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The Journal of Immunology, 2007, 178: 4667-4675.
Copyright © 2007 by The American Association of Immunologists, Inc.

Augmentation of NZB Autoimmune Phenotypes by the Sle1c Murine Lupus Susceptibility Interval1

Brendan M. Giles*, Svetlana N. Tchepeleva*, Julie J. Kachinski*, Katherine Ruff*, Byron P. Croker{dagger},{ddagger}, Laurence Morel{dagger} and Susan A. Boackle2,*

* Department of Medicine and Department of Immunology, University of Colorado at Denver and Health Sciences Center, Aurora, CO 80045; {dagger} Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, Gainesville, FL 32610; and {ddagger} North Florida South Georgia Veterans Health System, Gainesville, FL 32608


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The Sle1c lupus susceptibility interval spans a 7-Mb region on distal murine chromosome 1. Cr2 is the strongest candidate gene for lupus susceptibility in this interval, as its protein products are structurally and functionally altered. B6.Sle1c congenic mice develop Abs to chromatin by 9 mo of age with a 30% penetrance and do not develop GN. To determine whether the New Zealand White (NZW)-derived Sle1c interval would interact with New Zealand Black (NZB) genes to result in enhanced autoimmune phenotypes, NZB mice were bred with B6 or B6.Sle1c congenic mice and ~20 female offspring were selected from each breeding for longitudinal study. These mice differ only at the Sle1c locus at which they have either a NZB/B6 or NZB/NZW genotype. NZB x B6.Sle1c mice had an accelerated onset of anti-chromatin Abs (100 vs 68% at 6 mo, p = 0.006) and anti-dsDNA Abs (45 vs 5% at 9 mo, p = 0.0048). Furthermore, median titers of anti-chromatin and anti-dsDNA Abs were significantly higher in the NZB x B6.Sle1c group compared with the NZB x B6 group. This corresponded with a higher prevalence of proliferative GN at 12 mo (55 vs 16%, p = 0.0214) as well as increased glomerular deposition of C3 (p = 0.0272) and IgG (p = 0.032), although blood urea nitrogen remained normal and significant proteinuria was not identified in either group. These data show that the Sle1c interval accelerates and augments the loss of tolerance to chromatin and dsDNA induced by NZB genes and induces significantly greater end-organ damage.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Systemic lupus erythematosus (SLE)3 is an autoimmune disease characterized by the loss of tolerance to nuclear self-Ags that is manifested by the production of autoantibodies and the development of an immune complex-mediated glomerulonephritis (GN). Linkage analyses in SLE patients and in murine models have identified multiple loci containing potential susceptibility genes (1). No single gene is believed to be solely responsible for disease development; rather, it is likely that combinations of genes interact to predispose an individual to disease and determine their heterogeneous clinical manifestations.

Animal models of SLE have been extremely useful in confirming the genetic contributions to SLE. The NZM2410 mouse model is a recombinant inbred strain derived from the New Zealand Black (NZB) and New Zealand White (NZW) strains that evolves a severe form of lupus-like disease with the development of autoantibodies to nuclear Ags and death from renal disease by 1 year of age (2). Along with the MHC locus on chromosome 17, three major loci on chromosomes 1, 4, and 7, termed Sle1, Sle2, and Sle3, have been shown to contribute to lupus development in NZM2410 mice (3). Congenic mice have been created containing each of these intervals (4), allowing the unique immunologic phenotypes contributed by each interval to be demonstrated (5, 6, 7). Using these mice, it has been clearly shown that epistatic interactions between these intervals are required for the development of full-blown lupus-like disease (8). Identifying the specific genes within each interval that are responsible for the phenotypes contributed by that interval will provide a means of piecing together the pathogenesis of this disease.

The Sle1c interval is a subinterval of the larger Sle1 interval on distal chromosome 1 (9). We have previously identified Cr2, which encodes complement receptors (CR) 1 and 2 (CR1/CR2, CD35/CD21) in the mouse, to be a strong candidate gene for lupus susceptibility in this interval based on structural and functional alterations in its protein products (10). The primary autoimmune phenotype of congenic mice containing the Sle1c interval on a C57BL/6 (B6) background is a loss of tolerance to chromatin, resulting in the production of autoantibodies specific for chromatin. This phenotype is present in only 30% of B6.Sle1c congenics compared with 80% of B6 congenics containing the larger Sle1 interval (9). Although neither strain develops significant kidney disease, when crossed with NZW mice 35% of the offspring of a B6.Sle1 mouse cross developed proliferative GN compared with <5% of the offspring of a B6.Sle1c cross (9). Furthermore, when the Sle1 interval was transferred onto a homozygous lpr (Fas-deficient) background, 100% of the B6.Sle1 x lpr mice developed proliferative GN with 100% mortality by 9 mo of age (11). In comparison, only 25% of the B6.Sle1c x lpr mice developed proliferative GN with a 15% mortality at 9 mo. Therefore, although Sle1c had epistatic effects in combination with other genes that predispose to autoimmunity, these effects were modest.

