Immune dysregulation accelerates atherosclerosisand modulates plaque composition in systemiclupus erythematosusAleksandar K. Stanic*, Charles M. Stein†‡, Adam C. Morgan*, Sergio Fazio*§, MacRae F. Linton*‡, Edward K. Wakeland¶,Nancy J. Olsen�, and Amy S. Major*,**

Divisions of *Cardiovascular Medicine and †Rheumatology and Immunology, Department of Medicine, and Departments of ‡Pharmacology and§Pathology, Vanderbilt University School of Medicine, Nashville, TN 37232-6300; ¶Center for Immunology and Department of Pathology, Universityof Texas Southwestern Medical Center, Dallas, TX 75390; and �Division of Rheumatic Diseases, Department of Internal Medicine, University of TexasSouthwestern Medical School, Dallas, TX 75390

Communicated by Robert W. Mahley, The J. David Gladstone Institutes, San Francisco, CA, March 22, 2006 (received for review October 10, 2005)

Patients with systemic lupus erythematosus (SLE) have acceleratedatherosclerosis. The underlying mechanisms are poorly under-stood, and investigations have been hampered by the absence ofanimal models that reflect the human condition of generalizedatherosclerosis and lupus. We addressed this problem by transfer-ring lupus susceptibility to low-density lipoprotein (LDL) receptor-deficient (LDLr�/�) mice, an established model of atherosclerosis,creating radiation chimeras with NZM2410-derived, lupus-suscep-tible, B6.Sle1.2.3 congenic or C57BL�6 control donors (LDLr.Sle andLDLr.B6, respectively). LDLr.Sle mice developed a lupus-like diseasecharacterized by production of double-stranded DNA autoantibod-ies and renal disease. When fed a Western-type diet, LDLr.Slechimeras had increased mortality and atherosclerotic lesions. Theplaques of LDLr.Sle mice were highly inflammatory and containedmore CD3� T cells than controls. LDLr.Sle mice also had increasedactivation of CD4� T and B cells and significantly higher antibodyto oxidized LDL and cardiolipin. Collectively, these studies dem-onstrate that the lupus-susceptible immune system enhancesatherogenesis and modulates plaque composition.

autoimmunity � congenic mice

Three decades ago, Urowitz et al. (1) recognized that cardiovas-cular disease (CVD) and myocardial infarction were major

causes of mortality in patients with systemic lupus erythematosus(SLE). More recently, Manzi et al. (2) reported that premenopausalwomen with SLE, a population usually protected from atheroscle-rosis, had a 50 times greater risk of a fatal vascular event comparedwith age- and gender-matched controls. In addition, we showed anincreased prevalence of coronary atherosclerosis in SLE (3). De-spite the fact that CVD is the most common cause of death inpatients with SLE who survive the acute complications of theillness, little is known about the underlying mechanisms. It has beensuggested that a combination of traditional risk factors, includinghypertension, dyslipidemia, and lipid oxidation as well as nontra-ditional risk factors, such as autoantibodies and inflammation, maycontribute to advanced vascular disease in SLE (4). Therefore,defining the autoimmune mechanisms that promote atherosclerosisis essential to optimize risk reduction and develop targeted thera-peutics for prevention of CVD in SLE.

Atherosclerosis involves many cellular processes, and increasingevidence supports the role of inflammation and immunity in thepathogenesis of atherosclerosis (5). Macrophages and T cells makeup a large percentage of the cells present in the atheroscleroticplaque (6). These cells contribute to the inflammatory process byproducing cytokines that attract smooth muscle cells and lympho-cytes that compromise plaque stability. B cell responses and auto-antibodies to self-antigens such as oxidized LDL (oxLDL), heat-shock protein 60�65, and �-2-glycoprotein I have also beenidentified in humans with CVD and in animal models of athero-sclerosis (7, 8). These antibodies can also be detected in humans and

animals with autoimmune diseases such as SLE and the antiphos-pholipid antibody syndrome (9). However, whether autoantibodyproduction is causally related to atherosclerosis is not known.

