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. 1999 Jun 7;189(11):1799-814.
doi: 10.1084/jem.189.11.1799.

MRL-lpr/lpr mice exhibit a defect in maintaining developmental arrest and follicular exclusion of anti-double-stranded DNA B cells

L Mandik-Nayak  1 , S J Seo, C Sokol, K M Potts, A Bui, J Erikson
Affiliations

MRL-lpr/lpr mice exhibit a defect in maintaining developmental arrest and follicular exclusion of anti-double-stranded DNA B cells

L Mandik-Nayak et al. J Exp Med. .

Abstract

A hallmark of systemic lupus erythematosus and the MRL murine model for lupus is the presence of anti-double-stranded (ds)DNA antibodies (Abs). To identify the steps leading to the production of these Abs in autoimmune mice, we have compared the phenotype and localization of anti-dsDNA B cells in autoimmune (MRL+/+ and lpr/lpr) mice with that in nonautoimmune (BALB/c) mice. Anti-dsDNA B cells are actively regulated in BALB/c mice as indicated by their developmental arrest and accumulation at the T-B interface of the splenic follicle. In the MRL genetic background, anti-dsDNA B cells are no longer developmentally arrested, suggesting an intrinsic B cell defect conferred by MRL background genes. With intact Fas, they continue to exhibit follicular exclusion; however, in the presence of the lpr/lpr mutation, anti-dsDNA B cells are now present in the follicle. Coincident with the altered localization of anti-dsDNA B cells is a follicular infiltration of CD4 T cells. Together, these data suggest that MRL mice are defective in maintaining the developmental arrest of autoreactive B cells and indicate a role for Fas in restricting entry into the follicle.

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Figures

Figure 1
Figure 1
The VH3H9 Tg does not change the seroconversion time point or titer of ANAs but does increase serum λ ANA frequency. An age-matched cohort of Tg− (○) and VH3H9 (•) MRL-lpr/lpr mice was bled weekly from the age of 6 to 16 wk. Sera were tested for the presence of ANAs by immunofluorescence. (A) Symbols represent individual mice at the age when anti-HN ANAs first appear. Tg− MRL-lpr/lpr mice became serum ANA+ at approximately the same time (10.1 ± 3.0 wk) as VH3H9 MRL-lpr/lpr mice (9.1 ± 2.0 wk; P = 0.2). n = 25 mice of each genotype. (B) ANA titers were determined for the seroconversion time point (10 wk) of a subset of mice (n = 6 mice of each genotype). The serum titer is defined as the reciprocal of the last dilution at which positive staining was seen. No difference was detected between Tg− and VH3H9 MRL-lpr/lpr mice. (C) Serum samples were tested for the appearance of λ ANAs. Symbols placed above the dotted line represent mice at the age when λ ANAs were first detected. Below the dotted line are symbols representing mice that failed to show λ ANAs by 15 wk of age. n = 17 Tg− MRL-lpr/lpr, n = 18 VH3H9 MRL-lpr/lpr mice.
Figure 2
Figure 2
VH3H9/λ anti-dsDNA B cells are present with a reduced Ig density: (A) BM and (B) spleen in (left) MRL-lpr/lpr and (right) MRL+/+ mice. Dot plots are graphed on a log scale, and mean fluorescence intensity (MFI) is given for the λ+ cells in the boxed region. n = 7 mice of each genotype.