Genetic background is known to have a major influence on the manifestations of disease susceptibility genes (12). In addition to the studies cited above, similar analyses of the NZB-derived Nba2 lupus susceptibility interval on distal chromosome 1, which partially overlaps with the Sle1c interval, have been performed. In these studies, mice congenic for the Nba2 interval did not develop significant renal disease on a B6 background but developed substantial GN when crossed with NZW mice (13). Because the offspring of NZW and NZB mice develop substantial autoimmune disease, we hypothesized that crossing the NZW-derived Sle1c interval onto an NZB background may better reveal the phenotypes contributed by this interval. A more robust phenotype would be useful in evaluating the contribution to autoimmunity of recombinant congenic Sle1c subintervals as well as candidate Sle1c genes introduced into genetically engineered mouse models. Furthermore, these results may suggest a mechanism(s) for the potential lupus susceptibility genes contained within the Sle1c interval and thereby move us closer to their identification.

We bred B6 or B6.Sle1c mice with NZB mice and followed the female offspring for 1 year for the development of autoantibodies and kidney disease. We found that the offspring of B6.Sle1c mice crossed with NZB mice developed higher titers of autoantibodies at an earlier time point compared with the offspring of B6 and NZB mice. In addition, the prevalence of proliferative GN was significantly higher in NZB x B6.Sle1c mice. These results suggest that the Sle1c gene(s) significantly accelerates and augments murine lupus when it acts in epistasis with NZB genes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

The production of the B6.Sle1c subcongenic strain from B6.Sle1 has been previously described (9). C57BL/6 and NZB mice were obtained from The Jackson Laboratory. Mice were bred and maintained at the University of Colorado Health Sciences Center for Laboratory Care (Aurora, CO). All of the experimental procedures performed on these animals were reviewed and approved by the University of Colorado Health Sciences Center Institutional Animal Care and Use Committee.

Detection of autoantibodies

Anti-chromatin and anti-dsDNA Abs were detected by a sandwich ELISA. Briefly, Costar high binding microtiter plates (Corning) were incubated with 10 µg/ml methylated BSA (Sigma-Aldrich) in PBS overnight at 4°C. For anti-chromatin ELISAs, plates were incubated with 50 µg/ml calf thymus dsDNA (Sigma-Aldrich) in PBS for 5–6 h at room temperature followed by 10 µg/ml histone (Sigma-Aldrich) in 0.06 M bicarbonate buffer overnight at 4°C. For anti-dsDNA ELISAs, plates were incubated with 10 µg/ml calf thymus dsDNA (Sigma-Aldrich) in PBS overnight at 4°C. Plates were washed three times with PBS and 0.05% Tween 20, three times with PBS, and blocked overnight at 4°C with 3% BSA, 3 mM EDTA, and 0.1% gelatin. Plates were washed as described above, and standards, samples, controls, and blanks were added. Standards for the anti-chromatin ELISA were supernatants from the anti-chromatin-producing hybridoma 2B1, and standards for the anti-dsDNA ELISA were supernatants from the anti-dsDNA-producing hybridoma 1D12 (gifts from B. Kotzin, University of Colorado Health Sciences Center, Denver, CO). Positive controls were pooled sera from aged SNF1 or MRL/lpr mice, and negative controls were pooled sera from young B6 mice. Samples and controls were diluted 1/100 and plated in duplicate. After a 2-h incubation with agitation at room temperature, plates were washed as described above and incubated with alkaline phosphatase-conjugated goat anti-mouse IgG (Caltag Laboratories) overnight at 4°C. After a final series of washes, plates were developed with a p-nitrophenyl phosphate substrate (Sigma-Aldrich) and read at 405/490 nm. Analysis was performed using SoftMax Pro software (Molecular Devices). The reactivities of a 1/400 dilution of the anti-chromatin standard and a 1/100 dilution of the anti-dsDNA standard were arbitrarily set to 10 U/ml. The reactivities of the positive control sera were consistent across the experiments, allowing valid comparison of data gathered at different time points. Sera with reactivities that did not lie within the linear portion of the standard curve were diluted further and reassayed.

Measurement of urine protein and blood urea nitrogen (BUN)

Mice were monitored monthly for the development of proteinuria by dipstick analysis (Roche). In addition, at 12 mo of age the albumin/creatinine ratio was determined as a more sensitive measure of proteinuria. Total albumin in urine was measured by albumin ELISA (Bethyl Laboratories), and urine creatinine was determined with a Beckman autoanalyzer (Beckman Coulter). As a measure of renal function, BUN was determined for each mouse at 12 mo of age using a Beckman autoanalyzer (Beckman Coulter).