A factor that has limited understanding the relationship betweeninflammation and atherosclerosis in SLE is that animal models oflupus are genetically resistant to diet-induced atherosclerosis. Thedevelopment of the NZM2410-derived congenic B6.Sle mousestrains made it feasible to examine lupus and atherosclerosistogether on the susceptible C57BL�6 background. Morel et al. (10)described three major chromosome intervals in the NZM2410mouse strain termed Sle1, Sle2, and Sle3 that are highly associatedwith lupus susceptibility. The investigators made a series of com-bined and single congenic mice on the C57BL�6 background. Ingeneral, Sle1 mediates loss of tolerance to nuclear antigens (11);Sle2 lowers the activation threshold of B cells leading to expansionof B-1 B cells and polyclonal IgM (12); and Sle3 is associated withdecreases in the activation threshold of T cells, a concomitantincrease in T cell-dependent polyclonal IgG production, and re-duced activation-induced cell death (13). In bone marrow transferstudies to normal C57BL�6 animals, it was demonstrated that lupussusceptibility was carried and could be transferred by cells ofhematopoietic origin (13, 14). Therefore, we exploited this ability totransfer lupus and made radiation chimeras of B6.Sle1.2.3 triplecongenics with lethally irradiated, atherosclerosis-susceptible LDLreceptor-deficient (LDLr�/�) mice and used this animal model toaddress the hypothesis that lupus-associated immune dysregulationpromotes atherosclerosis.

ResultsDevelopment of SLE in LDLr�/� Radiation Chimeras. We made lupus-susceptible animals in the setting of atherosclerosis by transplantinglethally irradiated LDLr�/� mice with bone marrow from either B6controls (LDLr.B6) or lupus-susceptible B6.Sle1.2.3 animals (LD-Lr.Sle). Sixteen weeks after transplantation, mice were placed on ahigh-fat Western diet. Animals were killed at 8 weeks after theinitiation of diet (Fig. 1A). At the time of killing, there was nodifference in body weight (Fig. 1B). However, the majority ofLDLr.Sle mice had a urinary protein grade of 2� or greater,significantly higher than the LDLr.B6 group (89% vs. 14%, respec-tively, P � 0.001) (Fig. 1C). In addition, although many of theLDLr.Sle mice had serum creatinine and urea levels similar to thoseof controls, the mean concentrations were significantly increased in

Conflict of interest statement: No conflicts declared.

Abbreviations: CVD, cardiovascular disease; LDL, low-density lipoprotein; LDLr, LDL recep-tor; LDLr.B6, LDLr and C57BL�6 chimeras; LDLr.Sle, LDLr and B6.Sle1.2.3 chimeras; oxLDL,oxidized LDL; SLE, systemic lupus erythematosus.

**To whom correspondence should be addressed at: Division of Cardiovascular Medicine,Department of Medicine, Vanderbilt University School of Medicine, Room 383, PRB, 2220Pierce Avenue, Nashville, TN 37232-6300. E-mail: amy.major@vanderbilt.edu.

© 2006 by The National Academy of Sciences of the USA

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LDLr.Sle mice (Fig. 1 D and E), indicating a decline of renalfunction. The anti-dsDNA antibody titer was also substantiallyincreased in the LDLr.Sle mice compared with controls (Fig. 1F).These results indicate that the lupus disease state was successfullytransferred to the LDLr�/� mice by bone marrow transplantation.

Susceptibility to Lupus Exacerbates Atherosclerosis in LDLr.Sle Radi-ation Chimeras. Next, we studied the size and composition ofatherosclerotic lesions in the aortic sinus. After 8 weeks of aWestern diet, the atherosclerotic lesion area was significantlyincreased in LDLr.Sle chimeras compared with control LDLr.B6mice (Fig. 2A).

The levels of serum cholesterol and triglycerides at time of killingwere slightly, but significantly, decreased (Fig. 2B) in LDLr.Sle micecompared with controls. FPLC analysis of lipoprotein distributionshowed that the difference was primarily because of a decrease in

the non-high-density lipoprotein cholesterol fractions (Fig. 2C).Serum creatinine and urea concentrations (see Fig. 1) did notcorrelate with atherosclerosis lesion size (Fig. 2 D and E). Also, theLDLr.Sle mice were not hypertensive compared with LDLr.B6controls (Fig. 2F). Therefore, mechanisms other than dyslipidemia,renal disease, and hypertension contribute to the advanced athero-sclerosis in the lupus-susceptible animals.

