Abstract

Methods

Statistics

Proteinuria and survival data for the entire group are shown in Figure 1 and were analyzed using Kaplan-Meier curves and log-rank test. Data in bar graphs are shown as mean + 1 SD. Comparisons in Figure 2 and Table 1 were performed using Mann-Whitney test. p values ≤ 0.05 were considered significant.

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Figure 1

Panel A shows accelerated proteinuria onset and death comparable to male mice in the IFNα group vs. 22w controls (p < 0.0001 treated vs. control). Kidneys of IFNα treated mice had more intense IgG deposits in the glomeruli (1B, green) and more infiltrates of F4/80+ macrophages (1B, red) and CD11c+ dendritic cells (1D, red) than control 22w mice (1C, 1E). Images are representative of 5 mice examined per group. 17-22w IFNα treated mice had significantly more glomerular (p<0.001) and interstitial (p<0.01) damage than the control group (1F). Cardiac damage occurred in a few IFNα treated mice (1G, p < 0.09). Thrombocytopenia was not found in the IFNα treated mice (1H).

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Figure 2

IFNα treated mice had higher titers of autoantibodies than age matched untreated controls (2A, 2B). ELISpot analysis of spleens showed an increase in the frequency (2C) and total number (2D) of IgG and IgG anti-cardiolipin producing B cells in IFNα treated mice compared with 22w controls. Graphs show mean +/- 1 SD. Spleens of male mice stained with PNA (green) and IgD (red) showed very large germinal centers at 14w with disorganization and loss of follicular architecture and germinal centers by 17w when nephritis develops (2E and 2F respectively). Germinal centers appeared earlier in the spleens of 17w female IFNα mice (2G) than in 17w untreated controls (2H). Spleens of 22w IFNα treated mice (2I) became disorganized compared with 22w controls (2J), similar to those of sick males (2F).

Table 1

Spleen cell phenotypes

Cell number per spleen Percent cells per spleen	Control 17w	IFNα 17w	Control 22w	IFNα 22w
Cell count × 107	5.9 ± 1.9	26.7 ± 12.3†	9.6 ± 1.3	24.7 ± 8.8†
CD4 T cells × 107	1.4 ± 0.4	6.3 ± 3.7†	2.0 ± 0.6	8.5 ± 1.8†
Activated × 107	0.2 ± 0.1	2.0 ± 1.5§	0.3 ± 0.1	2.6 ± 1.2†
Activated (%)	11.6 ± 1.9	31.8 ± 9.0§	13.9 ± 4.2	30.2 ± 12.2§
CD8 T cells × 107	0.8 ± 0.2	1.0 ± 0.5	1.1 ± 0.2	1.5 ± 1.1
B cells × 107	3.1 ± 1.1	10.7 ± 3.8†	4.4 ± 1.0	11.4 ± 6.2†
Activated × 107	0.2 ± 0.1	1.2 ± 1.1§	0.2 ± 0.1	1.3 ± 1.0†
Activated (%)	8.1 ± 3.4	11.2 ± 6.5	5.6 ± 1.6	10.9 ± 3.0†
Marginal zone	0.4 ± 0.1	1.8 ± 1.1†	0.5 ± 0.1	1.0 ± 0.7‡
Follicular × 107	2.3 ± 0.9	7.6 ± 0.2‡	2.9 ± 0.7	6.8 ± 3.6†
Isotype switched × 106	0.2 ± 0.1	1.0 ± 0.6‡	0.2 ± 0.1	1.1 ± 0.5†
Isotype switched (%)	4.9 ± 2.2	8.7 ± 4.0	3.6 ± 1.5	9.8 ± 3.1†
Plasma cells	0.01 ± 0.01	0.3 ± 0.3†	0.04 ± 0.02	0.5 ± 0.7§
Plasma cells (%)	0.3 ± 0.1	2.7 ± 1.3‡	0.9 ± 0.4	5.4 ± 2.1§
CD11b × 106	0.2 ± 0.1†	0.7 ± 0.2	0.3 ± 0.1	0.9 ± 0.5§