Renal histology and immunofluorescence

Mice were euthanized at 12 mo of age and the kidneys were fixed, sectioned, and stained with H&E and periodic acid-Schiff (PAS). Multiple sections were examined in a blinded fashion by light microscopy for indications of inflammation and tissue damage as described previously (14). Briefly, GN was quantified on a 0–4 scale in which the grades 1, 2, 3, and 4 are accorded when 1–10, 11–25, 26–50, and >50% of the glomeruli are affected, respectively. Kidneys were also snap frozen and embedded in OCT compound (Miles Diagnostics) for the evaluation of immune complex deposition in glomeruli. Sections (8 µm) were incubated for 30 min with pretitered dilutions of the appropriate FITC-conjugated Abs: anti-mouse C3 (ICN Pharmaceuticals), anti-IgG {gamma}-chain (Jackson ImmunoResearch Laboratories), anti-IgG1 (Southern Biotechnology Associates), anti-IgG2a (Southern Biotechnology Associates), anti-IgG3 (ICN Pharmaceuticals/Cappel), or anti-IgM (Igh-6b; BD Biosciences). After the removal of excess Ab, sections were examined in a blinded fashion and scored by multiplying the extent of distribution of fluorescent staining (1, 2, 3, and 4 assigned for involvement of 1–25, 26–50, 51–75, and >75% of the glomeruli) by the intensity (0–3) of staining.

Sequencing

RNA was extracted from the spleen of an NZB mouse using Trizol reagent (Invitrogen Life Technologies). cDNA was generated by reverse transcription using random hexamers (Applied Biosystems), and PCR products were generated from cDNA using primer sets described previously (10). PCR, performed in a GeneAmp PCR System 9700 (Applied Biosystems), generally used touchdown PCR cycling parameters (94°C for 2 min, 20 cycles of 94°C for 30 s, 65°C to 55°C for 30 s, and 72°C for 1 min, followed by 15 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 1 min, and then 72°C for10 min). PCR products were purified with the QIAquick PCR product purification kit (Qiagen) and sequenced with the same primers by the University of Colorado Cancer Center DNA Sequencing and Analysis Core. ABI Prism kits from Applied Biosystems were used for sequencing with the AmpliTaq DNA polymerase FS and the BigDye Terminator cycle sequencing ready reaction kit, version 1.1 (part no. 4337451). In the case of difficult templates, the PCR product was mixed with an aliquot of the dGTP BigDye Terminator cycle sequencing ready reaction kit (part number 4307175). The standard sequencing thermocycling parameters were as follows: denaturation for 5 min at 94°C followed by 30 cycles of denaturation at 96°C for 10 s, annealing at 50°C for 5 s, and extension/termination at 60°C for 4 min followed by incubation at 4°C until the samples were processed. Residual dye-labeled dideoxynucleotides (dye terminators) were removed from the cycle-sequencing reaction products using the paramagnetic bead technology (CleanSEQ, part number 000136) from Agencourt Bioscience and a modification of the manufacturer’s recommended protocol. The products were sequenced on a fluorescent capillary automated sequencer with a 50-cm-long, 48-capillary array containing the POP7 polymer (Hitachi 3730 Genetic Analyzer; Applied Biosystems).

Construction and expression of the recombinant C3dg expression vector

The human C3dg and biotin signal peptide sequences were amplified from the pET11d-C3dg biotin signal peptide BirA plasmid (15) with the primers 5'-GCGCGGTACCGAAGGAGTGCAGAAAGAGGAC-3' and 5'-CGGCTCGAGTATCACGCAGTTCCATTTTCATTGC-3'. This adds a KpnI site at the 5' end of the cDNA and an XhoI site at the 3' end. The fragment was directly cloned into the pSecTag2B vector (Invitrogen Life Technologies), which allows expression in eukaryotic cells. The plasmid was transfected into FreeStyle 293-F cells (Invitrogen Life Technologies) per the manufacturer’s protocol, and supernatant containing expressed protein was concentrated and dialyzed into loading buffer consisting of 50 mM NaH2PO4 and 300 mM NaCl (pH 7.4); 20 mM Imidazole and 0.1 mM PMSF were also added. The supernatant was loaded on a 2-ml Ni-NTA column (Qiagen, Valencia) and washed with loading buffer until the eluate was protein-free by OD260. Recombinant C3dg was eluted with loading buffer containing 250 mM imidazole (pH 7.4). The eluate was dialyzed into biotinylation buffer consisting of 40 mM Tris and 200 mM K-glutamate (pH 7.4) and biotinylated in vitro using BirA (Avidity) per the manufacturer’s protocol. The purity and appropriate biotinylation of the protein were assessed by PAGE followed by Coomassie blue staining and Western blotting with streptavidin-HRP.

Flow cytometry

Splenic cell suspensions were depleted of RBC with Gey’s solution and Fc{gamma}RII receptors blocked with 2.4G2 (American Type Tissue Collection). B cells were incubated with saturating amounts of biotinylated rat anti-mouse CR1 (clone 8C12) or rat anti-mouse CR1/CR2 (clone 7E9), followed by PE-streptavidin (Southern Biotechnology Associates) and FITC-B220 (BD Biosciences). Cells were analyzed on a FACScan device (BD Biosciences) and the mean amount of fluorescence bound was determined.

