Type-I interferon receptor deficiency reduces lupus-like disease in NZB mice

Abstract

Indirect evidence suggests that type-I interferons (IFN-alpha/beta) play a significant role in the pathogenesis of lupus. To directly examine the contribution of these pleiotropic molecules, we created congenic NZB mice lacking the alpha-chain of IFN-alpha/betaR, the common receptor for the multiple IFN-alpha/beta species. Compared with littermate controls, homozygous IFN-alpha/betaR-deleted NZB mice had significantly reduced anti-erythrocyte autoantibodies, erythroblastosis, hemolytic anemia, anti-DNA autoantibodies, kidney disease, and mortality. These reductions were intermediate in the heterozygous-deleted mice. The disease-ameliorating effects were accompanied by reductions in splenomegaly and in several immune cell subsets, including B-1 cells, the major producers of anti-erythrocyte autoantibodies. Decreases of B and T cell proliferation in vitro and in vivo, and of dendritic cell maturation and T cell stimulatory activity in vitro were also detected. Absence of signaling through the IFN-alpha/betaR, however, did not affect increased basal levels of the IFN-responsive p202 phosphoprotein, encoded by a polymorphic variant of the Ifi202 gene associated with the Nba2 predisposing locus in NZB mice. The data indicate that type-I IFNs are important mediators in the pathogenesis of murine lupus, and that reducing their activity in the human counterpart may be beneficial.

Figure 1.

Serum concentrations of IFN-α induced by poly (I:C). WT NZB (n = 3) and control BALB/c (n = 4) mice were bled before and, at different time points, after intraperitoneal injection of poly (I:C) and IFN-α levels were determined by ELISA.

Figure 2.

Mortality rates and hemolytic anemia. (a) Cumulative survival of IFN-α/βR KO (n = 11), HT (n = 9), and WT (n = 9) NZB mice followed up to 12 mo; P = 0.015 for KO vs. WT; P > 0.05 for HT vs. WT. (b) Cumulative incidence of direct Coomb's test. Mouse groups were the same as in panel a. HT mice were examined at 12 mo only. (c) FACS^® analysis of RBC-bound IgG autoantibodies in 10–12-mo-old mice. Each point represents the mean fluorescence intensity unit (FIU) from a single mouse and horizontal lines indicate the mean FIU; P = 0.004 for KO vs. WT; P = 0.04 for HT vs. WT mice. (d) Frequency of erythroblasts in spleens of 12-mo-old mice. RBC-depleted splenocyte suspensions were stained with an anti-erythroblast Ab (TER119) and analyzed by FACS^®. Results of a representative mouse from each group are depicted. Numbers indicate percent of erythroblasts (mean ± SE) of 3–4 mice/group; P = 0.02 for KO or HT vs. WT mice; P = 0.05 for KO vs. HT.

Figure 3.

Kidney immunohistology. Representative sections from 12-mo-old IFN-α/βR WT and KO NZB mice (n = 4 to 10) stained with PAS or FITC-conjugated anti–mouse IgG are shown.

Figure 4.

Serum concentrations of (a) polyclonal Ig and (b) anti-ss and dsDNA autoantibodies. BALB/c mice (a pool of 3 individuals) and IFN-α/βR WT (n = 8), or KO (n = 7) NZB mice were analyzed at 10–12 mo. Bars indicate mean ± SE; *P < 0.05.

Figure 5.

Frequency of splenic polyclonal Ig and anti-DNA AFCs. ELISPOT results for total IgM, total IgG, and corresponding anti-dsDNA or anti-ssDNA AFC in 12-mo-old BALB/c and IFN-α/βR WT or KO NZB mice are depicted. Data result from two separate experiments and each dot represents a single mouse. In all measurements, P < 0.0001 for WT vs. KO or BALB/c mice.

Figure 6.

Frequency of peritoneal B-1 cells. Representative density plots of total peritoneal lymphocytes are shown, with numbers indicating percentages (mean ± SE) of gated B220⁺CD5⁺ cells in 12-mo-old IFN-α/βR WT and KO NZB mice (n = 3–6 mice/group); P = 0.01.

Figure 7.

In vivo lymphocyte proliferation. (a) Spontaneous proliferation of splenic B and T cell subsets in 12 mo-old BALB/c, IFN-α/βR WT, and KO mice (n = 2–3/group) defined by BrdU-incorporation. Representative histograms and percentage of BrdU^hi cells (mean ± SE) are shown. (b) Lymphopenia-induced homeostatic T cell proliferation. LN cells from 2–3 mo-old WT and KO mice were labeled with CFSE and infused into sublethally-irradiated WT NZB recipients (n = 3/group). Representative histograms at 8 d post-transfer and percentage (mean ± SE) of cells that had undergone 1 to 7 divisions are shown, with peaks of decreasing CFSE-intensity identifying sequential cellular divisions. Reduced in vivo proliferation of KO T cells was also observed 5 and 14 d after transfer (data not depicted).

Figure 7.

In vivo lymphocyte proliferation. (a) Spontaneous proliferation of splenic B and T cell subsets in 12 mo-old BALB/c, IFN-α/βR WT, and KO mice (n = 2–3/group) defined by BrdU-incorporation. Representative histograms and percentage of BrdU^hi cells (mean ± SE) are shown. (b) Lymphopenia-induced homeostatic T cell proliferation. LN cells from 2–3 mo-old WT and KO mice were labeled with CFSE and infused into sublethally-irradiated WT NZB recipients (n = 3/group). Representative histograms at 8 d post-transfer and percentage (mean ± SE) of cells that had undergone 1 to 7 divisions are shown, with peaks of decreasing CFSE-intensity identifying sequential cellular divisions. Reduced in vivo proliferation of KO T cells was also observed 5 and 14 d after transfer (data not depicted).

Figure 8.

Maturation and function of DCs. (a) Effect of IFN-α on DC maturation. BM cells from IFN-α/βR WT and KO NZB mice were incubated with GM-CSF and IL-4 for 6 d, then with or without IFN-α for another 24 h to induce final maturation. The frequency of cells that had up-regulated MHC and B7 costimulatory molecules was determined by FACS^® on gated live, CD11c⁺ cells. Basal levels (horizontal lines) for MHC class II, B7.1, and B7.2 were set on the basis of parallel stainings in the absence of the specific antibodies, and for MHC class I on the basis of constitutive expression defined with specific antibody. Percentages are the mean ± SE of three separate experiments with pooled cells from 3 mo-old mice (n = 2–4/pool); *P = 0.05. (b) DCs (3 × 10⁴) from IFN-α/βR WT, KO, and BALB/c mice, derived as described in panel a using either medium, IFN-α or LPS, were incubated with allogenic T cells (10⁵) for 3 d and proliferative responses determined by H³-thymidine incorporation. Patterns of allostimulation remained similar when lower numbers (10⁴) of DCs were used (data not depicted). Results are the mean ± SE of three separate experiments; *P < 0.05.

Figure 9.

Expression of Ifi202-encoded p202. Protein extracts from spleens of BALB/c, IFN-α/βR WT, and KO NZB mice (n = 3) were analyzed by immunoblotting using an anti-p202 polyclonal antiserum. Representative results are shown. Expression levels (mean ± SE) of p202, calculated as percent of actin expression, were 13.6 ± 3.4% for WT mice, 12.2 ± 1.7% for KO mice, and 0.7 ± 0.2% for BALB/c mice. PC, positive control (recombinant p202).