Breaking tolerance to double stranded DNA, nucleosome, and other nuclear antigens is not required for the pathogenesis of lupus glomerulonephritis - PubMed (original) (raw)

Breaking tolerance to double stranded DNA, nucleosome, and other nuclear antigens is not required for the pathogenesis of lupus glomerulonephritis

Samuel T Waters et al. J Exp Med. 2004.

Abstract

In lupus-prone NZM2328 mice, a locus Cgnz1 on chromosome 1 was linked to chronic glomerulonephritis, severe proteinuria, and early mortality in females. A locus Adnz1 on chromosome 4 was linked to antinuclear antibody (ANA) and anti-double stranded DNA (dsDNA) antibody (Ab) production. In this investigation, two congenic strains, NZM2328.C57L/Jc1 (NZM.C57Lc1) and NZM2328.C57L/Jc4 (NZM.C57Lc4), were generated by replacing the respective genetic intervals containing either Cgnz1 or Adnz1 with those from C57L/J, a nonlupus-prone strain. The NZM.C57Lc1 females had markedly reduced incidence of chronic glomerulonephritis and severe proteinuria. NZM.C57Lc4 females had chronic glomerulonephritis and severe proteinuria without circulating ANA, anti-dsDNA, and antinucleosome Ab. These data confirm the linkage analysis. Unexpectedly, NZM.C57Lc1 females had little anti-dsDNA and related Ab, suggesting the presence of a second locus Adnz2 on chromosome 1. The diseased NZM.C57Lc4 kidneys had immune complexes by immunofluorescence and electron microscopy. The eluates from these kidneys did not contain ANA, anti-dsDNA, and antinucleosome Ab, indicative of the presence of non-anti-dsDNA nephritogenic Ab. Thus, breaking tolerance to dsDNA and chromatin is not required for the pathogenesis of lupus nephritis. These results reaffirm that anti-dsDNA and related Ab production and chronic glomerulonephritis are under independent genetic control. These findings have significant implications in the pathogenesis of systemic lupus erythematosus.

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Figures

Figure 1.

Figure 1.

NZM.C57Lc1 and NZM.C57Lc4 congenic lines were derived by replacing the genetic intervals in NZM2328 with those from C57L/J (hatched bars). The genetic intervals with SLE susceptibility genes in NZM2328 delineated by informative microsatellite markers are shown (open bars). Chromosome intervals are not drawn to scale.

Figure 2.

Figure 2.

Development of severe proteinuria in females of NZM2328 and NZM.C57Lc4 but not in those of NZM.C57Lc1. (A) Incidence of severe proteinuria in each strain of mice. (B) Kinetics of proteinuria development.

Figure 3.

Figure 3.

Histological, immunofluorescence, and EM studies of representative kidneys from NZM.C57Lc1 and NZM.C57Lc4 female mice. (A) Normal glomeruli (hematoxylin and eosin staining, ×200) are seen in NZM.C57Lc1. (B) In contrast, in the NZM.C57Lc4 congenic, enlarged glomeruli with mesangial proliferation, hypercellularity, obliterated capillary loops, and glomerulosclerosis are evident. (C) Immunofluorescence studies show some mesangial IgG deposits in NZM.C57Lc1, similar to the pattern seen in aged C57L/J. (D) A coarsely granular staining pattern of IgG deposits in both the mesangia and peripheral capillary walls of the glomeruli of NZM.C57Lc4. (E) Staining of the Bowman capsule and mesangia with anti-C3 Ab are seen in NZM.C57Lc1. (F) Coarsely granular staining by anti-C3 Ab throughout the glomeruli is seen in NZM.C57Lc4. (G) EM study shows normal glomeruli without electron-dense deposits in the subepithelial or subendothelial spaces (×10,000) in the kidney of NZM.C57Lc1. (H) In comparison, electron-dense deposits in both subendothelial space (arrow) and the mesangia (arrowheads) in the glomeruli of NZM.C57Lc4 are readily detected.

Figure 4.

Figure 4.

Marked reduction of circulating anti-dsDNA, antinuclear, and antinucleosome Ab in NZM.C57Lc1 and NZM.C57Lc4 congenic lines in comparison with NZM2328. Staining of HeLa cell nuclei by DAPI are seen in A, C, and E. The right side of the figure shows the presence of ANA in the serum of NZM2328 (B) but not in the sera of NZM.C57Lc1 (D) and NZM.C57Lc4 (F). Although not shown, the majority of the sera from C57L/J were not positive for ANA. On the bottom, frequencies of the presence of anti-dsDNA, antinuclear, and antihistone/DNA Ab in these strains are summarized.

Figure 5.

Figure 5.

Lack of RF in sera of NZM2328 and its congenics. Human IgG was used as the substrate in A and C to detect anti-IgG activities. Mouse sera were used at a dilution of 1:50 for A and 1:250 for C. Rabbit IgG was used as the substrate in B and D with mouse sera diluted at 1:50 and 1:250, respectively. A pool of MRL/_lpr+/+_sera was used as a positive control showing readily detectable RF in this strain of mice.

Figure 6.

Figure 6.

Western blot analysis to detect autoantibodies to cellular constituents in sera of NZM2328 and its congenics. Cell lysate of WEHI 7.1 lymphoma cell line was used as the substrate with sera at the dilution of 1:50. On the top, sera from mice at 4–5 mo of age were used with MRL/lpr+/+(MRL) pooled sera as the positive control. Lanes 1–8, NZM.C57Lc1; lanes 9–15, NZM.C57Lc4; lanes 16–22, NZM2328. On the bottom, sera at death or at death at the age of 12 mo were used. Lanes 1–6, NZM.C57Lc1; lanes 7–13, NZM.C57Lc4; lanes 14–20, NZM2328; lanes 21–24, C57L/J.

Figure 7.

Figure 7.

Anti-dsDNA Ab in sera and kidney eluates from NZM2328, NZM.C57Lc1, NZM.C57Lc4, and C57L/J females at 11–12 mo. Abs to dsDNA were present in sera of NZM2328 and they were enriched in their kidney eluates. These Abs were rarely detected in the sera and the kidney eluates of the other three strains. P-values indicate significant differences of the respective strain as compared with NZM2328.

Figure 8.

Figure 8.

Abs eluted from nephritic kidneys of NZM2328 and NZM.C57Lc4 mice are reactive with proteins within the kidney and liver extracts. Proteins in the kidney (left) and liver (right) extracts were separated on a 12% SDS-PAGE, transferred to nitrocellulose paper, and used for analyzing the reactivity of Abs eluted from the nephritic kidneys of NZM2328 (lanes 2 and 3) and NZM.C57Lc4 (lanes 4 and 5) mice. Each lane is equivalent to 60 μg total protein. Abs were used at a concentration of 30 (lanes 2 and 4) and 10 μg/ml (lanes 3 and 5). Lane 1 in both panels represents reactivity of the goat anti–mouse IgG–horseradish peroxidase conjugate with the extracts. Numbers on the left represent molecular weights in kilodaltons.

Figure 9.

Figure 9.

Interactive model for the pathogenesis of SLE. Pathway I, autoantibody production and activation of effector T cells and pathway II, activation of susceptibility genes and end organ damage, can be initiated independently while they interact at different levels as indicated by pathways III and IV. The interactions between these pathways lead to end organ damage.

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