Intracellular calcium measurements

Splenic cell suspensions from 10- to 12-wk-old female mice were depleted of RBC with Gey’s solution. Cells were loaded with Indo-1AM (Molecular Probes) and B cells labeled with FITC-B220. The sufficiency of the Indo-1AM loading was assessed by stimulation with 10 µg of F(ab')2 polyclonal goat anti-mouse surface Ig (sIg) (Southern Biotechnology Associates). Tetramers prepared by incubating 2 µg of rC3dg and 0.25 µg of biotinylated rat anti-mouse IgM (clone b-7-6; provided by Dr. J. Cambier, University of Colorado Health Sciences Center, Denver, CO) with 0.2 µg of streptavidin (BioSource International) were added to 2 x 106 cells, and calcium flux of B220-positive cells was measured on a BD LSR cytometer (BD Biosciences). Results were analyzed using FlowJo software (Tree Star).

Statistical analysis

All data were analyzed using GraphPad Prism and InStat software. To compare the phenotypes of the (NZB x B6)F1 and (NZB x B6.Sle1c)F1 mice when the data were normally distributed, an unpaired t test with a Welch correction was performed. When the data were not normally distributed, Mann-Whitney tests were used. Data are represented as median ± interquartile ranges. The penetrance of various phenotypes was evaluated using the one-sided Fisher’s exact test. p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Generation of (NZB x B6)F1 and (NZB x B6.Sle1c)F1 mice

To evaluate whether the Sle1c congenic interval exacerbates the autoimmune phenotypes contributed by NZB genes, NZB mice were crossed with either B6 or B6.Sle1c mice. Only female offspring were evaluated because of the higher penetrance of autoimmune disease in female (NZB x NZW)F1 mice compared with males. Nineteen (NZB x B6)F1 and 22 (NZB x B6.Sle1c) mice were evaluated. These mice differed only at the 7-Mb Sle1c interval on distal chromosome 1, at which they have either a B6/NZW or NZB/NZW genotype. The mice were followed monthly beginning at 3 mo of age for the development of autoantibodies or proteinuria and sacrificed at 1 year for the analysis of kidney pathology.

(NZB x B6.Sle1c)F1 mice produced higher titer and earlier onset anti-chromatin autoantibodies

B6.Sle1c mice develop anti-chromatin Abs at a 30% penetrance by 9 mo of age. To determine whether the Sle1c congenic interval contributed to the production of anti-chromatin Abs in the NZB crosses, anti-chromatin Ab levels were determined by ELISA at 3, 6, 9, and 12 mo. Beginning at the assessment at 6 mo, median titers were higher in (NZB x B6.Sle1c)F1 mice compared with (NZB x B6)F1 mice (98 ± 1526 vs 38 ± 69, p = 0.0033) and remained significantly higher at both 9 mo (679 ± 5492 vs 63 ± 92, p < 0.0001) and 12 mo of age (3114 ± 8438 vs 393 ± 1530, p = 0.008) (Fig. 1). (NZB x B6.Sle1c)F1 mice also developed significant titers of autoantibodies to chromatin more rapidly, with 100% positive by 6 mo of age compared with 68% in the (NZB x B6)F1 group (p = 0.006) (Fig. 2). This difference in penetrance was maintained at 9 mo of age (100 vs 79%, p = 0.0383), whereas by 12 mo of age nearly all of the mice in both groups were producing anti-chromatin Abs. These data demonstrate that the NZW-derived Sle1c interval both accelerated the onset and augmented the production of anti-chromatin Abs in NZB x B6 mice.


Figure 1
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  		FIGURE 1. (NZB x B6.Sle1c)F1 mice produce significantly higher titers of anti-chromatin Abs compared with (NZB x B6)F1 mice. Sera were analyzed by ELISA for the presence of anti-chromatin Abs at 3 (A), 6 (B), 9 (C), and 12 mo of age (D). Each point represents a single mouse and the bar marks median titers for that cohort. The dotted line represents the cutoff for positive and negative titers, set at the mean titer +2 SD of anti-chromatin Abs measured in four B6 mice analyzed in the same experiment.

 

Figure 2
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  		FIGURE 2. Significant titers of anti-chromatin Abs appear earlier in (NZB x B6.Sle1c)F1 mice. Using the cutoff for positive values determined in Fig. 1, the percentage of mice expressing anti-chromatin Abs in each cohort was calculated at 3 (A), 6 (B), 9 (C), and 12 mo of age (D). Statistics were performed using the one-sided Fisher’s exact test.

