PMC

Identification of BAFF-R-mutant MRL-lpr mice. Representative PCR results from mice with the following genotypes are shown: BAFF-R^wt/wt Fas^lpr/lpr, BAFF-R^wt/mut Fas^lpr/lpr and BAFF-R^mut/mutFas^lpr/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.

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.

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.

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.

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.

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 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.