Figure 3
Figure 3
Phenotypic analysis of Tg− and VH3H9 (A–C) splenic and (D) BM B cells in age-matched BALB/c, MRL+/+, and MRL-lpr/lpr mice. (A) Tg− mice. Histograms show staining of total B cell population (gated on B220+Ig+ cells). Percentages are given for the indicated gates on representative plots. There is an exaggerated population of CD21high cells (MFI > 200) present in MRL+/+ (32.9 ± 11.5%) and MRL- lpr/lpr (40.1 ± 9.9%) compared with BALB/c (7.9 ± 3.1%) mice (P < 0.0001). CD23 levels decrease on MRL-lpr/lpr B cells as the mice age (percentage of CD23low B cells at 4–7 wk, 48 ± 10.6% vs. at 8–14 wk, 70.4 ± 8.7%; P = 0.03). There is also an increased population of CD23low B cells in MRL+/+ mice (MRL+/+, 48 ± 11.3% vs. BALB/c, 16.4 ± 3.4%; P = 0.016), although not to the extent in 8–14 wk MRL- lpr/lpr mice (70.4 ± 8.7%; P = 0.0003). The proportion of L-selectinlow B cells is increased in MRL-lpr/lpr (38.8 ± 13.6%) and MRL+/+ (38.4 ± 13.9%) compared with BALB/c (23.5 ± 4.8%) mice (P = 0.009). VH3H9/λ B cells in (B) MRL+/+, BALB/c, and (C) MRL-lpr/lpr spleen. Histograms are gated on the total B cell population (thin line) and Igλ+ population (bold line). The underlaid histograms (thin line) were scaled down to allow for comparison to the λ+ B cells (which comprise ∼10% of the total B cell population). The majority of markers show that VH3H9/λ B cells in MRL+/+ and MRL-lpr/lpr mice, unlike in BALB/c mice, are no longer developmentally arrested; however, CD21/35 levels are decreased. VH3H9 BALB/c, VH3H9 MRL+/+, and VH3H9 MRL-lpr/lpr mice at 4–7 wk are λ ANA−, whereas VH3H9 MRL-lpr/lpr mice at 8–14 wk are λ ANA+. VH3H9/λ B cells in MRL-lpr/lpr and MRL+/+ spleens are developmentally mature regardless of the mouse's ANA status. (D) BM B cells were stained with CD22 and Igλ. VH3H9/λ B cells are Iglow at the CD22low stage (VH3H9/ Igλ MFI, 50 vs. Tg− Igλ MFI, 200) and do not decrease as the B cells mature (VH3H9/Igλ MFI, 68 vs. Tg− Igλ MFI, 480), suggesting an encounter with Ag at an early developmental stage. Note that there is a decrease in the CD22high population (23 to 6%) in MRL-lpr/lpr mice, with and without the VH3H9 Tg. This is consistent with a previous report that documents a decrease in the frequency of mature B cells in the BM of MRL-lpr/lpr mice (reference 87). All graphs are plotted on a log scale. n = 7 mice at each age range for each genotype.
Figure 3
Figure 3
Phenotypic analysis of Tg− and VH3H9 (A–C) splenic and (D) BM B cells in age-matched BALB/c, MRL+/+, and MRL-lpr/lpr mice. (A) Tg− mice. Histograms show staining of total B cell population (gated on B220+Ig+ cells). Percentages are given for the indicated gates on representative plots. There is an exaggerated population of CD21high cells (MFI > 200) present in MRL+/+ (32.9 ± 11.5%) and MRL- lpr/lpr (40.1 ± 9.9%) compared with BALB/c (7.9 ± 3.1%) mice (P < 0.0001). CD23 levels decrease on MRL-lpr/lpr B cells as the mice age (percentage of CD23low B cells at 4–7 wk, 48 ± 10.6% vs. at 8–14 wk, 70.4 ± 8.7%; P = 0.03). There is also an increased population of CD23low B cells in MRL+/+ mice (MRL+/+, 48 ± 11.3% vs. BALB/c, 16.4 ± 3.4%; P = 0.016), although not to the extent in 8–14 wk MRL- lpr/lpr mice (70.4 ± 8.7%; P = 0.0003). The proportion of L-selectinlow B cells is increased in MRL-lpr/lpr (38.8 ± 13.6%) and MRL+/+ (38.4 ± 13.9%) compared with BALB/c (23.5 ± 4.8%) mice (P = 0.009). VH3H9/λ B cells in (B) MRL+/+, BALB/c, and (C) MRL-lpr/lpr spleen. Histograms are gated on the total B cell population (thin line) and Igλ+ population (bold line). The underlaid histograms (thin line) were scaled down to allow for comparison to the λ+ B cells (which comprise ∼10% of the total B cell population). The majority of markers show that VH3H9/λ B cells in MRL+/+ and MRL-lpr/lpr mice, unlike in BALB/c mice, are no longer developmentally arrested; however, CD21/35 levels are decreased. VH3H9 BALB/c, VH3H9 MRL+/+, and VH3H9 MRL-lpr/lpr mice at 4–7 wk are λ ANA−, whereas VH3H9 MRL-lpr/lpr mice at 8–14 wk are λ ANA+. VH3H9/λ B cells in MRL-lpr/lpr and MRL+/+ spleens are developmentally mature regardless of the mouse's ANA status. (D) BM B cells were stained with CD22 and Igλ. VH3H9/λ B cells are Iglow at the CD22low stage (VH3H9/ Igλ MFI, 50 vs. Tg− Igλ MFI, 200) and do not decrease as the B cells mature (VH3H9/Igλ MFI, 68 vs. Tg− Igλ MFI, 480), suggesting an encounter with Ag at an early developmental stage. Note that there is a decrease in the CD22high population (23 to 6%) in MRL-lpr/lpr mice, with and without the VH3H9 Tg. This is consistent with a previous report that documents a decrease in the frequency of mature B cells in the BM of MRL-lpr/lpr mice (reference 87). All graphs are plotted on a log scale. n = 7 mice at each age range for each genotype.