 
(NZB x B6.Sle1c)F1 mice produced higher titer and earlier onset anti-dsDNA autoantibodies

Although B6.Sle1c mice do not develop anti-dsDNA Abs, we hypothesized that the NZW-derived Sle1c interval may induce these Abs in epistasis with other lupus susceptibility genes contributed by the NZB strain. To test this hypothesis, anti-dsDNA Abs were measured by ELISA at 3, 6, 9, and 12 mo. Only one mouse, from the (NZB x B6.Sle1c)F1 group, developed significant titers of anti-dsDNA Abs at 6 mo of age. At 9 mo, median titers of anti-dsDNA Abs were higher in (NZB x B6.Sle1c)F1 mice compared with (NZB x B6)F1 mice (6.5 ± 12 vs 4 ± 4, p = 0.0079) and remained significantly higher at 12 mo of age (35 ± 52 vs 19 ± 11, p = 0.0016) (Fig. 3). In addition, the penetrance of anti-dsDNA Abs was higher in the (NZB x B6.Sle1c)F1 cohort, present in 45% by 9 mo of age compared with 5% of the (NZB x B6)F1 mice (p = 0.0041) (Fig. 3). By 12 mo of age there were still more (NZB x B6.Sle1c)F1 mice producing significant titers of anti-dsDNA than (NZB x B6)F1 mice, but this difference was no longer statistically significant (59 vs 32%; p = 0.0734). Thus, the NZW-derived Sle1c interval is able to accelerate the onset and augment the production of anti-dsDNA Abs on a strain background that is predisposed to develop these Abs.


Figure 3
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  		FIGURE 3. (NZB x B6.Sle1c)F1 mice produce significantly higher titers of anti-dsDNA Abs at an earlier time point compared with (NZB x B6)F1 mice. Sera were analyzed by ELISA for the presence of anti-dsDNA Abs at 9 (A) and 12 mo of age (B). Each point represents a single mouse and the bar marks median titers for that cohort. The dotted line represents the cutoff for positive and negative titers, set at the mean titer +2 SD of anti-dsDNA Abs measured in four B6 mice analyzed in the same experiment. Using the cutoff for positive values, the percentage of mice expressing anti-dsDNA Abs in each cohort was calculated at 9 (C) and 12 mo of age (D). Statistics were performed using the one-sided Fisher’s exact test.

 
Absence of renal dysfunction in (NZB x B6.Sle1c)F1

Glomerular damage due to immune complex deposition is a common manifestation of SLE. To determine whether the NZW-derived Sle1c interval induced glomerular damage resulting in proteinuria, we monitored the mice monthly by urine dipstick. None of the mice in either group had sustained levels of proteinuria over 1+. In addition, the levels of albumin and creatinine in the urine at 12 mo of age were measured and an albumin/creatinine ratio was determined for each mouse. There were no significant differences in albumin/creatinine ratios between the two groups (p = 0.1468; Fig. 4A). BUN levels were also normal in both groups at 12 mo (p = 0.7338; Fig. 4B). Therefore, despite the presence of high titer anti-chromatin and anti-dsDNA Abs in both groups at 12 mo of age, there was no clinical evidence of renal damage in either group.


Figure 4
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  		FIGURE 4. The absence of evidence of renal damage as measured by proteinuria and BUN. At 12 mo of age, urinary albumin/creatinine (Cr) ratios (A) and serum BUN (B) were measured for each mouse in the study. Each point on the graph represents the value for an individual mouse, and the bar shows the median value for each cohort.

 
Increased immune complex deposits in glomeruli of (NZB x B6.Sle1c)F1 mice

One of the hallmarks of renal disease due to SLE is the glomerular deposition of immune complexes containing all isotypes of Ig as well as complement. Immunofluorescence of kidney sections from the (NZB x B6.Sle1c)F1 mice revealed significantly greater mesangial deposits of C3 (p = 0.0365; Fig. 5A) and IgG (p = 0.032; Fig. 5B) compared with the (NZB x B6)F1 mice, although the deposition of IgM was equivalent in the two groups (p = 0.38, Fig. 5C). This demonstrates that although there was no evidence of overt renal damage clinically, immune complex deposition involving IgG, IgM, and C3 was present in both cohorts, with increased deposition of IgG and C3 in the cohort expressing the NZW-derived Sle1c interval. Furthermore, although the deposition of IgG1 and IgG2a did not differ in the two groups (data not shown), the deposition of IgG3 was found in 41% of the (NZB x B6.Sle1c)F1 mice compared with 16% of the (NZB x B6)F1 mice (p = 0.0768). Representative staining for glomerular C3 and IgG3 is shown in Fig. 7.


Figure 5
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  		FIGURE 5. Increased deposition of immune complexes in (NZB x B6.Sle1c)F1 mice compared with (NZB x B6)F1 mice. Kidneys harvested from 12-mo-old mice were sectioned and stained with FITC anti-mouse C3 (A), FITC anti-mouse IgG (B), and FITC anti-mouse IgM (C). Scores were assigned as described in Materials and Methods.