Lupus Susceptibility Results in Changes in Lesion Composition. Weconducted histochemical and immunohistochemical analyses of thecellular content of the atherosclerotic plaques in the aortic sinus and

Fig. 1. The lupus disease phenotype of B6.Sle1.2.3 mice can be transferred toLDLr�/� mice. (A) Lethally irradiatedfemaleLDLr�/� micewerereconstitutedwitheither B6 or B6.Sle1.2.3 bone marrow. Sixteen weeks after transplantation, allanimalswereplacedonaWestern-typediet for8weeks.After this time(24weeksafter 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 asmeasured by specific ELISA in LDLr.B6 (squares) and LDLr.Sle (circles) mice. Barsrepresent the mean � SEM of 12 LDLr.B6 and 9 LDLr.Sle mice. Shown is one of atleast 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 �2 analysis (see text).

Fig. 2. Increased atherosclerosis in LDLr.Sle mice. (A) Oil-red-o analysis of theatherosclerotic 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 aWestern-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 ofcholesterol 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 serumurea 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 notused in the calculations. (F) Measurement of systolic blood pressure by tail cuffingin LDLr.B6 (circles) and LDLr.Sle (triangles) mice. Data are the mean � SEM of nineLDLr.B6 and seven LDLr.Sle mice. Depicted P values were calculated by Mann–Whitney analysis. Correlation was determined by Spearman’s analysis.

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observed no significant difference in macrophage content in LD-Lr.Sle and LDLr.B6 mice (Fig. 3 A and B). There was a 3-foldincrease in the number of CD3� T cells in lesions of the LDLr.Slemice compared with controls (Fig. 3 A and B). Similar increaseswere observed in LDLr.Sle mice when lesions were stained for theCD4 T cell marker. Fluorescent staining for CD4 and CD3 revealedcolocalization of these markers (Fig. 3B). We determined theexpression of the MHC class II molecule I-Ab in lesions. Consistentwith the chronic inflammatory phenotype of lupus, we observed adramatic increase in I-Ab expression in the LDLr.Sle mice com-pared with controls (Fig. 3B). Collagen content did not differbetween LDLr.Sle and LDLr.B6 mice (Fig. 3 A and B) and SMC(�-SMA) were present in the media but were not found in the lesionitself (data not shown). Collectively, these data demonstrate thatthe LDLr.Sle mice have an atherosclerotic plaque phenotype thatis more cellular and contains more activated T cells.

LDLr.Sle Mice Exhibit Hyperactive Immunity in the Periphery. Wedetermined whether LDLr.Sle mice showed increased peripheralimmune activation. At the time of killing, the spleen weights inLDLr.Sle chimeras were significantly greater than those of controls(Fig. 4A). Increased spleen weights corresponded to a slight, butsignificant, increase in cell numbers (Fig. 4B). Flow cytometricanalysis of LDLr.Sle splenocytes demonstrated an increase in thepercentages (LDLr.B6 � 19.5 � 1.9%; LDLr.Sle � 25.7 � 0.3%,P � 0.03) and absolute numbers of CD4� T cells compared withcontrols (Fig. 4 C and D). Numbers of CD8� T cells, B cells(B220�,CD11c�), macrophages (Mac-3�), and NK1.1� cells werenot significantly affected. In addition, total CD11c� dendritic cells(DC) were not different between the two groups. Although theplasmacytoid DC (B220�, CD11c�) cells were increased 2-fold inthe LDLr.Sle mice (2.0 � 0.6 � 106 cells) compared with theLDLr.B6 mice (1.0 � 0.1 � 106 cells), the difference did not quitereach statistical significance (P � 0.06). Increases in these types ofDC are consistent with the reported B6.Sle1.2.3 phenotype (15).

Autoimmunity may be considered a disorder of decreased acti-vation thresholds of specific immune populations and excessiveactivation during lymphocyte selection or in their mature state.

