Increased atherosclerosis in LDLr.Sle mice. (A) Oil-red-o analysis of the atherosclerotic lesion area in the aortic sinus of LDLr.B6 (squares) and LDLr.Sle (circles) mice; n = 17 for both groups. (B) Serum cholesterol (Left) and triglyceride (Right) levels before bone marrow transplantation (0), at the initiation of a Western-type diet (16 weeks), and at the time of killing (24 weeks) in LDLr.B6 (squares) and LDLr.Sle (circles) mice; n = 17 for both groups. (C) FPLC analysis of cholesterol lipoprotein distribution in serum of LDLr.B6 (squares) and LDLr.Sle (circles) mice. Shown are data from a pool of five to six mice in each group. Analysis of individual mice showed similar results. (D and E) Correlation of serum urea and creatinine levels and atherosclerotic lesion area in LDLr.Sle mice (circles). For comparison, LDLr.B6 mice (squares) are included on the graph but were not used in the calculations. (F) Measurement of systolic blood pressure by tail cuffing in LDLr.B6 (circles) and LDLr.Sle (triangles) mice. Data are the mean ± SEM of nine LDLr.B6 and seven LDLr.Sle mice. Depicted P values were calculated by Mann–Whitney analysis. Correlation was determined by Spearman’s analysis.

Analysis of splenic and thymic cells. (A and B) Comparison of wet spleen weights and cellularity in LDLr.B6 (open bars) and LDLr.Sle (filled bars) mice. (C) Contour plots of CD4⁺ T lymphocytes in the spleens of LDLr.B6 (Left) and LDLr.Sle (Right) mice. Cells were gated on total lymphocytes based on the forward vs. side scatter plot. Shown is one mouse from one experiment of two containing three mice per group. (D) Absolute numbers of designated spleen cells in LDLr.B6 (open bars) and LDLr.Sle (filled bars) mice. Numbers were calculated based on the total spleen cell count and the percent of each cell type. (E) Expression of CD69 on CD8α⁺CD4⁻ single-positive and CD8α⁺CD4⁺ double-positive T lymphocytes isolated from thymi of LDLr.B6 (Left) and LDLr.Sle (Right) mice. (F) Representative contour plots of the T lymphocyte compartments of the thymus in LDLr.B6 (Left) and LDLr.Sle (Right) mice. Cells were gated on total lymphocytes based on the forward vs. side scatter plot. (G) Expression of the marker of activation CD69 on CD4⁺ T lymphocytes isolated from the spleen and thymus in LDLr.B6 (Left) and LDLr.Sle (Right) mice. Shown is one mouse from one experiment of two containing three mice per group. (H) Mean fluorescence intensity of Bcl-2 expression (Left) and percentage of Ki67⁺ cells (Right) in B cells isolated from LDLr.B6 (filled bars) and LDLr.Sle (open bars) mice. Shown is one experiment of two each containing three mice per group. Bars in graphs are the mean ± SEM. P values for flow cytometry experiments were calculated by using Student’s t test. P values for spleen weight and cellularity were calculated by using a Mann–Whitney test.

The lupus disease phenotype of B6.Sle1.2.3 mice can be transferred to LDLr^−/− mice. (A) Lethally irradiated female LDLr^−/− mice were reconstituted with either B6 or B6.Sle1.2.3 bone marrow. Sixteen weeks after transplantation, all animals were placed on a Western-type diet for 8 weeks. After this time (24 weeks after transplant), mice were killed and analyzed. (B) Body weight of LDLr.B6 (open bars) and LDLr.Sle (filled bars) mice. (C) Percentage of LDLr.B6 (open bars) and LDLr.Sle (filled bars) mice exhibiting protein in urine (1+, 30 mg/dl; 2+, 30–100 mg/dl; 3+, 100–300 mg/dl). (D) Levels of serum creatinine in LDLr.B6 (squares) and LDLr.Sle (circles) mice. (E) Levels of serum urea in LDLr.B6 (squares) and LDLr.Sle (circles) mice. (F) Levels of serum antibody specific for dsDNA as measured by specific ELISA in LDLr.B6 (squares) and LDLr.Sle (circles) mice. Bars represent the mean ± SEM of 12 LDLr.B6 and 9 LDLr.Sle mice. Shown is one of at least three experiments. In C–F, the P values were calculated by using a Mann–Whitney analysis. In D, the P value was calculated by using a χ² analysis (see text).

Atherosclerotic plaque composition. (A) Quantitation of macrophages (CD3⁺ T and CD4⁺ T lymphocytes) in atherosclerotic plaques of LDLr.B6 (open bars) and LDLr.Sle (filled bars) mice. Macrophages are expressed as the percent macrophage staining area of total lesion area; n = 5 for both groups. CD3⁺ and CD4⁺ T lymphocytes are expressed as the percentage positive cells of total cells in the lesion; n = 3–5 for both groups. Collagen content was measured by Masson’s trichrome and expressed as a percentage of positive staining of total lesion area; n = 18 for the LDLr.B6 group and 19 for the LDLr.Sle group. Bars are the mean ± SEM from the average of four sections per mouse. (B) Immunohistochemical and immunofluorescent staining for macrophages (MOMA-2, magnification, ×50), Masson’s trichrome staining (magnification, ×50), MHC class II I-A^b (magnification, ×200), and colocalization of CD3 and CD4 (arrowheads) on T lymphocytes (magnification, ×400) in LDLr.B6 (Upper) and LDLr.Sle (Lower) mice. In the CD3/CD4 figures, the dotted line represents the internal elastic lamina, and the solid line designates the edge of the lesion. Shown are sections from one representative mouse of three to five in each group.

Anti-oxLDL and anticardiolipin antibodies. Total anti-oxLDL (A) and anticardiolipin (B) Ig in LDLr.B6 (squares) and LDLr.Sle (circles) mice at baseline, before bone marrow transplantation and at postdiet or time of killing. In both A and B, n = 10–14 mice per group. (C) Anti-oxLDL Ig isotypes in serum of LDLr.B6 (squares) and LDLr.Sle (circles) mice. (D) Anticardiolipin Ig isotypes in serum of LDLr.B6 (squares) and LDLr.Sle (circles) mice. Shown are the means ± SEM. Calculated P values were obtained by Mann–Whitney analysis.