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. 2013 Mar 27;15(2):R47.
doi: 10.1186/ar4206.

Germline deletion of β2 microglobulin or CD1d reduces anti-phospholipid antibody, but increases autoantibodies against non-phospholipid antigens in the NZB/W F1 model of lupus

Ram Raj Singh, Jun-Qi Yang, Peter J Kim, Ramesh C Halder

Germline deletion of β2 microglobulin or CD1d reduces anti-phospholipid antibody, but increases autoantibodies against non-phospholipid antigens in the NZB/W F1 model of lupus

Ram Raj Singh et al. Arthritis Res Ther. .

Abstract

Introduction: β2-microglobulin (β2m) is required for the surface expression of MHC class I and class I-like proteins such as CD1d, Qa1 and neonatal Fc receptor (FcRn), all of which may impact the development of autoimmunity. Since CD1d is known to bind and present phospholipid antigens to T cells, we asked if the deficiency of β2m or CD1d will impact the development of anti-phospholipid antibodies as compared to other aspects of lupus autoimmunity.

Methods: We introgressed the β2m-null genotype onto the NZB and NZW backgrounds for 12 to 14 generations to generate genetically lupus-susceptible (NZB/NZW)F1 (BWF1) mice that are β2m-deficient (β2m°). Circulating immunoglobulins (Ig), rheumatoid factor (RF), anti-DNA and anti-cardiolipin (anti-CL) antibodies, and renal disease were analyzed in these and CD1d-deficient (CD1d°) BWF1 mice that we had previously generated.

Results: Whereas β2m° BWF1 mice had reduced serum IgG, they had increased mortality, nephritis, serum IgG anti-DNA antibody and RF as compared to heterozygous and wild-type littermates. These effects were recapitulated in CD1d° BWF1 mice, except that they also had increased serum IgG as compared to control littermates. Intriguingly, both β2m° and CD1d° mice had lower serum anti-CL antibody levels than in control littermates. Such CD1d dependence of anti-CL antibody production is not mediated by CD1d/glycolipid-reactive iNKT cells, as these cells reduced the production of RF and anti-DNA antibodies but had no effect on anti-CL antibodies.

Conclusions: We report a novel dichotomous role of β2m and CD1d, whereby these molecules differently regulate autoimmunity against phospholipid versus non-phospholipid autoantigens.