Therefore, we investigated whether increased CD4� T cell numbersin the spleen were the result of increased selection in the thymus.Analysis of surface levels of CD69, which is up-regulated on thymicCD4�CD8� double-positive (DP) cells after successful positiveselection, showed no differences on either DP cells or CD8� cellsbetween the groups (Fig. 4E). Interestingly, total CD4� T cellnumbers were lower in thymi of LDLr.Sle mice (Fig. 4F; LDLr.B6 �18.5 � 3.2%; LDLr.Sle � 8.3 � 1.2%, P � 0.038) but weresignificantly more active (Fig. 4G; CD69 staining LDLr.B6 �30.0 � 6.1%; LDLr.Sle � 59.5 � 4.3%, P � 0.017). Once in theperiphery, CD4� T cells retained this overactive phenotype char-acterized by the expression of CD69 (Fig. 4G; LDLr.B6 � 18.5 �1.8%; LDLr.Sle � 31.7 � 3.6%, P � 0.03). We observed nodifference in CD4� T cell expression of the proliferation markerKi67 (LDLr.B6 � 8.3 � 1.0%; LDLr.Sle � 10.6 � 0.6%, P � 0.13)but increased expression of the active apoptosis marker caspase 3in LDLr.Sle mice (LDLr.B6 � 4.1 � 0.6%; LDLr.Sle � 6.6 � 0.6%,P � 0.03), perhaps indicating greater turnover of the CD4� T cells.

In addition to increased CD4� T cell activation, we also observedincreased B cell activity. As reported in B6.Sle animals (12, 14),there were increases in the activation markers CD80 and CD86 onB cells isolated from spleens of LDLr.Sle mice compared withcontrols (data not shown). Increased B cell activation in LDLr.Slemice was accompanied by increased proliferation, measured byKi67 expression (Fig. 4H Right) and an increase in Bcl-2 expression(Fig. 4H Left) compared with controls. Collectively, these dataindicate that LDLr.Sle mice have an increased B cell and T helpercell activation state characteristic of the B6.Sle1.2.3 congenic ani-mals (16) and that exacerbation of atherosclerosis in LDLr.Sle miceoccurs in the setting of dysregulated and overactivated B and CD4�

T cell populations.

Lupus-Susceptible Mice Have Higher Titers of Antiphospholipid Anti-bodies. We measured serum antibody titers directed against oxLDLand cardiolipin and observed that LDLr.Sle mice exhibited signif-icant increases in anti-oxLDL (Fig. 5A) and anticardiolipin (Fig. 5B)antibodies compared with controls. There was little to no detectableantibody against oxLDL or cardiolipin in either group at baseline

Fig. 3. Atherosclerotic plaquecomposition. (A) Quantitation ofmacrophages (CD3� T and CD4� Tlymphocytes) in atheroscleroticplaques of LDLr.B6 (open bars)and LDLr.Sle (filled bars) mice.Macrophages are expressed as thepercent macrophage stainingarea of total lesion area; n � 5 forboth groups. CD3� and CD4� Tlymphocytes are expressed as thepercentage positive cells of totalcells in the lesion; n � 3–5 for bothgroups. Collagen content wasmeasured by Masson’s trichromeand expressed as a percentage ofpositive staining of total lesionarea; n � 18 for the LDLr.B6 groupand 19 for the LDLr.Sle group. Barsare the mean � SEM from the av-erage of four sections per mouse.(B) Immunohistochemical and im-munofluorescent staining formacrophages (MOMA-2, magnifi-cation, �50), Masson’s trichromestaining (magnification, �50),MHC class II I-Ab (magnification,�200), and colocalization of CD3and CD4 (arrowheads) on T lym-phocytes (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.

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(Fig. 5 A and B). Although it was not statistically significant, we didobserve an increase in the anti-oxLDL IgM titer (Fig. 5C Left). Inaddition, there was a significant increase in the anti-cardiolipin IgMtiters (Fig. 5D Left). There was no clear indication of either a Th-1or Th-2 bias, because both IgG1 and IgG2a were increased in theLDLr.Sle mice compared with LDLr.B6 control animals (Fig. 5 Cand D).

