Involvement of a tissue-specific autoantibody in skin disorders of murine systemic lupus erythematosus and autoinflammatory diseases

Abstract

Human systemic lupus erythematosus (SLE) and its murine model, MRL lpr/lpr mice, are well known to develop a wide range of symptoms, such as glomerulonephritis, dermatitis, and arthritis, as an immune-complex disease. However, the deposition of circulating immune complex does not fully explain the tissue specificity of disease. Tissue-specific autoantigens may also be involved in tissue inflammation. In this study, desmoglein 3 (Dsg3), a major component of epidermal desmosomes, was identified as a skin-specific autoantigen. Several murine models of skin inflammation were found to develop autoantibodies to Dsg3 tightly correlated with disease aggravation, especially in MRL lpr/lpr mice. Furthermore, SLE-prone skin disease-free FcgammaRIIb-deficient mice developed skin inflammation upon immunization with Dsg3. Taken together with histological studies, we concluded that skin-specific Dsg3 serves as an autoantigen in chronic skin inflammatory diseases accompanied by mast cell degranulation, including both murine SLE and other autoinflammatory diseases.

Fig. 1.

Dsg3 detected with sera from MRL lpr/lpr mice. Whole-skin extract (100 μg) from wild type (+/+) and Dsg3-deficient (−/−) mice were subjected to Western blotting with the indicated sera: Seven sera from 5- to 6-month-old MRL lpr/lpr (MRL lpr) and two from 6-month-old C57BL/6 mice were tested. Representative pictures are shown. MRL lpr(1) and MRL lpr(2) are sera from different individuals. D indicates a lane with 100 ng of mouse rDsg3 (open arrowhead). Filled arrowheads indicate the size of a protein recognized by sera from five of seven MRL lpr/lpr mice. Filled dots indicate IgH recognized by goat anti-mouse IgG (second antibody, data not shown). The numbers on the left indicate molecular sizes in kDa. All of the whole-extract-transferred membranes were checked by ponceau S staining to ensure equivalent loading of skin extracts.

Fig. 2.

Time course of appearance of anti-Dsg3, anti-dsDNA, total isotypes, and skin inflammation. (A) Sera from MRL lpr/lpr (Left) (n = 13) and C57BL/6 (Center) (n = 7) at the indicated age were examined by ELISA for relative intensity (%) of anti-Dsg3. All mice were observed from 2–3 months to 12 months of age. Values from each individual mouse are connected with lines that are discontinued at the death of that mouse. (Right) Sera from several murine models of skin inflammation were tested: strain 1, six fsn at the age of 14 weeks; strain 2, six BALB/By at 14 weeks; strain 3, two A/J at 14 weeks; strain 4, four me^v at 8 weeks; strain 5, four scurfy at 4 weeks; and strain 6, four C57BL/6 at 8 weeks. Relative intensity (%) was determined by the following formula: (values from sera)/(a value obtained by binding of HRP-conjugated anti-E tag to coated E-tagged rDsg3) × 100. In some cases, values for individual mice overlapped. (B) Isotypes of anti-Dsg3 (Left), anti-dsDNA (Center), and total amount of isotypes (Right) were determined with sera from seven skin-diseased MRL lpr/lpr mice (Upper) and one non-skin-diseased mice (Lower) at the indicated months of age. The values are expressed as means ± SEM in Upper. (C) The logarithms of the ratios of two isotypes of anti-Dsg3 or anti-dsDNA and total isotype amount in B were calculated to examine the correlation and specificity between the logarithmic values and skin inflammation. Of 10 combinatorial profiles, 3 are shown and the remaining 7 are not shown. In the logarithm of anti-Dsg3 IgA/IgG3, a sharp peak was present at the age of 5 months, and a cluster of these values had little overlap with those of anti-dsDNA or total isotypes, whereas the other logarithmic profiles had significant overlaps. Values from each individual mouse are connected with lines. Arrows in Top indicate the values from the single non-skin-diseased MRL lpr/lpr mouse; arrows are not shown in Middle and Bottom, where these values were buried in the clusters of values.

Fig. 3.

Macroscopic, histological, and immunohistological analysis of skin inflammation in C57BL/6-FcγRIIb-deficient mice immunized with rDsg3. (A1 and A2) FcγRIIb-deficient mice immunized with rDsg3 developed tiny erosions on the paw (A1), which generally expanded into scab formation on the entire paw (A2). (A3) Some mice developed tiny vesicles, pustules, and erosion on the tail. (A4 and A5) Erosions also occurred in the skin on the mammary gland (A4), and a solitary ulcer occurred on the back (A5). (B1–B12) Skin tissues were from C57BL/6 mice (B1 and B4) and C57BL/6-FcγRIIB-deficient mice (B2, B3, and B5–B12) immunized with human rDsg3. Representative stainings are shown from tails (B1–B3) or back skin (B4–B12). (B1–B7) Hematoxylin/eosin staining. (B8 and B9) Toluidine blue and methylene green staining. (B10–B12) Sections were stained with mouse anti-CD3 (B10), anti-B220 (B11), and anti-F4/80 (B12) (brown-stained cells), followed by counterstaining with hematoxylin. (Magnifications: B1, B2, B4–B6, and B10–B12, ×80; B3, B7, and B8, ×160; and B9, ×504.)