these mice.

BAFF-R mutation impaired germinal centre formation, antibody response and B-cell proliferation in MRL-lpr mice

It previously was reported that BAFF-R is required for germinal centre (GC) maintenance, but not for the formation of GC in non-autoimmune mice.29 Our data showed that GC could be formed in the BAFF-R-mutant lupus mice at 10 days post-challenge with the T-cell-dependent antigen NP-KLH. However, the staining intensity and number of GCs were significantly less than in the control mice (Fig. 4 and data not shown). To test whether BAFF-R mutation affects the antibody production in MRL-lpr mice, we measured the serum level of NP-specific IgG. As shown in Fig. 5(a), A/WySnJ mice showed severe impairment in the production of NP-specific IgG compared to A/J mice, which is consistent with a previous report.21 Mutation of BAFF-R also impaired the production of anti-NP IgG in MRL-lpr mice. Next, we purified the B cells and cultured the cells with LPS, anti-CD40, anti-IgM, IL-4 as indicated (Fig. 5b). B cells from mice with the BAFF-R mutation exhibited strongly decreased proliferation in response to all the stimuli tested. These results indicated that BAFF-R plays a significant role in the B-cell dysfunction observed.

Figure 5.

Figure 5

Open in a new tab

Decreased antibody responses and B-cell proliferation in BAFF-R-mutant MRL-lpr mice. (a) Relative anti-NP IgG antibody titres. Sera from mice immunized with 100 μg NP-KLH were collected at 10 days. NP-specific IgG was determined by ELISA. Data are shown as mean ± SD. The framed numbers show the relative reduction of the antibody titres between control and BAFF-R-mutant mice in each group. (b) Purified splenic B220+ cells (2 × 105/ml) were cultured in triplicate with the indicated stimuli for 3 days. The cultures were pulsed with 1 μCi methyl-[3H]thymidine for the last 12 hr of culture. B-cell proliferation was determined by isotope incorporation, measured by liquid scintillation counting. The mean and SD from five mice in each group indicated are shown. *P < 0·001; **P < 0·05 as compared with the BAFF-R wild-type lupus mice.

BAFF-R-mutant lpr splenic B cells produced increased immunoglobulin in vitro

The defects in splenic B-cell maturation and function observed in the BAFF-R-mutant MRL-lpr mice were discordant with the fact that these mice develop lupus-like phenotypes. Therefore, we assayed the ability of BAFF-R-mutant lpr B cells to produce immunoglobulin in vitro. We cultured the purified B cells with LPS, anti-CD40, anti-IgM and IL-4 as indicated in the Materials and methods. As shown in Fig. 6, the secretion of IgG1 was not reduced significantly under the various stimulation conditions, but rather was increased significantly by stimulation with LPS and anti-CD40 plus IL-4 in BAFF-R homozygous mutant MRL-lpr mice. Moreover, the supernatant level of IgG2a was also significantly increased in response to LPS stimulation. These results indicated that the BAFF-R mutation did not impair the ability of lpr splenic B cells to produce immunoglobulin in vitro. In contrast, BAFF-R-mutant lpr B cells could produce higher amounts of immunoglobulin, especially in response to the polyclonal activator LPS.

Figure 6.

Figure 6

Open in a new tab

BAFF-R-mutant lpr splenic B cells produced increased immunoglobulin in vitro. Purified splenic B cells (1 × 106/ml) were cultured with the indicated stimuli for 7 days. Supernatants were collected for measurement of IgG1 and IgG2a. The immunoglobulin isotypes were determined by ELISA. Data from five mice of each genotype indicated are shown. The statistically significant differences are indicated.

