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. 2010 Jan 15;184(2):775-86.
doi: 10.4049/jimmunol.0901322. Epub 2009 Dec 16.

Development of murine lupus involves the combined genetic contribution of the SLAM and FcgammaR intervals within the Nba2 autoimmune susceptibility locus

Trine N Jørgensen  1 , Jennifer Alfaro, Hilda L Enriquez, Chao Jiang, William M Loo, Stephanie Atencio, Melanie R Gubbels Bupp, Christina M Mailloux, Troy Metzger, Shannon Flannery, Stephen J Rozzo, Brian L Kotzin, Mario Rosemblatt, María Rosa Bono, Loren D Erickson
Affiliations

Development of murine lupus involves the combined genetic contribution of the SLAM and FcgammaR intervals within the Nba2 autoimmune susceptibility locus

Trine N Jørgensen et al. J Immunol. .

Abstract

Autoantibodies are of central importance in the pathogenesis of Ab-mediated autoimmune disorders. The murine lupus susceptibility locus Nba2 on chromosome 1 and the syntenic human locus are associated with a loss of immune tolerance that leads to antinuclear Ab production. To identify gene intervals within Nba2 that control the development of autoantibody-producing B cells and to determine the cellular components through which Nba2 genes accomplish this, we generated congenic mice expressing various Nba2 intervals where genes for the FcgammaR, SLAM, and IFN-inducible families are encoded. Analysis of congenic strains demonstrated that the FcgammaR and SLAM intervals independently controlled the severity of autoantibody production and renal disease, yet are both required for lupus susceptibility. Deregulated homeostasis of terminally differentiated B cells was found to be controlled by the FcgammaR interval where FcgammaRIIb-mediated apoptosis of germinal center B cells and plasma cells was impaired. Increased numbers of activated plasmacytoid dendritic cells that were distinctly CD19+ and promoted plasma cell differentiation via the proinflammatory cytokines IL-10 and IFNalpha were linked to the SLAM interval. These findings suggest that SLAM and FcgammaR intervals act cooperatively to influence the clinical course of disease through supporting the differentiation and survival of autoantibody-producing cells.