 

Figure 7
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  		FIGURE 7. Increased proliferative GN and immune complex deposition in (NZB x B6.Sle1c)F1 mice. Sections from an (NZB x B6)F1 kidney are shown in A–D and sections from an (NZB x B6.Sle1c)F1 kidney are shown in E–H. A, Low-power view of intact cortex with four glomeruli showing mild mesangial expansion (PAS stain); bar, 100 µm. B, High-power view of one glomerulus with patent capillaries and mild thickening of stalk (PAS stain); bar, 50 µm. C, C3 immunofluorescence stain showing mesangial pattern; bar, 100 µm. D, IgG3 immunofluorescence stain showing negligible staining; bar, 100 µm. E, Low-power view showing inflamed, hypercellular, and swollen glomeruli (PAS stain); bar, 100 µm. F, High-power view showing expanded mesangium and congestion of many capillary loops (PAS stain); bar, 50 µm. G, C3 immunofluorescence stain showing mesangial and capillary wall deposits (6 and 12 o’clock); bar, 100 µm. H, IgG3 immunofluorescence stain showing primarily mesangial deposits, bar, 100 µm.

 
(NZB x B6.Sle1c)F1 mice have a higher prevalence of severe proliferative GN

To further investigate the renal pathology in these mice, a histological examination of fixed kidney tissue was performed. Despite the absence of evidence of overt renal damage as measured by BUN or proteinuria, there was greater penetrance of proliferative GN at 12 mo of age in (NZB x B6.Sle1c)F1 compared with (NZB x B6)F1 mice (55 vs 16%, p = 0.0113; Fig. 6A). In addition, the (NZB x B6.Sle1c)F1 cohort had higher renal scores compared with the (NZB x B6)F1 mice (2.5 vs 0, p = 0.0233; Fig. 6B). Representative histological changes are shown in Fig. 7. These data show that in addition to inducing higher titers of autoantibodies, the NZW-derived Sle1c interval also contributed to enhanced renal damage as determined histologically.


Figure 6
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  		FIGURE 6. (NZB x B6.Sle1c)F1 mice develop proliferative GN at higher rates and greater severity than (NZB x B6)F1 mice. The presence of proliferative GN was determined by light microscopy. The penetrance of proliferative GN is expressed as a percentage (A). Renal scores are also shown (B), with each point on the graph representing the value for an individual mouse and the bar showing the median value for each cohort.

 
Sequence of Cr2 in NZB mice is identical to that of B6

We had previously demonstrated that NZB shared the NZW allele for the single-nucleotide polymorphism identified at nucleotide 1700 in the Cr2 gene, based on restriction fragment length polymorphism analysis (10). To evaluate whether the NZB Cr2 gene contained other polymorphisms that might affect the function of its gene products, we determined the sequence of the coding region for the entire NZB Cr2 gene. We found that the sequence was identical with that of B6 Cr2, including the allele for the single-nucleotide polymorphism at position 1700 (data not shown). Because this allele was characterized based on the absence of a BsrI restriction enzyme site, it is likely that our previous results reflected the inefficiency of the restriction enzyme digestion rather than the presence of the NZW allele at this location. Therefore, alterations in the coding domains of the NZB Cr2 gene would not be expected to contribute to the phenotypes identified in these studies.

CD35/CD21-mediated signaling is diminished in (NZB x B6.Sle1c)F1 B cells

Signaling via CD35/CD21 is impaired in B6.Sle1c mice (10), which are homozygous for the NZW-derived Cr2 allele. To evaluate whether CD35/CD21 signals are also altered in mice heterozygous for NZW Cr2, we measured calcium flux induced by co-crosslinking CD35/CD21 and sIgM with streptavidin-linked complexes of b-rC3dg and biotinylated anti-sIgM (clone b-7-6). As shown previously (15), at subthreshold doses of b-rC3dg and biotinylated anti-sIgM, a response could only be seen if both reagents were combined in the complexes, consistent with the known function of CD21 in lowering the threshold for B cell activation through sIgM (16). Calcium flux induced by these complexes requires CR1/CR2 expression, as it does not occur when stimulating Cr2–/– B cells (data not shown). When B cells from (NZB x B6.Sle1c)F1 mice were stimulated with these complexes, the mean number of cells responding was reduced by 20% (Fig. 8A) and the mean amplitude of the response was decreased by 25% (Fig. 8B). In addition, the time to peak response was increased. When either a strong (F(ab')2 goat anti-mouse Ig; Fig. 8, C and D) or intermediate (tetramers containing a higher dose of biotinylated anti-sIgM; data not shown) anti-sIgM stimulus was used to crosslink the BCR alone, B cells from both strains responded similarly in the numbers of cells responding and the amplitude of the response. These findings were similar to those demonstrated previously for the parental B6 and B6.Sle1c strains (10). To ensure that the decreased signaling mediated by CD35/CD21 was not due to altered expression of CD35/CD21 on (NZB x B6.Sle1c)F1 B cells, the binding of mAbs to CD35 (8C12; Fig. 8E) and CD35/CD21 (7E9; Fig. 8F) was assessed. The expression of both CD35 and CD21 on the cell surface was equivalent in both groups. These results demonstrate that B cells from the two strains generate comparable signals through the BCR but that the NZW Cr2 allele, even in a heterozygous state, is unable to function optimally to lower the threshold for B cell activation through sIgM.