BAFF-R-mutant MRL-lpr mice had more antibody-secreting cells in their bone marrow

Furthermore, we examined B-cell development in the bone marrow of BAFF-R-mutant MRL-lpr mice. In contrast with the observations in the spleen, the number of immature (B220+ IgM+) and mature (B220+ IgD+) B cells was not decreased in BAFF-R-mutant MRL-lpr mice. Conversely, increased B220+ IgM+ cells were observed (Fig. 7a, Table 2). Interestingly, the BAFF-R mutation led to a marked increase in B220+ B cells in MRL-lpr mice (Fig. 7a, Table 2). Therefore, we investigated the function of antibody-secreting cells in the bone marrow using ELISPOT. As shown in Fig. 7(b), with the same number of bone marrow cells, the number of IgG-secreting cells was significantly increased in BAFF-R-mutant MRL-lpr mice compared with the BAFF-R-intact lupus mice. Thus, these IgG-secreting cells probably contributed to the development of hypergammaglobulinaemia and autoantibodies in BAFF-R-mutant MRL-lpr mice.

Figure 7.

Figure 7

Open in a new tab

BAFF-R-mutant MRL-lpr mice had increased B cells and IgG-secreting cells in the bone marrow. (a) Bone marrow cells from mice representing each genotype were double-stained with antibodies as indicated and analysed by flow cytometry. The percentage of positive cells for single or double antibody staining is given. (b) Bone marrow IgG-secreting cells were determined by ELISPOT. Data shown are the mean and SD for five mice analysed in each group. *P < 0·005; **P < 0·001 as compared with the BAFF-R wild-type lupus mice. Representative pictures for each group are shown.

Table 2.

Bone marrow B cells in BAFF-R mutant and intact MRL-lpr mice1

B cells (CD19+) Immature B cells (CD19+ IgM+) Mature B cells (CD19+ IgD+)
BAFF-Rwt/wt Faslpr/lpr 36 ± 3·3 11 ± 1·9 7·3 ± 1·1
BAFF-Rwt/mut Faslpr/lpr 47 ± 4·2 15 ± 1·3 8·9 ± 1·9
BAFF-Rmut/mut Faslpr/lpr 49 ± 4·6 19 ± 4·3 9·6 ± 1·2
Open in a new tab
1

Values shown indicate the mean cell number and SD for six mice in each group.

Discussion

The present study presented evidence that mutation of BAFF-R could not prevent or impair the development of autoimmunity in MRL-lpr mice. The BAFF-R-mutant MRL-lpr mice produced anti-dsDNA antibody, developed hypergammaglobulinaemia and exhibited immune complex-mediated glomerulonephritis. Discordantly, BAFF-R homozygous mutant MRL-lpr mice had severely defective B-cell development, including strongly reduced numbers of CD19+, CD19+ IgM+, CD19+ IgD+ and MZ B cells in their spleens, which would be predicted to affect the development of hypergammaglobulinaemia and autoimmunity. However, the BAFF-R mutation did not impair the ability of lpr B cells to produce immunoglobulin in response to the stimuli tested. Further studies found markedly increased numbers of B cells in the bone marrow of BAFF-R-mutant MRL-lpr mice. Moreover, ELISPOT analysis demonstrated that the bone marrow of BAFF-R-mutant MRL-lpr mice contained more antibody-secreting cells than the bone marrow of BAFF-R-intact lupus mice. Therefore, the antibody-secreting cells could compensate for the defective splenic B-cell differentiation and contribute to the development of hypergammaglobulinaemia and autoimmunity in BAFF-R-mutant MRL-lpr mice.

The essential role of BAFF-R in late-stage B-cell maturation has been well established through studies using A/WySnJ and BAFF-R null mice.13,21–26 Consistent with these data, our results demonstrated that BAFF-R is critical for splenic B-cell development in autoimmune MRL mice with the Fas mutation. Conversely, our results indicated that the Fas mutation cannot rescue the impaired splenic B-cell maturation caused by BAFF-R mutation, suggesting that Fas is dispensable for BAFF-R-mediated splenic B-cell development. Interestingly, the ability of BAFF-R mutant lpr splenic B cells to produce immunoglobulin was not impaired although their antibody response and proliferative function were significantly decreased. MZ B cells may contribute to the development of autoimmunity and lupus.30 However, BAFF-R-mutant MRL-lpr mice that develop lupus-like phenotypes had impaired MZ B cells. These results may reflect the multifactorial and complicated mechanism of autoimmune development in MRL-lpr mice.