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Figures

FIGURE 1
FIGURE 1
Map of telomeric chromosome 1 encoding Nba2 and the congenic strains generated to express various intervals. Distal chromosome 1 with the positions of known immune regulatory genes is shown. Dashed lines show microsatellite markers used to determine B6 or NZB origin. Congenic strains are identified according to inheritance of proximal (A), central (B), and distal (C) regions that encompass the Nba2 locus.
FIGURE 2
FIGURE 2
FcγR and SLAM gene clusters within Nba2 are sufficient for autoantibody production and renal failure. Sera from female, 7-mo-old congenic strains (A) and F1 mice from congenic strains crossed with NZW (B) was analyzed for IgG autoantibodies to chromatin, total histones, and dsDNA by ELISA. Statistical differences between congenic strains and control mice are indicated as #p < 0.05, ##p < 0.01, and ###p < 0.001. Statistical differences between each group and B6.Nba2-ABC or (ABC × NZW)F1 mice are indicated as *p < 0.05, **p < 0.01, and ***p < 0.001. C, Proteinuria levels were determined in female F1 mice on a monthly basis for 12 mo. Data are shown as the percentage of mice within each group whose proteinuria levels were ≥100 mg/dl protein. Each group consisted of at least 13 mice. The difference in frequency of disease in the (B6.Nba2-ABC × NZW)F1 and (B6.Nba2-A’B × NZW)F1 mice compared with other groups by 8 mo of age was significant, p < 0.001.
FIGURE 3
FIGURE 3
Expression levels of Ifi202 correspond with the Nba2 genotype of congenic strains. A, Real-time PCR analysis of Ifi202 expression in spleen cells from 4-mo-old congenic strains and B6 control mice. Results are expressed as the mean ± SEM from six mice. B, Extracts prepared from individual age-matched female mice were analyzed by immunoblotting using Abs specific to the indicated proteins. Densitometry values of protein expression relative to β-actin are shown. Data are representative of two experiments.
FIGURE 4
FIGURE 4
Reduced levels of FcγRIIb expression on PCs and GC B cells are associated with Nba2 genotype. Spleen cells were prepared from 4-mo-old female mice immunized with 100 μg NP-KLH Ag for 7 d. A, The levels of FcγRIIb membrane expression on DAPI−B220+GL7+ gated GC B cells (open black histograms), DAPI−B220low/−CD138+ gated PCs (solid red histograms), and DAPI−B220+GL7− gated B cells (solid gray histograms) were determined by flow cytometry. B, Total numbers were quantified for the indicated cell populations from immune animals shown in A. Data shown are expressed as the mean ± SEM for six mice per group. Statistical differences between congenic strains and B6 control mice are indicated as *p < 0.05. C, T cell-depleted spleen cells were cultured 4 h with plate-bound rat IgG (Fab’)2 (gray histograms), anti-FcγRIIb (2.4G2; red histograms) alone, or with the caspase-3 inhibitor ZVAD (blue histograms). Apoptosis was determined by intracellular caspase-3 staining of GC B cells and PCs. Numbers represent the percentage of apoptotic cells. Open black histograms represent isotype-matched control staining. Cells cultured 4 h in the presence of 10 μM DMSO were stained for intracellular caspase-3 (bottom row; red histograms) as a positive control for apoptosis. Solid gray histograms represent isotype-matched Ab staining.
FIGURE 5
FIGURE 5
Nba2 controls the frequency and subset distribution of pDCs in an age-dependent manner. A, Total numbers of pDCs (DAPI−B220+CD11cint) and cDCs (DAPI−B220−CD11chigh) from spleens of 4-mo-old congenic strains were determined by flow cytometry. Data are expressed as the mean ± SEM for nine mice per group. Statistical differences between congenic strains and B6 control mice are denoted by *p < 0.05 and **p < 0.01. B, Total B220+CD11cint gated pDCs from spleens of 2- and 4-mo-old congenic strains were further segregated into CD19− and CD19+ pDC subsets and absolute numbers were determined. Statistical differences between congenic strains and control mice are indicated as **p < 0.01. C, The expression of Siglec-H and PDCA1 was determined on cDCs, CD19− pDCs, and CD19+ gated pDCs from spleens of the indicated strains.
FIGURE 6
FIGURE 6
CD19+ pDCs exhibit hallmark DC markers but have an altered activation phenotype that is associated with combined expression of FcγR and SLAM regions of Nba2. A, Ig gene rearrangement in sort-purified splenic B cell, cDC, CD19− pDC, and CD19+ pDC populations from B6.Nba2-ABC congenic mice was determined by PCR analysis; transcripts for 5′ JH1 and 5′ DFL16.1 to determine D-J and V to D-J rearrangement, respectively, indicate Ig rearrangement in B cells where intron fragments have been deleted yet are retained in DC subsets. B, Cytospins of sort-purified cells were morphologically assessed by Giemsa staining. Arrows indicate dendrite formation. Images were taken at original magnification ×40. C, Spleen cells from 4-mo-old female mice were analyzed by FACS for the expression of indicated markers on cDCs, CD19− pDC, and CD19+ pDCs using the same gating strategy described in Fig. 5. Results show the mean fluorescence intensity for each population. Statistical differences in the mean fluorescence intensity of a marker between congenic strains and B6 control mice are denoted by *p < 0.05. Data shown represent three independent experiments. Representative expression levels of all DC subset markers and SLAM receptors are shown in Supplemental Fig. 4.
FIGURE 7
FIGURE 7
The Nba2 locus controls TLR hypersensitivity of DCs to produce proinflammatory cytokines. A, Total CD11c+ DCs were sort-purified from spleens of 4-mo-old B6.Nba2-ABC and B6 control mice, and were cultured 24 h in the absence (no treatment [NT]) or presence of 100 ng/ml LPS, 10 μg/ml CpG, and 10 μg/ml R837. Supernatants were collected and the amount of cytokine was determined by ELISA. Statistical differences between TLR-stimulated groups and NT are indicated by *p < 0.05, **p < 0.01, and ***p < 0.001. B, Using identical culture conditions, levels of IL-10 and IFNα were determined from purified total pDCs or CD19− and CD19+ pDCs. Data are expressed as the mean ± SEM from 6–9 mice per group. NT, no treatment.
FIGURE 8
FIGURE 8
Combined expression of FcγR and SLAM intervals control TLR hypersensitivity of pDCs to promote the generation of Ab-secreting cells. A, Purified B220+CD11cint pDCs from spleens of congenic ABC, A’B, B, and C strains as well as B6 control mice were cultured with an equal number (1 × 104) of purified control B cells in medium alone (NT), with 10 μg/ml CpG, or with 10 μg/ml nonstimulatory GpC oligonucleotide. B cells and pDCs cultured alone served as negative controls. After 72 h, the number of total Ig-secreting cells was determined by ELISPOT. Statistical differences between CpG stimulated groups and control GpC groups are indicated by ***p < 0.001. B, B cells and pDCs were also cocultured in the presence of neutralizing mAb specific for IL-10 (5 μg/ml) and IFNα (10 μg/ml) throughout the 72 h, followed by ELISPOT. Statistical differences between CpG-stimulated groups containing pDCs from congenic strains and control mice are indicated as **p < 0.01. CpG-stimulated groups with IL-10 and IFNα neutralizing mAb that had statistically fewer Ab-secreting cells compared with CpG alone are designated *p < 0.05. Data are expressed as the mean ± SEM from three replicates per group that represent two independent experiments. NT, no treatment.
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