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Figures

Figure 1
Figure 1
β2 microglobulin (β2m) deficiency reduces survival and accelerates lupus nephritis in BWF1 mice. We monitored 33 β2m-/-(β2m°) (18 female and 15 male, open circles), 66 β2m+/- (30 female and 36 male, triangles) and 31 β2m+/+ (18 female and 13 male, closed circles) littermates for the development of lupus. (a) Cumulative percent survival in β2m° BWF1 mice was compared with control β2m+/- and β2m+/+ littermate mice (*P <0.01 to 0.05, log rank test). (b) Cumulative percent mice with severe proteinuria (≥300 mg/dl protein on two consecutive occasions) was increased in β2m° mice compared to control littermates (*P = 0.02 to 0.05, Fisher's exact test). Differences were more pronounced (P <0.01) when β2m° mice were compared with all controls (β2m+/- plus β2m+/+) mice. (c) Renal histological changes are expressed as the mean ± standard error of the composite kidney biopsy index and its components, glomerular activity and chronicity scores. Results from a representative experiment are shown (*P <0.05, β2m° vs control groups; n = 6 to 8 male 10-month-old mice per group, Student's t-test). (d) Representative kidney sections from these mice show severe kidney lesions with fibrosis (note increased aniline blue staining) in the glomeruli (glomerulosclerosis, GS), periglomerular region (fibrous crescent, FC) and interstitium (IF), interstitial inflammation, and large segmental lesions (SL) in β2m° mice, whereas the β2m+ mice showed relatively less advanced lesions with increased glomerular cellularity (GC) and typical immune deposits (ID, see pink-colored deposits), but minimal or no GS or IF. PAS, periodic acid Schiff.
Figure 2
Figure 2
Serum immunoglobulin (Ig) levels in β2 microgloblin-deficient (β2m°) BWF1 mice: reduced polyclonal total IgG, but not IgM. β2m+/+ (n = 18 to 22 female and 12 male, closed circles), β2m+/- (n = 30 to 35 female and 28 to 35 male, triangles) and β2m° (n = 11 to 16 female and 10 to 16 male, open circles) mice were bled and sera tested for total IgG, IgG2a and IgM. Results are expressed as the mean ± standard error. (a, b) Serum levels of total IgG and IgG2a isotype were lower in β2m° mice than in control animals (*P <0.05, **P <0.01 to <0.001; Student's t-test). (c) Serum levels of total IgM were higher in β2m° than in β2m+/+ mice (*P <0.05, Student's t-test).
Figure 3
Figure 3
Serum anti-dsDNA autoantibodies and rheumatoid factor (RF) are increased in β2 microglobulin-deficient (β2m°) BWF1 mice. β2m+/+ (n = 18 female and 12 male, closed circles), β2m+/- (n = 30 female and 28 male, triangles), and β2m-/- (n = 11 female and 10 male, open circles) mice were bled and serum samples tested for IgG anti-dsDNA antibody and RF. (a) Results are expressed as the percent of mice with autoantibodies, using a cutoff level of the mean + 4 SD optical density (OD) value in sera from six normal age-matched BALB/c mice. Results pooled from male and female mice are shown (*P <0.05, **P <0.01; Fisher's exact test). (b) Results of autoantibodies are shown as the mean ± standard error of log U/ml in male and female mice separately (*P <0.01 to 0.05, **P <0.001, Student's t-test).
Figure 4
Figure 4
Serum total immunoglobulin G (IgG) and rheumatoid factor (RF) are increased in CD1d-deficient (CD1d°) BWF1 mice. CD1d° BWF1 mice and control littermates (CD1d+/+ or CD1d+/-, designated as CD1d+) were bled and serum samples tested for total IgG and RF levels. (a) Eight-month-old CD1d+ (n = 7) and CD1d° (n = 13) mice (*P = 0.04). (b) CD1d+ (n = 8) and CD1d° (n = 15) mice (*P <0.01 and **P = 0.03 to 0.06). The negative control mean ± SD value in six normal BALB/c mice was 28.2 ± 8.6 U/ml for RF. Results are expressed as the mean ± standard error of the values.
Figure 5
Figure 5
Serum anti-cardiolipin (anti-CL) antibodies are decreased in β2 microglobulin-deficient (β2m°) and CD1d-deficient (CD1d°) BWF1 mice. (a-c) β2m° or CD1d° BWF1 mice and control littermates (β2m+/+ or β2m+/-, designated as β2m+; and CD1d+/+ or CD1d+/-, designated as CD1d+) were bled and serum samples tested for serum immunoglobulin G (IgG) anti-CL antibodies: (a) 4-month-old β2m+ (n = 88) and β2m° (n = 32); (b) 6- to 8-month-old β2m+ (n = 75) and β2m° (n = 14); (c) 7- to 8-month-old CD1d+ (n = 57) and CD1d° (n = 26) mice. Each symbol represents a single mouse, and horizontal bars represent the mean U/ml for the group. The negative control mean ± SD values for anti-CL antibody in six normal BALB/c mice was 15.1 ± 5.2 U/ml. *P <0.01, Student's t-test.
Figure 6
Figure 6
Activated invariant natural killer T (iNKT) cells reduce anti-DNA antibodies, but not anti-cardiolipin (anti-CL) antibodies. (a) In vitro studies. Spleen cells harvested from 5-month-old BWF1 mice were cultured with αGalCer for 5 days, and supernatants tested for immunoglobulin G (IgG) anti-DNA and anti-CL antibodies, shown as the mean ± SD optical density (OD); *P <0.05 to 0.01, n = 5. Results represent two similar experiments. (b) In vivo studies. BALB/c SCID mice (4-month-old) were reconstituted with purified B-cells from iNKT cell-deficient Jα18° BALB/c mice, and injected with lipopolysaccharide and αGalCer, as described in Methods. These mice were then implanted with T-cells from Vα14Tg BALB/c mice or with T-cells from Jα18° BALB/c mice. Four days after the reconstitution, spleen cells were harvested and cultured without any further stimulation for 6 days. Culture supernatants were tested for IgG anti-DNA and anti-CL antibodies. Results represent two independent experiments, each using three mice per group. *P <0.05.
Figure 7
Figure 7
Distinct roles of different β2 microglobulin (β2m)-associated glycoproteins in the regulation of humoral autoimmunity. The findings in this report and previous studies suggest that β2m may affect lupus-like autoimmunity via at least six possible mechanisms: 1) neonatal Fc receptor (FcRn) effects on immunoglobulin G (IgG) catabolism [17,18] (Figure 2); 2) Qa-1-restricted CD8+ regulatory or suppressor T-cells that can suppress autoimmunity [39]; 3) major histocompatability complex (MHC) class I-restricted CD8+ Ti cells (inhibitory T-cells) that suppress autoantibody production via production of transforming growth factor β [5,28]; 4) MHC class I-restricted CD8+ cytotoxic T lymphocytes (CTL) that can ablate autoreactive B-cells [6]; 5) protective role of CD1d-restricted glycolipid (GL)-reactive invariant natural killer T (iNKT)-cells in autoimmunity [8,24,31,42,44] (Figures 4 and 6a); and 6) the ability of CD1d to bind phospholipid (PL) antigens to induce anti-phospholipid autoimmunity (Figure 5). RF, rheumatoid factor; APC, antigen presenting cell.
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