Previous studies demonstrated profound abnormalities of B-cell development and function in BAFF-R mutant mice (A/WySnJ).21–24 Moreover, the phenotypes of BAFF-R null mice were similar to those of A/WySnJ mice.25,26 Importantly, our data showed that the BAFF-R mutation resulted in severely impaired B-cell maturation and function in the autoimmune MRL-lpr mice. Thus, the autoimmunity that developed in BAFF-R-mutant MRL-lpr mice was probably not mediated by residual BAFF-R-generated signalling.

MRL-lpr mice have an increased level of circulating BAFF, and overexpression of BAFF results in the development of lupus in mice.5,8,9 Therefore, BAFF could be associated with the development of autoimmunity in MRL-lpr mice. Although BAFF-R mutation severely impaired splenic B-cell development in MRL-lpr mice, the differentiation of bone marrow B cells was not diminished but rather enhanced. Thus, when BAFF-R is mutated, BAFF may interact with another known or unidentified receptor(s) and cause the breakdown of B-cell self-tolerance in bone marrow.

It has been reported that blocking of BAFF using BAFF receptor fusion proteins significantly impairs or delays the development of lupus-like phenotypes.6,31,32 However, these studies were undertaken either in BAFF transgenic mice or NZBW/F1 mice. Whether the decoy fusion proteins of BAFF receptors could be effective for treatment of MRL-lpr mice remains unclear. In spite of the mandatory role of BAFF-R in mediating BAFF signalling, our results indicate that BAFF-R is dispensable for the development of autoimmunity in MRL-lpr mice.

Acknowledgments

We thank Dr Yuan Zhuang for critically reading the manuscript. This work is supported by grants from the National Natural Science Foundation of China (No. 30471583, to J.S.) and the Shanghai Science and Technology Committee (No. 04JC14048, to J.S.), and by the Shanghai Leading Academic Discipline Project (T0206).