DiscussionPatients with SLE have a marked increase in the prevalence ofatherosclerosis and its complications (4). However, the cellular andmolecular mechanisms underlying this accelerated atherosclerosisare poorly understood and cannot be attributed to traditional CVDrisk factors (3, 17). Mechanistic studies of accelerated atheroscle-rosis in lupus are difficult due in part to the lack of an appropriateanimal model. Vasculitis and early fatty streak lesions are increasedin MRL�lpr mice (18, 19), and fatal myocardial infarction due tononcellular vessel occlusion occurs in male BXSB mice (20, 21).However, because of their genetic background, mice traditionallyused to study the pathogenesis of lupus are largely resistant todeveloping the large, multicellular atherosclerotic lesions observedin humans. To address this problem, we used lupus-susceptiblecongenic mice, B6.Sle1.2.3, originally described by Morel et al. (10),that are on the atherosclerosis-susceptible C57BL�6 background.The phenotype of autoimmune dysregulation characteristic of thismodel of SLE has been shown to transfer with bone marrow

transplantation; thus, we designed transplantation experiments inLDLr�/� mice to examine the impact of SLE on atherogenesis. Wecreated radiation chimeras with either C57BL�6 or B6.Sle1.2.3donors to LDLr�/� recipients. Studies from our laboratory havedemonstrated that LDLr expression on hematopoietic cells inLDLr�/� mice contributes significantly to foam cell formation (22);however, because both control and experimental groups receivedLDLr-sufficient bone marrow, the interpretation of the resultsshould not be affected. We found that LDLr�/� mice reconstitutedwith the B6.Sle1.2.3 bone marrow developed not only hallmarks ofautoimmune dysregulation and decreased renal function consistentwith SLE but also large atherosclerotic lesions in the aortic sinuscharacterized by accumulation of lipid-filled macrophages andincreased numbers of CD3� T lymphocytes.

Traditionally, dyslipidemia correlates directly with the extent andprogression of atherosclerosis in many mouse models and inhumans. Chronic renal failure, an independent complication oflupus, has also been associated with increased atherosclerosis (23,24). These studies of atherosclerosis and renal failure in apoE�/�

and LDLr�/� mice have shown that decreased renal function resultsin significant increases in very (V)LDL and LDL cholesterolcompared with controls. Therefore, in the setting of renal impair-ment, it is difficult to ascertain whether the increase in atheroscle-rosis is a secondary effect of atherogenic lipids. In contrast, wefound that LDLr.Sle mice have increased atherosclerosis despitelower levels of pathogenic non-high-density lipoprotein (HDL) and

Fig. 4. 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 forwardvs. 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 CD69on CD8��CD4� single-positive and CD8��CD4� double-positive T lymphocytes isolated from thymi of LDLr.B6 (Left) and LDLr.Sle (Right) mice. (F) Representativecontour 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 theforward 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) andLDLr.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 eachcontaining 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 valuesfor spleen weight and cellularity were calculated by using a Mann–Whitney test.

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triglycerides (Fig. 2C). Not only were LDL and triglyceride con-centrations lower, but the extent of renal compromise did notcorrelate with the size of atherosclerotic lesions in LDLr.Sle mice(Fig. 2 D and E). These data support the hypothesis that immunemechanisms are primarily responsible for increased plaque forma-tion in the LDLr.Sle mice. Interestingly, we have reported accel-erated coronary artery atherosclerosis in lupus patients whose LDLand HDL cholesterol concentrations were not higher that those ofcontrol subjects (3). Similar observations regarding carotid arteryatherosclerosis were made by Roman et al. (25). Therefore, itappears that traditional associations between LDL cholesterol andseverity of atherosclerosis do not apply in the case of mice orpatients with SLE.

A central question in lupus is whether abnormal immune acti-vation results in progression of atherosclerotic lesions. We observedsignificant differences in atherosclerotic plaque composition inLDLr.Sle mice, consistent with increased immune activation. Spe-cifically, there were increased areas of intense cellularity in theLDLr.Sle plaques that were not present in the LDLr.B6 controls(Fig. 3). These cells were not MOMA-2� macrophages but stainedfor CD3, a T cell marker. In fact, CD3� and CD4� T cell numberswere 3-fold higher in the lupus-susceptible animals compared withcontrols. Double-staining experiments showed that the majority ofCD4� cells were CD3� T cells. Antigen-presenting cells corre-sponding to areas of MOMA-2 staining appeared to be moreactivated in the LDLr.Sle mice, as exemplified by the markedoverexpression of I-Ab, an MHC class II molecule that is up-regulated by IFN-� production. Previous studies have implicated Tcell-rich lesions as more unstable and prone to rupture, leading tovessel occlusion and myocardial infarction (26). Therefore, not onlyis atherosclerosis increased in LDLr.Sle animals, but, also, theplaques are typified by a cellular composition of a more vulnerablephenotype.

