Reduction of atherosclerosis in low-density lipoprotein receptordeficient mice by passive administration of antiphospholipid antibody.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 48, No. 10, October 2003, pp 2974–2978 DOI 10.1002/art.11255 © 2003, American College of Rheumatology Reduction of Atherosclerosis in Low-Density Lipoprotein Receptor–Deficient Mice by Passive Administration of Antiphospholipid Antibody Danielle Nicolo, Bruce I. Goldman, and Marc Monestier Objective. Patients with antiphospholipid syndrome (APS) are at a high risk of developing atherosclerotic complications. Conversely, individuals with primary atherosclerosis have an increased prevalence of antiphospholipid antibodies (aPL) and antibodies to oxidized low-density lipoproteins (ox-LDL). Several studies suggest that these two antibody populations may in fact overlap, although it is unclear how aPL contribute to pathogenesis. In this study, we characterized an IgG monoclonal aPL and assessed its ability to modulate atherosclerosis in low-density lipoprotein receptor– deficient (LDLRⴚ/ⴚ) mice. Methods. The cardiolipin-reactive monoclonal antibody FB1 was obtained from an (NZW ⴛ BXSB)F1 mouse, a strain with APS features that make it prone to fatal myocardial infarctions. Using an enzyme-linked immunosorbent assay, we investigated the binding of this antibody to phospholipid and LDL antigens. We also passively administered FB1 to atherosclerosisprone mice to determine its effect on atherogenesis. Results. In contrast to earlier studies of aPL that were specific for oxidized forms of LDL, FB1 crossreacted with both native LDL and ox-LDL. In vivo, passive administration of FB1 significantly reduced plaque formation in atherosclerosis-prone LDLRⴚ/ⴚ mice. Conclusion. These results indicate that some aPL may play a protective role in atherogenesis and suggest a novel approach to the prevention of atherosclerosis. Autoantibodies that react with negatively charged phospholipids are encountered in a variety of systemic autoimmune diseases, including systemic lupus erythematosus and antiphospholipid syndrome (APS). Several studies indicate that some antiphospholipid antibodies (aPL) can react with oxidized low-density lipoproteins (ox-LDL) (1). Some authors have suggested that similar antigenic determinants can be created by the oxidation of phospholipids and LDLs (2,3). Because the oxidation process can create a great diversity of epitopes, it is likely that aPL possess different specificities. Antibodies to ox-LDL are seen in patients with atherosclerosis, a condition associated with a progressive oxidation of lipoprotein particles (4). It is unclear how autoantibodies affect the progression of the disease. Some investigators have suggested that antibodies to ox-LDL may contribute to the pathogenesis of atherosclerosis (5). In contrast, recent studies suggest that these antibodies may protect against the disease, possibly because they can scavenge the ox-LDL particles (6,7). To further explore the relationship between aPL and atherosclerosis, we studied an IgG monoclonal antibody (mAb) from an (NZW ⫻ BXSB)F1 mouse, a cross that develops autoimmune and pathologic manifestations that are reminiscent of APS (8). This mAb was originally selected for its ability to bind cardiolipin (CL), but it also reacts with both native and oxidized forms of LDL. Administration of this mAb to atherosclerosisprone mice paradoxically resulted in a decrease in plaque formation. These data suggest that the manipulation of humoral immunity represents a therapeutic approach to atherosclerosis. Supported by grants from the NIH and the American Heart Association. Danielle Nicolo, PhD, Bruce I. Goldman, MD, Marc Monestier, MD, PhD: Temple University School of Medicine, Philadelphia, Pennsylvania. Address correspondence and reprint requests to Marc Monestier, MD, PhD, Temple University School of Medicine, Department of Immunology and Microbiology, 3400 North Broad Street, Philadelphia, PA 19140. E-mail: firstname.lastname@example.org. Submitted for publication August 27, 2002; accepted in revised form June 13, 2003. MATERIALS AND METHODS FB1 antiphospholipid monoclonal antibody. FB1 is a CL-reactive IgG2b mAb that was obtained from a 5-month-old 2974 ROLE OF aPL IN THE PREVENTION OF ATHEROSCLEROSIS female (NZW ⫻ BXSB)F1 mouse (9). FB1 was purified from supernatant by affinity chromatography on protein G– Sepharose (Amersham Biosciences, Piscataway, NJ). Enzyme-linked immunosorbent assay (ELISA). An ELISA was used to assess the binding of FB1 to CL (Avanti Polar Lipids, Alabaster, AL) or to LDL and its oxidized derivatives. Briefly, polyvinyl chloride microtiter plates (Becton Dickinson, Franklin Lakes, NJ) were coated with 20 g/ml of native LDL, minimally modified LDL, or ox-LDL. When native LDL was used as the antigen, EDTA (0.1 mg/ml) and butylated hydroxytoluene (20 M) (both from Sigma, St. Louis, MO) were included throughout to prevent oxidation. When CL was used as the antigen, the plates were coated with 20 g/ml of CL in ethanol. After blocking, purified FB1 (diluted in phosphate buffered saline [PBS] containing 1% bovine serum albumin [Sigma] and 0.05% Tween 20 [Sigma]), was added to the wells at varying concentrations and incubated for 2 hours at room temperature. After washing, binding was detected with goat anti-mouse IgG–alkaline phosphatase (Southern Biotechnology, Birmingham, AL) followed by color development with the appropriate substrate. Animal studies. LDL receptor–deficient (LDLR⫺/⫺) mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained in our animal facilities. All animal research