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Control 17w compared with INFα 17w and Control 22w compared with IFNα 22w

^‡p< 0.05;

^§p<0.02;

^†p<0.002

Comparable data for males can be found in (11)

Results

Administration of a small dose of Ad-IFNα markedly accelerated disease onset in female NZW/BXSB mice and proteinuria occurred 4-12 weeks after virus injection, followed rapidly by death (IFNα vs. controls p<0.0001 for proteinuria and death - Figure 1A). Serum IFNα levels were below detectable levels in treated mice. However two weeks after virus injection there was a significant increase in the expression of several IFN inducible genes in the spleens of treated mice to levels comparable with those of proteinuric male mice (data not shown). Imiquimod had no effect on autoantibody production, proteinuria onset or mortality and these mice were not studied further.

The kidneys of IFNα treated mice contained intense IgG deposits. Infiltrating F4/80 positive macrophages were located in the interstitium and around glomeruli, whereas CD11c positive dendritic cells were within the glomerular tuft (Figure 1B, 1D). This pattern is similar to that observed in male NZW/BXSB mice (11). In contrast, few glomerular or interstitial infitrates were present in 22 week control mice (Figure 1C, 1E). 17-22w IFNα treated mice had significant renal damage compared with untreated controls (Figure 1F - p<0.001 for glomerular score and p<0.01 for interstitial score). Mild cardiac damage was also observed in the IFNα treated mice but not in the untreated controls (Figure 1G). In contrast, thrombocytopenia was not found (Figure 1H), despite the presence of high titers of anti-phospholipid autoantibodies in the serum.

Serum IgG2a levels increased significantly in the IFNα treated mice compared with controls (1036.2 +/- 452.9 vs. 433.4 +/- 258.9 ug/ml; p < 0.0072 at 12w). The difference was no longer significant at 17w. In contrast, serum IgG1 levels were significantly lower in the IFNα group than in the controls at both 12 and 17w (59.8 +/- 31.5 vs. 142.3 +/-71.5 ug/ml; p< 0.001 and 116.1 +/- 91.0 vs. 209.6 +/- 95.5ug/ml; p < 0.02 respectively). Autoantibodies to both cardiolipin (p < 0.004 at 14w and p < 0.03 at 18-20w) and Sm/RNP (p < 0.02 at 12w, p < 0.04 at 16w) arose earlier in the IFNα treated mice than in age matched untreated controls; the maximal autoantibody titers reached were comparable to male mice (Figure 2A, 2B). Low titer anti-dsDNA antibodies arose late in both treated and untreated mice (not shown). In accordance with these data, ELISpot analysis of spleens revealed an increase in both the frequency (p < 0.04 at 17w and p < 0.0001 at 22w) and total number of IgG (p < 0.003 at 17w and p < 0.0001 at 22w) and IgG anti-cardiolipin antibody producing B cells (p < 0.0001 at 22w) in IFNα treated mice compared with controls (Figure 2C, 2D). Anti-cardiolipin producing B cells also increased in the bone marrows (3.93 +/-.99 per 10⁵ cells in IFNα treated vs. 1.37 +/- 0.87 in controls at 17w; p < 0.02; 9.93 +/-4.48 in IFNα treated vs.3.52 +/- 2.92 in controls at 22w; p < 0.03)

Phenotypic analysis of spleen cells revealed a marked increase in spleen cell number in the treated mice compared with controls, with a significant increase in CD11b positive cells and activated CD4 T cells and B cells (Table 1). Circulating monocytes in the blood were also twofold increased (not shown). All B cell subsets were expanded in the treated mice. This is in contrast to male NZW/BXSB mice in which the overexpression of TLR7 results in loss of the marginal zone subset and a marked increase in the follicular to marginal zone B cell ratio (Kahn and Davidson, unpublished). There was also a significant increase in the number of class switched B cells and plasma cells in the spleens of IFNα treated mice (Table 1). Germinal centers appeared earlier in the spleens of IFNα treated mice compared with untreated controls, but the spleens became disorganized with loss of follicular architecture and germinal centers as the mice aged, similar to males (Figure 2E-J).