Figure 3
Figure 3
Phenotypic analysis of Tg− and VH3H9 (A–C) splenic and (D) BM B cells in age-matched BALB/c, MRL+/+, and MRL-lpr/lpr mice. (A) Tg− mice. Histograms show staining of total B cell population (gated on B220+Ig+ cells). Percentages are given for the indicated gates on representative plots. There is an exaggerated population of CD21high cells (MFI > 200) present in MRL+/+ (32.9 ± 11.5%) and MRL- lpr/lpr (40.1 ± 9.9%) compared with BALB/c (7.9 ± 3.1%) mice (P < 0.0001). CD23 levels decrease on MRL-lpr/lpr B cells as the mice age (percentage of CD23low B cells at 4–7 wk, 48 ± 10.6% vs. at 8–14 wk, 70.4 ± 8.7%; P = 0.03). There is also an increased population of CD23low B cells in MRL+/+ mice (MRL+/+, 48 ± 11.3% vs. BALB/c, 16.4 ± 3.4%; P = 0.016), although not to the extent in 8–14 wk MRL- lpr/lpr mice (70.4 ± 8.7%; P = 0.0003). The proportion of L-selectinlow B cells is increased in MRL-lpr/lpr (38.8 ± 13.6%) and MRL+/+ (38.4 ± 13.9%) compared with BALB/c (23.5 ± 4.8%) mice (P = 0.009). VH3H9/λ B cells in (B) MRL+/+, BALB/c, and (C) MRL-lpr/lpr spleen. Histograms are gated on the total B cell population (thin line) and Igλ+ population (bold line). The underlaid histograms (thin line) were scaled down to allow for comparison to the λ+ B cells (which comprise ∼10% of the total B cell population). The majority of markers show that VH3H9/λ B cells in MRL+/+ and MRL-lpr/lpr mice, unlike in BALB/c mice, are no longer developmentally arrested; however, CD21/35 levels are decreased. VH3H9 BALB/c, VH3H9 MRL+/+, and VH3H9 MRL-lpr/lpr mice at 4–7 wk are λ ANA−, whereas VH3H9 MRL-lpr/lpr mice at 8–14 wk are λ ANA+. VH3H9/λ B cells in MRL-lpr/lpr and MRL+/+ spleens are developmentally mature regardless of the mouse's ANA status. (D) BM B cells were stained with CD22 and Igλ. VH3H9/λ B cells are Iglow at the CD22low stage (VH3H9/ Igλ MFI, 50 vs. Tg− Igλ MFI, 200) and do not decrease as the B cells mature (VH3H9/Igλ MFI, 68 vs. Tg− Igλ MFI, 480), suggesting an encounter with Ag at an early developmental stage. Note that there is a decrease in the CD22high population (23 to 6%) in MRL-lpr/lpr mice, with and without the VH3H9 Tg. This is consistent with a previous report that documents a decrease in the frequency of mature B cells in the BM of MRL-lpr/lpr mice (reference 87). All graphs are plotted on a log scale. n = 7 mice at each age range for each genotype.