Abbreviations

APRIL

A Proliferation-Inducing Ligand

BAFF

B-cell-activating factor belonging to the TNF family

BCMA

B-cell maturation antigen

GC

germinal centre

MZ

marginal zone

SLE

systemic lupus erythematosus

T1/2

transitional type1/2

TACI

transmembrane activator and calcium modulator and cyclophilin ligand interactor

References

  • 1.Schneider P, MacKay F, Steiner V, et al. BAFF, a novel ligand of the tumor necrosis factor family, stimulates B cell growth. J Exp Med. 1999;189:1747–56. doi: 10.1084/jem.189.11.1747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Shu HB, Hu WH, Johnson H. TALL-1 is a novel member of the TNF family that is down-regulated by mitogens. J Leukoc Biol. 1999;65:680–3. [PubMed] [Google Scholar]
  • 3.Mukhopadhyay A, Ni J, Zhai Y, Yu GL, Aggarwal BB. Identification and characterization of a novel cytokine, THANK, a TNF homologue that activates apoptosis, nuclear factor-kappaB, and c-Jun NH2-terminal kinase. J Biol Chem. 1999;274:15978–81. doi: 10.1074/jbc.274.23.15978. [DOI] [PubMed] [Google Scholar]
  • 4.Moore PA, Belvedere O, Orr A, et al. BLyS: member of the tumor necrosis factor family and B lymphocyte stimulator. Science. 1999;285:260–3. doi: 10.1126/science.285.5425.260. [DOI] [PubMed] [Google Scholar]
  • 5.Gross JA, Johnston J, Mudri S, et al. TACI and BCMA are receptors for a TNF homologue implicated in B-cell autoimmune disease. Nature. 2000;404:995–9. doi: 10.1038/35010115. [DOI] [PubMed] [Google Scholar]
  • 6.Gross JA, Dillon SR, Mudri S, et al. TACI-Ig neutralizes molecules critical for B cell development and autoimmune disease. Impaired B cell maturation in mice lacking BLyS. Immunity. 2001;15:289–302. doi: 10.1016/s1074-7613(01)00183-2. [DOI] [PubMed] [Google Scholar]
  • 7.Schiemann B, Gommerman JL, Vora K, Cachero TG, Shulga-Morskaya S, Dobles M, Frew E, Scott ML. An essential role for BAFF in the normal development of B cells through a BCMA-independent pathway. Science. 2001;293:2111–14. doi: 10.1126/science.1061964. [DOI] [PubMed] [Google Scholar]
  • 8.Mackay F, Woodcock SA, Lawton P, Ambrose C, Baetscher M, Schneider P, Tschopp J, Browning JL. Mice transgenic for BAFF develop lymphocytic disorders along with autoimmune manifestations. J Exp Med. 1999;190:1697–710. doi: 10.1084/jem.190.11.1697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Khare SD, Sarosi I, Xia XZ, et al. Severe B cell hyperplasia and autoimmune disease in TALL-1 transgenic mice. Proc Natl Acad Sci USA. 2000;97:3370–5. doi: 10.1073/pnas.050580697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Groom J, Kalled SL, Cutler AH, et al. Association of BAFF/BLyS overexpression and altered B cell differentiation with Sjögren's syndrome. J Clin Invest. 2002;109:59–68. doi: 10.1172/JCI14121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Cheema GS, Roschke V, Hilbert DM, Stohl W. Elevated serum B lymphocyte stimulator levels in patients with systemic immune-based rheumatic diseases. Arthritis Rheum. 2001;44:1313–19. doi: 10.1002/1529-0131(200106)44:6<1313::AID-ART223>3.0.CO;2-S. [DOI] [PubMed] [Google Scholar]
  • 12.Yan M, Marsters SA, Grewal IS, Wang H, Ashkenazi A, Dixit VM. Identification of a receptor for BLyS demonstrates a crucial role in humoral immunity. Nat Immunol. 2000;1:37–41. doi: 10.1038/76889. [DOI] [PubMed] [Google Scholar]
  • 13.Thompson JS, Bixler SA, Qian F, et al. BAFF-R, a newly identified TNF receptor that specifically interacts with BAFF. Science. 2001;293:2108–11. doi: 10.1126/science.1061965. [DOI] [PubMed] [Google Scholar]
  • 14.Yan M, Brady JR, Chan B, et al. Identification of a novel receptor for B lymphocyte stimulator that is mutated in a mouse strain with severe B cell deficiency. Curr Biol. 2001;11:1547–52. doi: 10.1016/s0960-9822(01)00481-x. [DOI] [PubMed] [Google Scholar]
  • 15.Smith SH, Cancro MP. Cutting edge: B cell receptor signals regulate BLyS receptor levels in mature B cells and their immediate progenitors. J Immunol. 2003;170:5820–3. doi: 10.4049/jimmunol.170.12.5820. [DOI] [PubMed] [Google Scholar]