Consistent with increased immune activity in the atheroscleroticplaque, both the peripheral CD4� T cell and B cell compartmentof the LDLr.Sle mice exhibited immune volatility. We observed anincrease in the absolute numbers and activation state of splenicCD4� T cells in LDLr.Sle mice compared with controls (Fig. 4). Wealso observed the characteristic increase in B cell activation andautoantibody production associated with SLE (27). Increased an-

tibody production in LDLr.Sle mice was not restricted to lupus-associated antigens such as dsDNA; there were also significantincreases in anti-oxLDL and anticardiolipin antibodies (Fig. 5). Theincrease of anti-oxLDL and anticardiolipin antibody correspondedto a significant increase in the T cell-dependent IgG1 and IgG2aisotypes. Given the fact that Sle2 mediates expansion of B-1 B cells,a major source of IgM antibody, we were surprised that there werenot even greater differences between LDLr.Sle and LDLr.B6 micefor anti-oxLDL IgM. Of note, some aspects of the induced antibodyresponse may have been affected by the bone marrow transplan-tation protocol. It has been shown that bone marrow transplanta-tion does not replace the B-1 B cell compartment in the peritoneum(28). Much of the literature suggests that much of the ox-LDLantibody is B-1 B cell-derived (29, 30); thus, it is possible that ourcurrent experimental protocol did not transfer the hyperactivephenotype to this cell type. Additional experiments designed tospecifically look at B-1 B cells in this compound disease model willbe necessary in the future. It is possible that increased ox-LDL-specific IgG1 and IgG2a, which can modulate inflammation viaimmune complex binding of Fc� receptors (31), may act to exac-erbate atherosclerotic disease in the LDLr.Sle mice.

Interestingly, we also observed an as-yet-unreported decrease inthe number of CD4�CD8� T cells in the thymi of the LDLr.Sle micecompared with controls. However, a larger percentage of thethymic CD4� T cells expressed the CD69 surface antigen, indicat-ing that they were more activated compared with control animals.It is not known why CD4� T cells are decreased in the LDLr.Sleanimals. Because we observed an increase in the numbers of CD4�

T cells in the periphery or LDLr.Sle mice compared with LDLr.B6animals, it is possible that the CD4� T cells are being exported fromthe thymus at a faster rate in the lupus-susceptible animals. Anotherpossibility is that the thymic CD4� T cells are undergoing a higherrate of activation-induced cell death.

In conclusion, in a mouse model of atherosclerosis in SLE, wehave shown that an overactive immune system participates in theprogression of atherosclerotic lesions and leads to modification ofplaque cellular composition. These studies shed light on the patho-genic role of the SLE-affected immune system in atherosclerosis. Byusing this model, it will be possible to examine the effects of lupusdrugs on the progression of atherosclerosis. Also, future studies

Fig. 5. Anti-oxLDL and anticardiolipin antibodies. Total anti-oxLDL (A) and anticardiolipin (B) Ig in LDLr.B6 (squares) and LDLr.Sle (circles) mice at baseline, beforebone 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 wereobtained by Mann–Whitney analysis.

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using the Sle1, Sle2, and Sle3 single congenic mice will allow us todetermine the contribution of different immune components on thechanges in atherosclerotic lesion size and composition observed inthis study. Such studies will facilitate the identification and devel-opment of therapeutic interventions to treat SLE and minimize therisk of accelerated atherosclerosis.

Materials and MethodsMice. C57BL�6 and LDLr�/� mice were originally obtained fromThe Jackson Laboratory and maintained in our colony. TheLDLr�/� mice have been backcrossed to the C57BL�6 backgroundat least 10 times. Lupus-susceptible B6.Sle1.2.3 triple congenic micehave been described and extensively characterized (10, 14, 32, 33).The mice are homozygous for the three lupus-susceptibility chro-mosome intervals on chromosomes 1, 4, and 7, respectively. Allmice were maintained in microisolator cages and used according tothe guidelines of the Vanderbilt University Institutional AnimalCare and Use Committee. Unless otherwise stated, mice were feda normal chow diet.

