Gestational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin disrupts B-cell lymphopoiesis and exacerbates autoimmune disease in 24-week-old SNF1 mice

Abstract

Female SNF(1) hybrid mice spontaneously develop an immune complex-mediated glomerulonephritis as early as 24 weeks of age, whereas the disease onset in males is much slower. Further, a rise in concentration of glomerulus-specific autoantibodies via autoreactive B cells is critical to progression of the disease in this strain. Environmental factors contributing to the onset or degree of such autoimmunity are of interest yet poorly understood. In the present study, time-pregnant SWR x NZB dams (10/treatment) were gavaged on gestational 12 with 40 or 80 mg/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and the SNF(1) offspring were evaluated at 24 weeks of age. Bone marrow B220(low)CD24(-)AA4.1(+) committed B lineage progenitors were increased in female offspring by TCDD, however, committed progenitors and pro-B cells were decreased in males. Splenic marginal zone B cells (CD21(hi)CD24(low-int)) were decreased and follicular B cells (CD21(int)CD24(low)) were increased across sex by prenatal TCDD, whereas transitional-2 B cells (CD21(int)CD24(hi)) and (CD23(low-int) CD1(low-int)) were decreased in males only. Antibodies to double-stranded DNA were significantly increased across sex by TCDD. Anti-IgG and anti-C3 immune complex renal deposition was visibly worsened in females, and present in TCDD-treated males. These data suggest that developmental exposure to TCDD permanently and differentially alters humoral immune function by sex, and exacerbates a type III hypersensitivity lupus-like autoimmune disease in genetically predisposed mice.

FIG. 1.

Computer-generated diagram showing the key cell phenotypes highlighting B-cell lymphopoiesis in the bone marrow (A) and B-cell maturation in the spleen (B). The cell-surface markers identified under the specific cell types were the ones evaluated in this study.

FIG. 2.

Representative dot plots showing the gating technique employed to differentiate select B-cell subsets associated with lymphopoiesis in the bone marrow. Bone marrow cells were adjusted based on size (FS) and granularity (SS) to elucidate the total cell population (A). Filters were employed to gate out dead cells and debris. Using the FloJo software (Tree Star), cells were then gated on for their ability to express CD45R_B220 (B). The cell populations were then sub divided based on low fluorescence to CD45R_B220 (B220 low) and high fluorescence to CD45R_B220 (B220 high) (C). These two subpopulations of CD45R_B220 cells were then evaluated based on their ability to express CD24 (D and E). Bone marrow cells with a CD45R_B220lowCD24⁻ phenotype were costained with AA4.1 (CD93, C1qRp) to identify the earliest B lineage cells (F).

FIG. 3.

Sera from 24-week-old SNF₁ mice that were prenatally exposed to 0, 40, or 80 μg/kg TCDD were analyzed for the presence of autoantibodies to dsDNA (A), ssDNA (B), and cardiolipin (C). The data are arranged by sex and are based on five mice per treatment per sex (*p ≤ 0.05, Dunnett's test).

FIG. 4.

The kidneys from 24-week-old SNF₁ mice that were prenatally exposed to 0, 40, or 80 μg/kg TCDD were collected, fixed, sectioned, and stained with H&E. Images are representative of renal sections by treatment and sex. All sections were scored for number of fibrinoid necrosis cells or crescents/100 glomeruli and number of inflammatory foci in the parenchyma. The data are based on five mice per treatment per sex (*p ≤ 0.05, Dunnett's test).

FIG. 5.

The kidneys from 24-week-old SNF₁ mice that were prenatally exposed to 0, 40, or 80 μg/kg TCDD were collected, fixed, section, and stained with FITC-labeled anti-IgG and anti-C3. The above are representative images of kidneys stained with FITC-anti-IgG based on treatment and sex. The data are based on five mice per treatment per sex (*p ≤ 0.05, Dunnett's test).

FIG. 6.

The spleens from 24-week-old SNF₁ mice that were prenatally exposed to 0, 40, or 80 μg/kg TCDD were collected, fixed, sectioned, and stained with H&E. Images are representative of splenic sections presented by treatment and sex. Spleens from male mice prenatally exposed to 40 μg/kg TCDD frequently displayed follicles with small germinal centers (arrows). In the 80 μg/kg TCDD male mice, the germinal centers were few and discrete with coalescing of follicles and a loss of follicular architecture (arrows).