Discussion

The association of the IFN signature with SLE in humans and the ability to induce accelerated SLE in some murine SLE models with IFNα has led to the hypothesis that IFNα is an important pathogenic cytokine in SLE and a target for therapy (3). IFNα accelerates SLE in female NZB/W mice in which it induces early development of anti-dsDNA antibodies and nephritis (2). We have shown in NZB/W mice that IFNα rapidly induces germinal centers that generate short-lived plasma cells producing pathogenic IgG2a autoantibodies in a T cell-dependent fashion, whereas pathogenic IgG3 autoantibodies are generated in a T cell-independent fashion (Liu, submitted). In other mouse models however, Type I interferons have protective effects with respect to SLE initiation, indicating strain heterogeneity (12).

NZW/BXSB mice develop autoantibodies to cardiolipin and to RNA-associated antigens. Together with immune mediated thrombocytopenia, inflammatory glomerulonephritis and a thrombotic vasculopathy that affects the small coronary arteries leading to myocardial infarcts, myocardial fibrosis and a dilated cardiomyopathy (8, 9, 13). Male mice have markedly accelerated disease because they carry the Yaa gene, a reduplication of a portion of the X chromosome that includes the TLR7 gene (7). TLR7 recognizes ssRNA, is required for the production of autoantibodies to RNA associated autoantigens (14), and appears to be responsible for much of the phenotype associated with the Yaa locus (4).

Ligation of TLR7 on plasmacytoid dendritic cells is a potent stimulus for secretion of IFNα as well as other cytokines including TNFα, IL-12 and IL-6 (14). We therefore wished to know whether the accelerating effects of TLR7 overexpression could be mimicked by administration of IFNα. We first showed that the TLR7 agonist Imiquimod did not accelerate disease in female NZW/BXSB mice, consistent with the finding that optimal TLR7 agonism requires the presence of IFNα (15). In contrast, IFNα accelerated the onset of anti-cardiolipin and anti-Sm/RNP autoantibodies and caused early mortality due to nephritis. However, the IFNα treated female mice developed only mild cardiac disease and they did not become thrombocytopenic. Like male NZW/BXSB mice, the IFNα treated females had marked splenomegaly, an increase in activated B and T cells and an increase in plasma cells; however they did not lose their marginal zone B cells. Thus the loss of marginal zone B cells in male NZW/BXSB mice and in female TLR7-overexpressing mice (4) is not mediated through IFNα and may be due to a TLR7 mediated intrinsic defect in B cell selection. The differences in B cell selection between male and female mice may be one reason for the failure to develop anti-platelet antibodies and the milder anti-phospholipid syndrome in the treated females. Alternatively, TLR7 ligation induces many cytokines in addition to IFNα that may cooperatively contribute to disease phenotype and severity. Finally it is possible that other genes in the Yaa locus could contribute to a more severe disease phenotype in males.

The accelerating effects of IFNα are clearly different between NZB/W and NZW/BXSB mice. In both strains IFNα recapitulates the autoantibody profile of the spontaneous disease (2) with early onset of B and T cell activation. In NZB/W mice IFNα induces abundant germinal centers, large numbers of short-lived plasma cells in the spleens with lack of expansion of these cells in the bone marrow, and high serum levels of IgG2a and IgG3 that deposit in the kidneys (Liu, submitted). In NZW/BXSB mice it accelerates the development of large germinal centers followed by splenic disorganization, similar to what is seen in males, an increase in anti-cardiolipin producing B cells in the bone marrow and an increase only in serum levels of IgG2a.

These data show in sum that the effects of IFNα do not fully recapitulate the B cell or disease phenotype associated with TLR7 overexpression and that its effects on disease phenotype are strain dependent. Further characterization of effects of excess IFNα in different lupus-prone mice models and on responses of these mice to therapy will increase our understanding of the physiologic significance of the interferon signature in humans.

Acknowledgments

References