Production of Radiation Chimeras. Transfer of the lupus-susceptibility phenotype to female LDLr�/� mice was accom-plished by production of radiation chimeras as described in ref. 34.

Atherosclerosis Studies. Sixteen weeks after bone marrow trans-plantation, animals were placed on a high-fat Western-type diet(21% milk fat and 0.15% cholesterol) for 8 weeks (Fig. 1A). At theend of 8 weeks, animals were killed and analyzed for the extent ofatherosclerosis and the presence of SLE as described in ref. 34.

Immunohistochemistry. Staining of macrophages (MOMA-2) andMHC class II (I-Ab) was done as described by using 5 �m ofacetone-fixed frozen sections (35). CD3� T lymphocytes werestained by using rat anti-mouse monoclonal antibody to the CD3antigen (clone C363.29B; Southern Biotechnology Associates, Bir-mingham, AL) and FITC-conjugated anti-rat. CD4� T cells werevisualized by using biotin-conjugated rat anti-CD4 (clone RM4–5;BD Biosciences) and avidin Texas red (Vector Laboratories).�-SMA was detected by using a rabbit anti-�-SMA polyclonalantibody (Lab Vision, Fremont, CA). Staining was quantitatedby using KINETIC HISTOMETRIX 6 imaging and analysis software(Kinetic Imaging).

Flow Cytometry. For flow cytometric analyses, spleens and thymiwere removed and processed through a 0.70-�m mesh screen as

described in ref. 35. Cells were counted, resuspended in 1.0%FBS�PBS (0.1% sodium azide), and incubated with the appropriateantibody for 30 min at 4°C. Cells were washed and analyzed by usinga FacsCalibur flow cytometer (Becton Dickinson) and FLOWJOsoftware.

ELISAs. Serum antibody titers against oxLDL and cardiolipin weremeasured as described in ref. 34. Titers of dsDNA antibodies weremeasured by using the method of Morel et al. (33).

Serum Lipoprotein Analyses. Total serum cholesterol and triglycer-ide levels were measured by using a colorimetric assay as describedin ref. 34. Lipoprotein distribution was determined by using FPLC.

Determination of Renal Disease. Renal function was assessed bymeasuring protein in urine at the time of killing by using a urineMultistix 10SG (Bayer). Serum levels of creatinine and urea weremeasured by specific quantitative colorimetric assay as per manu-facturer’s specifications (BioAssay Systems, Hayward, CA).

Measurement of Systolic Blood Pressure. Systolic blood pressure wasmonitored by tail cuffing on conscious, preconditioned mice byusing a BP-2000 (Visitech Systems, Apex, NC) and accompanyingsoftware, available at the Mouse Metabolic Phenotype Core facilityat Vanderbilt University. A total of three measurements were doneon each animal, and the average reading was used.

Statistical Analyses. Statistical analyses were conducted by usingPRISM 3.03 software. Unless otherwise stated, differences betweengroups were determined by using a Mann–Whitney nonparametrict test. Statistically significant differences in urine protein grade weredetermined by �2 analysis. Correlation analyses were conducted byusing a Spearman analysis. A P value of �0.05 was consideredsignificant.

We thank Drs. Luc Van Kaer and Mirsanda Stanic for thoughtful readingof the manuscript and Tiffany N. Crouch, Jennifer L. McCaleb, andYoumin Zhang for technical assistance. This work was supported byNational Institutes of Health (NIH) Building Interdisciplinary ResearchCareers in Women’s Health (BIRCWH) Grant 5 K12 HD043483-04,American Heart Association Scientist Development Grant 0330412N, asmall project grant from the Nashville Chapter of the Lupus Foundationof America, a grant from the Lupus Research Institute (to A.S.M.), andNIH Grants HL57986 and HL65709 (to S.F.) and HL65405 and HL53989(to M.F.L). We also acknowledge the Vanderbilt Mouse MetabolicPhenotyping Centers (NIH Grant DK59637-01).

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