Figure 8
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  		FIGURE 8. Impaired CD35/CD21-mediated signaling in (NZB x B6.Sle1c)F1 mice. A and B, Indo-1AM-loaded B cells were stimulated with tetramers prepared with 2 µg of b-rC3dg plus 0.25 µg of biotinylated anti-sIgM (b-7-6) and 0.2 µg of streptavidin to cocrosslink CD35/CD21 and sIgM. B220-positive cells were analyzed for increases in the intracellular concentration of calcium. The percentage of cells with intracellular calcium at levels >10% above baseline (A) and the mean ratio of bound to unbound Indo-1AM (B) were determined. The use of b-rC3dg tetramers or biotinylated b-7-6 tetramers alone at these doses resulted in no effect on intracellular calcium levels (data not shown). Cells were also activated with a strongly crosslinking anti-sIg Ab (F(ab')2 goat anti-mouse Ig) with percentage of cells responding depicted in C and the mean response in D. RBC-depleted splenic cells were stained with anti-CD35 (clone 8C12) (E) and anti-CD35/CD21 (clone 7E9) (F) and B220-positive (NZB x B6.Sle1c)F1 cells (thick gray line) and (NZB x B6)F1 cells (thick black line) were analyzed for expression of these receptors by flow cytometry. The binding of an isotype control Ab (thin black line) is also indicated. These data are representative of staining performed on three separate mice in each group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We describe here the effects of the NZW-derived Sle1c interval on the autoimmune phenotypes contributed by the NZB strain. In these experiments, we generated mice that were heterozygous for NZB and B6 at every locus except for the Sle1c interval, which was heterozygous for NZB and NZW. We compared these to mice that were heterozygous for NZB and B6 at every locus, including Sle1c. We found that NZB/NZW heterozygosity at this 7-Mb locus significantly enhanced the autoimmune phenotypes in these mice, affecting both autoantibody production and renal disease. These data demonstrate an effect on autoimmune disease of the gene(s) in the Sle1c interval that is more pronounced than that described in previous studies, which showed only a mild epistatic effect of the Sle1c locus.

Although all of the mice in both groups develop anti-chromatin autoantibodies by 1 year of age, the (NZB x B6.Sle1c)F1 mice develop these Abs earlier and have higher median titers at every time point. This pattern is similar with respect to the development of anti-dsDNA Abs, although the penetrance does not reach 100% by 12 mo. Therefore, these data demonstrate that NZW-derived genes in the Sle1c interval induce higher titers and accelerate the onset of anti-chromatin and anti-dsDNA Abs in mice predisposed to develop these autoimmune phenotypes.

Diffuse proliferative GN was present in 55% of the mice in the (NZB x B6.Sle1c)F1 cohort at 12 mo of age, despite the fact that severe proteinuria was not identified. Acute diffuse proliferative GN without severe proteinuria is also common in 12-mo-old NZM2328 mice (17), suggesting that progression to chronic GN does not always occur in the first year of life. Interestingly, a recessive locus that contributed to development of acute GN in these mice was located on distal chromosome 1 (17), which is derived from the NZW strain in both NZM2328 and NZM2410 strains. Although subsequent studies have suggested that the genes that control acute GN, antinuclear Ab, and anti-dsDNA production are located at 182.5–184 Mb, centromeric to the Sle1c interval (18), our data demonstrate that the Sle1c locus does contain genetic elements that may also result in these phenotypes.

Forty-one percent of the (NZB x B6.Sle1c)F1 cohort had glomerular IgG3 deposits compared with 16% of the (NZB x B6)F1 cohort. Murine IgG3 Abs possess cryoglobulin activity due to the unique structure of the {gamma}3 constant region, which allows them to self-aggregate (19). IgG3 cryoglobulins can be highly nephritogenic, and although the mechanism by which they induce renal damage is not well understood, it is dependent on neutrophil infiltration and independent of complement activation (20). Consistent with this, IgG3 scores were positively correlated with C3 deposition in the (NZB x B6.Sle1c)F1 cohort (Spearman r = 0.5031, p = 0.0170), but not with proliferative GN (Spearman r = –0.1285, p = 0.5683). These data suggest that although the enhanced glomerular deposition of IgG3 in the (NZB x B6.Sle1c)F1 mice may be induced by genes within the Sle1c interval, it is not directly responsible for their increased glomerular inflammation.

