B-lymphocyte depletion reduces skin fibrosis and autoimmunity in the tight-skin mouse model for systemic sclerosis

Abstract

Systemic sclerosis (scleroderma) is an autoimmune disease characterized by excessive extracellular matrix deposition in the skin. A direct role for B lymphocytes in disease development or progression has remained controversial, although autoantibody production is a feature of this disease. To address this issue, skin sclerosis and autoimmunity were assessed in tight-skin mice, a genetic model of human systemic sclerosis, after circulating and tissue B-cell depletion using an anti-mouse CD20 monoclonal antibody before (day 3 after birth) and after disease development (day 56). CD20 monoclonal antibody treatment (10 to 20 microg) depleted the majority (85 to 99%) of circulating and tissue B cells in newborn and adult tight-skin mice by days 56 and 112, respectively. B-cell depletion in newborn tight-skin mice significantly suppressed (approximately 43%) the development of skin fibrosis, autoantibody production, and hypergammaglobulinemia. B-cell depletion also restored a more normal balance between Th1 and Th2 cytokine mRNA expression in the skin. By contrast, B-cell depletion did not affect skin fibrosis, hypergammaglobulinemia, and autoantibody levels in adult mice with established disease. Thereby, B-cell depletion during disease onset suppressed skin fibrosis, indicating that B cells contribute to the initiation of systemic sclerosis pathogenesis in tight-skin mice but are not required for disease maintenance.

Figure 1

Equivalent B-cell depletion after intravenous, subcutaneous, or intraperitoneal CD20 mAb treatments. A: Two-month-old wild-type mice (two to three mice per dose) were given CD20 mAb at the dose indicated or were treated with an isotype-matched control (CTL, 250 μg) mAb. Seven days after mAb administration, tissue B-cell (B220⁺) numbers were quantified after immunofluorescence staining with flow cytometry analysis. Bone marrow mature B-cell (IgM⁺B220^hi) numbers were from bilateral femurs. Blood B cells were B220⁺ cells/ml. Lymph node B cells were from bilateral inguinal and axillary node pairs. B: Equivalent B-cell depletion in Tsk^/+ and wild-type littermates after CD20 mAb treatment. Representative blood, spleen, and peripheral lymph node B-cell frequencies after CD20 or isotype-matched control (CTL) mAb treatment as determined by immunofluorescence staining with flow cytometry analysis. Mice were treated subcutaneously with mAb (20 μg) on day 3 after birth, with repeated treatment on days 17, 31, and 45. Numbers indicate the percentage of gated B220⁺CD19⁺ B cells present in 56-day-old mice. C: Duration of B-cell depletion in 2-month-old wild-type (closed circles and bars) and CD20^−/− (open circles and bars) littermates (five mice per group) given 5 μg of CD20 mAb intravenously on day 0. Tissue B-cell (B220⁺) numbers were quantified on the days indicated as in A. Significant differences between sample means are indicated: *P < 0.05; **P < 0.01. D: Representative CD20 expression by blood B, T, and NK cells from 2-month-old wild-type and CD20-deficient (CD20^−/−) mice (≥3 mice). B220⁺ B cells, CD4⁺ T cells, CD8⁺ T cells, and NK (NK1.1⁺CD3⁻) cells were assessed by two- or three-color immunofluorescence staining using the MB20-18 anti-mouse CD20 mAb (thick line) with flow cytometry analysis. Data are shown as representative dot plots (B220⁺ cells) and histograms (B, T, and NK cells). Isotype-matched control mAb staining (dashed line) or results from leukocytes of CD20^−/− mice stained with MB20-18 mAb (thin line) are shown as negative controls.

Figure 2

Fibrosis of dorsal skin from 56-day-old Tsk^/+ and wild-type littermates treated with CD20 or isotype-matched control (CTL) mAb. Mice were given mAb (20 μg) subcutaneously on day 3 after birth, with repeated treatment on days 17, 31, and 45. Representative H&E-stained histological sections. Dermis is indicated by (d), with the loose connective tissue layer (ie, the hypodermis or superficial fascia) beneath the panniculus carnosus (arrow) indicated by (h). Results represent those obtained with ≥5 mice of each group. Original magnifications, ×40.

Figure 3

Fibrosis of dorsal skin from Tsk^/+ and wild-type littermates treated with CD20 or isotype-matched control (CTL) mAb. A: Dermal and hypodermal thickness and hydroxyproline content of 6-mm skin punch biopsies from mice given mAb subcutaneously on day 3 after birth, with repeated treatment on days 17, 31, and 45 with tissues harvested on day 56 as described in Figure 2. B: Littermates were given mAb subcutaneously on days 14, 28, 42, and 56 after birth, with 6-mm skin punch biopsies harvested on day 70. C: Littermates were given mAb subcutaneously on days 28, 42, 56, and 70 after birth, with 6-mm skin punch biopsies harvested on day 84. D: Littermates were given mAb subcutaneously on days 56, 70, 84, and 98 after birth, with tissues harvested on day 112. All values represent results from individual mice with horizontal bars representing means. Significant differences between sample means are indicated: *P < 0.05; **P < 0.01.

Figure 4

B-cell depletion inhibits autoantibody production and hypergammaglobulinemia in Tsk^/+ mice. Mice were treated with CD20 mAb (20 μg) bi-weekly (A, C, E) since day 3 after birth (days 3, 17, 31, and 45) and serum was harvested on day 56, or (B, D, F) since day 56 after birth (days 56, 70, 84, and 98) with serum harvested on day 112. Relative serum antibody, fibrillin-, topoisomerase I-, and single-stranded DNA-specific antibody and rheumatoid factor levels were determined by ELISA, with assay background results (+2 SDs) indicated by a horizontal dashed line. Horizontal bars represent mean autoantibody and antibody levels for each group. Significant differences between sample means are indicated: *P < 0.05; **P < 0.01.

Figure 5

B-cell depletion alters cytokine production in the skin and spleens of Tsk^/+ mice. Cytokine mRNA levels in the dorsal skin (A) or among spleen lymphocytes (B) of 21-day-old Tsk^/+ or wild-type littermates after CD20 or control mAb treatment (20 μg) on days 3 and 17 of age. Transcript levels were quantified by real-time reverse transcriptase-PCR analysis and were normalized relative to endogenous GAPDH levels. Values represent mean percentages (±SEM) relative to transcript levels of wild-type littermates treated with CTL mAb (100%, broken horizontal lines), with ≥10 mice (A) or four mice (B) in each group. Values significantly different from controls are indicated: *P < 0.05; **P < 0.01.