Figure 3
Figure 3
Phenotypic analysis of Tg− and VH3H9 (A–C) splenic and (D) BM B cells in age-matched BALB/c, MRL+/+, and MRL-lpr/lpr mice. (A) Tg− mice. Histograms show staining of total B cell population (gated on B220+Ig+ cells). Percentages are given for the indicated gates on representative plots. There is an exaggerated population of CD21high cells (MFI > 200) present in MRL+/+ (32.9 ± 11.5%) and MRL- lpr/lpr (40.1 ± 9.9%) compared with BALB/c (7.9 ± 3.1%) mice (P < 0.0001). CD23 levels decrease on MRL-lpr/lpr B cells as the mice age (percentage of CD23low B cells at 4–7 wk, 48 ± 10.6% vs. at 8–14 wk, 70.4 ± 8.7%; P = 0.03). There is also an increased population of CD23low B cells in MRL+/+ mice (MRL+/+, 48 ± 11.3% vs. BALB/c, 16.4 ± 3.4%; P = 0.016), although not to the extent in 8–14 wk MRL- lpr/lpr mice (70.4 ± 8.7%; P = 0.0003). The proportion of L-selectinlow B cells is increased in MRL-lpr/lpr (38.8 ± 13.6%) and MRL+/+ (38.4 ± 13.9%) compared with BALB/c (23.5 ± 4.8%) mice (P = 0.009). VH3H9/λ B cells in (B) MRL+/+, BALB/c, and (C) MRL-lpr/lpr spleen. Histograms are gated on the total B cell population (thin line) and Igλ+ population (bold line). The underlaid histograms (thin line) were scaled down to allow for comparison to the λ+ B cells (which comprise ∼10% of the total B cell population). The majority of markers show that VH3H9/λ B cells in MRL+/+ and MRL-lpr/lpr mice, unlike in BALB/c mice, are no longer developmentally arrested; however, CD21/35 levels are decreased. VH3H9 BALB/c, VH3H9 MRL+/+, and VH3H9 MRL-lpr/lpr mice at 4–7 wk are λ ANA−, whereas VH3H9 MRL-lpr/lpr mice at 8–14 wk are λ ANA+. VH3H9/λ B cells in MRL-lpr/lpr and MRL+/+ spleens are developmentally mature regardless of the mouse's ANA status. (D) BM B cells were stained with CD22 and Igλ. VH3H9/λ B cells are Iglow at the CD22low stage (VH3H9/ Igλ MFI, 50 vs. Tg− Igλ MFI, 200) and do not decrease as the B cells mature (VH3H9/Igλ MFI, 68 vs. Tg− Igλ MFI, 480), suggesting an encounter with Ag at an early developmental stage. Note that there is a decrease in the CD22high population (23 to 6%) in MRL-lpr/lpr mice, with and without the VH3H9 Tg. This is consistent with a previous report that documents a decrease in the frequency of mature B cells in the BM of MRL-lpr/lpr mice (reference 87). All graphs are plotted on a log scale. n = 7 mice at each age range for each genotype.
Figure 4
Figure 4
Localization of Igλ+ B cells. (A) Spleen sections from Tg−, VH3H9 BALB/c, VH3H9 MRL+/+, and VH3H9 MRL-lpr/lpr mice were stained with Abs against (top) CD22 and Igλ or (bottom) CD4 and Igλ. In Tg− mice of all three backgrounds, the Igλ+ cells are scattered throughout the B cell follicle. The follicle shown in the figure is from a Tg− MRL+/+ spleen. Igλ+ cells in VH3H9 MRL+/+ mice, like those in VH3H9 BALB/c, accumulate at the T–B interface. However, the VH3H9/λ B cells in MRL-lpr/lpr mice are found in the B cell follicle. Interestingly, CD4+ cells are also scattered in the B cell area with the λ+ B cells. Mice shown are 5 wk of age. (B) Spleen sections from (top) Tg− MRL-lpr/lpr and (bottom) VH3H9 MRL-lpr/lpr mice stained with Abs against (left) CD22 and Igλ, (middle) CD4 and Igλ, or (right) CD22 and syndecan-1 (AFCs). In the majority of follicles, VH3H9/Igλ AFCs are found in the PALS, although some follicles did not have syndecan-1+ cells. AFCs in Tg− MRL-lpr/lpr mice are also found in the PALS. AFCs are first detectable at 8 wk, and mice shown are 10 wk of age. Original magnification: ×100 (n = 10 mice of each genotype).