We have recently shown that the Sle1c interval contains a minimum of three genes that could alter immune function (21) and potentially result in the phenotypes described here. Using congenic recombinants of the Sle1c interval, decreased humoral immune responses and impaired germinal center formation mapped to the NZW Cr2 allele, whereas B cell autoreactivity, T cell hyperactivity, and reduction in regulatory T cells mapped to two separate regions centromeric of Cr2. There are several potential candidate genes for lupus susceptibility in these latter two regions, including a cluster of four novel genes containing fibronectin type III domain fragments and the protein tyrosine phosphatase Ptpn14 (also known as PTP36). We have already identified Cr2 to be a strong candidate gene for lupus susceptibility in the Sle1c interval based on the presence of a single nucleotide polymorphism in the ligand binding domain of CD21, which introduces a novel N-linked glycosylation site and results in decreased binding of C3d ligands and impaired B cell signaling (10). Furthermore, we have found that the NZW complement receptor-related gene Y (Crry), which lies within 10 kb of Cr2 in the Sle1c interval and encodes a potent complement regulatory protein in mice (22), has alterations in its structure and function (S. N. Tchepeleva and S. A. Boackle, unpublished data). It is possible that several if not all of these genes interact to result in the autoimmune phenotypes that we describe here.

We have hypothesized that CD21 may be important in regulating autoimmune responses by lowering the threshold for induction of B cell tolerance, just as it lowers the threshold for B cell activation (16, 23, 24). In addition, it may be involved in the targeting of self-Ags to follicular dendritic cells in secondary lymphoid organs (25, 26), which may be required for the maintenance of peripheral B cell tolerance. In the studies we describe in this manuscript, alterations in either of these potential functions of CD21 could result in increased penetrance and amplitude of autoantibody production. To definitively prove the role of one or more of the Sle1c genes in lupus-like autoimmunity, however, it will be important to directly show their effects using recombinant congenics or, preferably, transgenic mice in which only one of the candidate NZW genes is expressed. These studies may also be relevant to human disease, because CD21, CD35, and DAF (decay accelerating factor; CD55), which is a human homologue for Crry (27), are all located within a 1.1-Mb interval at chromosome 1.q32 that has been linked and associated with lupus susceptibility in humans (28).

The gene or genes in the Sle1c interval are believed to be recessive based on linkage analyses (3). We sequenced both NZB Cr2 and Crry and found no alterations in their sequence compared with the B6 sequence for these genes. In addition, CD21/CD35 and Crry are expressed equivalently in both groups of mice studied (Fig. 8E and data not shown), suggesting that differential expression of these genes related to polymorphisms in regulatory domains does not explain our results. It is possible that heterozygosity of the NZW allele for these genes results in an intermediate phenotype that has a functional effect that is clinically relevant. Indeed, B cells from mice heterozygous for NZW Cr2 had impaired calcium responses to CD35/CD21-mediated signals (Fig. 8, C and D), consistent with this hypothesis.

In the studies we describe here, we found both increased titers of autoantibodies as well as increased immune complex deposition and glomerular damage in mice containing the NZW-derived Sle1c interval. Both anti-chromatin and anti-dsDNA Abs have been associated with the development of lupus GN, although their presence is not required (reviewed in Ref. 29). These data would suggest that at least two independent factors may be responsible for our results: one that results in increased autoantibody production and a second that predisposes to target organ involvement. By crossing transgenic or knock-in mice expressing the NZW alleles for Sle1c candidate genes with NZB mice, we can determine which, if any, of the NZB autoimmune phenotypes can be attributed to functional variants of these genes. Recombinant congenic Sle1c mice can be examined in a similar fashion. Because only a single cross is required, this strategy will provide a potent tool for the amplification of the effects of these genes and genetic intervals, enabling us to confirm and characterize their contributions to the pathogenesis of SLE.


   Acknowledgments
 
We thank Dr. Joshua Thurman for assistance in the analysis of renal function for these studies, and Daniel Perry for excellent technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   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 an Arthritis Foundation Arthritis Investigator Award (to S.A.B.) and National Institutes of Health Grants R0-1 AI52441 (to S.A.B.) and RO-1 AI045050 (to L.M.). The DNA samples were sequenced by the University of Colorado Cancer Center DNA Sequencing and Analysis Core, which is supported by the National Institutes of Health/National Cancer Institute Cancer Core Support Grant (P30 CA046934). Back

2 Address correspondence and reprint requests to Dr. Susan A. Boackle, Division of Rheumatology, University of Colorado at Denver and Health Sciences Center, Mail Stop B115, P.O. Box 6511, Aurora, Colorado 80045. E-mail address: susan.boackle{at}uchsc.edu Back

3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; BUN, blood urea nitrogen; CR, complement receptor; Crry, CR-related gene Y; GN, glomerulonephritis; NZB, New Zealand Black; NZW, New Zealand White; PAS, periodic acid-Schiff; sIg, surface Ig. Back

Received for publication January 25, 2006. Accepted for publication January 5, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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