  • 16.Schneider P, Tschopp J. BAFF and the regulation of B cell survival. Immunol Lett. 2003;88:57–62. doi: 10.1016/s0165-2478(03)00050-6. [DOI] [PubMed] [Google Scholar]
  • 17.von Bulow GU, van Deursen JM, Bram RJ. Regulation of the T-independent humoral response by TACI. Immunity. 2001;14:573–82. doi: 10.1016/s1074-7613(01)00130-3. [DOI] [PubMed] [Google Scholar]
  • 18.Yan M, Wang H, Chan B, et al. Activation and accumulation of B cells in TACI-deficient mice. Nat Immunol. 2001;2:638–43. doi: 10.1038/89790. [DOI] [PubMed] [Google Scholar]
  • 19.Xu S, Lam KP. B-cell maturation protein, which binds the tumor necrosis factor family members BAFF and APRIL, is dispensable for humoral immune responses. Mol Cell Biol. 2001;21:4067–74. doi: 10.1128/MCB.21.12.4067-4074.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.O'Connor BP, Raman VS, Erickson LD, et al. BCMA is essential for the survival of long-lived bone marrow plasma cells. J Exp Med. 2004;199:91–8. doi: 10.1084/jem.20031330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Miller DJ, Hayes CE. Phenotypic and genetic characterization of a unique B lymphocyte deficiency in strain A/WySnJ mice. Eur J Immunol. 1991;21:1123–30. doi: 10.1002/eji.1830210506. [DOI] [PubMed] [Google Scholar]
  • 22.Lentz VM, Cancro MP, Nashold FE, Hayes CE. Bcmd governs recruitment of new B cells into the stable peripheral B cell pool in the A/WySnJ mouse. J Immunol. 1996;157:598–606. [PubMed] [Google Scholar]
  • 23.Lentz VM, Hayes CE, Cancro MP. Bcmd decreases the life span of B-2 but not B-1 cells in A/WySnJ mice. J Immunol. 1998;160:3743–7. [PubMed] [Google Scholar]
  • 24.Amanna IJ, Dingwall JP, Hayes CE. Enforced bcl-xL gene expression restored splenic B lymphocyte development in BAFF-R mutant mice. J Immunol. 2003;170:4593–600. doi: 10.4049/jimmunol.170.9.4593. [DOI] [PubMed] [Google Scholar]
  • 25.Sasaki Y, Casola S, Kutok JL, Rajewsky K, Schmidt-Supprian M. TNF family member B cell-activating factor (BAFF) receptor-dependent and -independent roles for BAFF in B cell physiology. J Immunol. 2004;173:2245–52. doi: 10.4049/jimmunol.173.4.2245. [DOI] [PubMed] [Google Scholar]
  • 26.Shulga-Morskaya S, Dobles M, Walsh ME, Ng LG, MacKay F, Rao SP, Kalled SL, Scott ML. B cell-activating factor belonging to the TNF family acts through separate receptors to support B cell survival and T cell-independent antibody formation. J Immunol. 2004;173:2331–41. doi: 10.4049/jimmunol.173.4.2331. [DOI] [PubMed] [Google Scholar]
  • 27.Melchers F. Actions of BAFF in B cell maturation and its effects on the development of autoimmune disease. Ann Rheum Dis. 2003;62(Suppl. 2):25–7. doi: 10.1136/ard.62.suppl_2.ii25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Gorelik L, Cutler AH, Thill G, et al. Cutting edge: BAFF regulates CD21/35 and CD23 expression independent of its B cell survival function. J Immunol. 2004;172:762–6. doi: 10.4049/jimmunol.172.2.762. [DOI] [PubMed] [Google Scholar]
  • 29.Rahman ZS, Rao SP, Kalled SL, Manser T. Normal induction but attenuated progression of germinal center responses in BAFF and BAFF-R signaling-deficient mice. J Exp Med. 2003;198:1157–69. doi: 10.1084/jem.20030495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Mackay F, Sierro F, Grey ST, Gordon TP. The BAFF/APRIL system: an important player in systemic rheumatic diseases. Curr Dir Autoimmun. 2005;8:243–65. doi: 10.1159/000082106. [DOI] [PubMed] [Google Scholar]
  • 31.Kayagaki N, Yan M, Seshasayee D, et al. BAFF/BLyS receptor 3 binds the B cell survival factor BAFF ligand through a discrete surface loop and promotes processing of NF-kappaB2. Immunity. 2002;17:515–24. doi: 10.1016/s1074-7613(02)00425-9. [DOI] [PubMed] [Google Scholar]
  • 32.Pelletier M, Thompson JS, Qian F, et al. Comparison of soluble decoy IgG fusion proteins of BAFF-R and BCMA as antagonists for BAFF. J Biol Chem. 2003;278:33127–33. doi: 10.1074/jbc.M305754200. [DOI] [PubMed] [Google Scholar]

Articles from Immunology are provided here courtesy of British Society for Immunology

RESOURCES