Figure 5
Figure 5
Disrupted architecture in MRL- lpr/lpr mice. (A) Spleen sections from (top) Tg− BALB/c, (middle) Tg− MRL+/+, and (bottom) Tg− MRL-lpr/lpr mice were stained with Abs against MOMA-1 (MZ metallophilic macrophages) and (left) CD22 (B cells), (middle) CD4 (T cells), or (right) CD8. In MRL-lpr/lpr mice, CD4 T cells are present scattered in the B cell area. CD8 T cells remain in the PALS. Mice shown are 12 wk old. (B) Spleen sections from age-matched (top) Tg− MRL-lpr/lpr and (bottom) VH3H9 MRL-lpr/lpr mice. VH3H9 Tg accelerates the appearance of disrupted architecture in MRL-lpr/lpr mice. Mice shown are 6 wk old. Original magnification: ×100 (n = 15 mice of each genotype).
Figure 5
Figure 5
Disrupted architecture in MRL- lpr/lpr mice. (A) Spleen sections from (top) Tg− BALB/c, (middle) Tg− MRL+/+, and (bottom) Tg− MRL-lpr/lpr mice were stained with Abs against MOMA-1 (MZ metallophilic macrophages) and (left) CD22 (B cells), (middle) CD4 (T cells), or (right) CD8. In MRL-lpr/lpr mice, CD4 T cells are present scattered in the B cell area. CD8 T cells remain in the PALS. Mice shown are 12 wk old. (B) Spleen sections from age-matched (top) Tg− MRL-lpr/lpr and (bottom) VH3H9 MRL-lpr/lpr mice. VH3H9 Tg accelerates the appearance of disrupted architecture in MRL-lpr/lpr mice. Mice shown are 6 wk old. Original magnification: ×100 (n = 15 mice of each genotype).
Figure 6
Figure 6
AFCs in the spleen from ANA− (4–6 wk) and ANA+ (10–12 wk) Tg− MRL-lpr/lpr, VH3H9 MRL-lpr/lpr, and BALB/c mice. The number of (A) total Ig and (B) Igλ AFCs were determined by ex vivo ELISpot. The data are presented as the mean number of AFCs per 104 cells ± the SD of triplicate wells. Total Ig+ and Igλ+ AFC numbers were calculated by normalizing for total B220+Ig+ and B220+Igλ+ numbers as determined by flow cytometry, respectively. Presented data are from a representative mouse of each genotype. Total number of mice from five experiments: n = 5 Tg− BALB/c; n = 4 VH3H9 BALB/c; n = 4 ANA+ Tg− and VH3H9 MRL-lpr/lpr; and n = 2 ANA− Tg− and VH3H9 MRL-lpr/lpr mice. Note that a higher number of Igλ+ AFCs than total Ig+ AFCs were detected for the ANA+ VH3H9 MRL-lpr/lpr mice in this assay. This difference is attributed to a sensitivity difference in the reagents used to detect total Ig versus Igλ. Therefore, although comparisons can be made for the relative number of AFCs of a given type (i.e., between Igλ+ numbers), they cannot be used to compare absolute frequencies between types (i.e., total Ig vs. Igλ).
Figure 6
Figure 6
AFCs in the spleen from ANA− (4–6 wk) and ANA+ (10–12 wk) Tg− MRL-lpr/lpr, VH3H9 MRL-lpr/lpr, and BALB/c mice. The number of (A) total Ig and (B) Igλ AFCs were determined by ex vivo ELISpot. The data are presented as the mean number of AFCs per 104 cells ± the SD of triplicate wells. Total Ig+ and Igλ+ AFC numbers were calculated by normalizing for total B220+Ig+ and B220+Igλ+ numbers as determined by flow cytometry, respectively. Presented data are from a representative mouse of each genotype. Total number of mice from five experiments: n = 5 Tg− BALB/c; n = 4 VH3H9 BALB/c; n = 4 ANA+ Tg− and VH3H9 MRL-lpr/lpr; and n = 2 ANA− Tg− and VH3H9 MRL-lpr/lpr mice. Note that a higher number of Igλ+ AFCs than total Ig+ AFCs were detected for the ANA+ VH3H9 MRL-lpr/lpr mice in this assay. This difference is attributed to a sensitivity difference in the reagents used to detect total Ig versus Igλ. Therefore, although comparisons can be made for the relative number of AFCs of a given type (i.e., between Igλ+ numbers), they cannot be used to compare absolute frequencies between types (i.e., total Ig vs. Igλ).
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