protocols were approved by the Temple University Institutional Animal Care and Use Committee. In experiment 1, 16-week-old mice received intraperitoneal injections of purified FB1 twice a week for 12 weeks (0.75 mg/injection), whereas control animals received PBS. Mice were fed a high-fat Western-type diet containing 20% fat with 1.25% cholesterol and 0.5% cholic acid. In experiment 2, 10-week-old mice received an identical regimen of FB1 for 12 weeks, but control animals received equivalent injections of mAb 51A9, an isotype-matched mAb to the influenza virus (a gift of Dr. T. Moran, Mount Sinai School of Medicine, New York, NY). These mice were also fed a Western-type diet. In both experiments, total serum cholesterol levels were determined at the beginning and end of the experiments. Quantification of atherosclerotic lesions. At the end of the experiments, each mouse was killed, and the aorta was perfused with PBS via the left ventricle of the heart and then removed. Dissections were performed in a blinded manner using a Leica MZ 12.5 dissecting microscope (Leica Instruments, Allendale, NJ). The heart and ascending and descending aorta were dissected to the iliac bifurcation. Adventitial and adipose tissues were removed, and the outer curvature of the arch was cut longitudinally (10). Aortas were briefly rinsed with 70% ethanol, stained with Sudan IV for 5 minutes, and then destained for 5 minutes (10). Aortas were pinned, with the intimal surface up, on a black wax surface. Images were captured using a Leica DC 200 digital camera, and the surface area of the red-stained lesions was quantified with Image Pro Plus 4.1 software (Media Cybernetics, Silver Spring, MD). Results were expressed as the percentage of intimal surface involved. Immunohistochemistry. The aortic arch was transected at the level of the aortic sinuses, snap-frozen in embedding compound, and stored at ⫺80°C. Serial 6 m–thick transverse cryosections from the aortic arch through the aortic root were taken at 300-m intervals. Sections were air-dried, stained with oil red O, counterstained with hematoxylin, and 2975 Figure 1. Cross-reaction of antiphospholipid monoclonal antibody FB1 with native and oxidized low-density lipoproteins (LDLs). Enzyme-linked immunosorbent assay was performed with 20 g/ml of antigen and varying concentrations of FB1, as described in Materials and Methods. OD ⫽ optical density; MM-LDL ⫽ minimally modified LDL; CL ⫽ cardiolipin; ox-LDL ⫽ oxidized LDL. examined with a light microscope to assess the overall architecture and severity of atherosclerosis. RESULTS Binding of FB1 to LDLs at various stages of oxidation. To determine the binding specificity of FB1, we tested the mAb against LDL at various stages of oxidation. The process of oxidation occurred continuously, affecting both the lipid and protein moieties of LDL (7). FB1 reacted with all forms of LDL in a dose-dependent manner (Figure 1). However, the level of FB1 binding was inversely correlated with the oxidation of LDL, since FB1 reacted best with native LDL and least with ox-LDL. For purposes of comparison, Figure 1 also shows FB1 binding to CL. Reduction of atherosclerosis in LDLRⴚ/ⴚ mice by FB1. Because FB1 reacts with LDL in various forms, we investigated whether FB1 could modulate disease in atherosclerosis-prone mice. FB1 was administered to groups of LDLR⫺/⫺ mice consuming a Western-type diet as described above. In experiment 1, the passive administration of FB1 reduced the extent of atherosclerosis from 28% lipid lesions in PBS-treated mice to 20% in FB1-treated mice (P ⫽ 0.03), representing a 28% reduction in atherosclerosis (Figure 2A). The total serum cholesterol levels were unaffected by FB1 treatment (Figure 2B). To exclude any nonspecific effect of FB1, we repeated this experiment while administering an isotypematched irrelevant mAb to the control group. In experiment 2, treating mice with mAb FB1 again reduced the 2976 NICOLO ET AL Figure 2. Reduction of atherosclerosis in low-density lipoprotein receptor–deficient (LDLR⫺/⫺) mice by antiphospholipid monoclonal antibody FB1. A, In experiment 1, 16-week-old LDLR⫺/⫺ mice were injected intraperitoneally for 12 weeks with FB1 at a dosage of 1.5 mg/week, while control mice were injected with an equal volume of sterile phosphate buffered saline (PBS). Values are the mean and SEM percentage of atherosclerosis in the aortas of both groups of mice that were fed a Western-type diet beginning at the start of the injections. Lesions in the aortic root area were quantitated morphometrically, as described in Materials and Methods. The difference in the percentage of intimal involvement in lipid lesions between the PBS- and FB1-treated mice was statistically significant (P ⫽ 0.03). B, Scatterplot showing total serum cholesterol levels in the same mice as in A. Cholesterol levels were not significantly different between the 2 groups of mice. Each symbol represents an individual mouse. C, In experiment 2, 10-week-old LDLR⫺/⫺ mice were injected intraperitoneally for 12 weeks with FB1 at a dosage of 1.5 mg/week, while control mice received injections of 51A9, an irrelevant isotype-matched control antibody. The difference in lipid lesions between the 51A9- and FB1-treated mice was statistically significant (P ⫽ 0.007). Mice in experiment 1, which compared FB1 and PBS treatments, were 1.5 months older and, consequently, had a higher percentage of lipid lesions than the mice used in this experiment. Values are the mean and SEM. D, Scatterplot of total serum cholesterol levels in the same mice as in C. extent of atherosclerosis compared with mice treated with the anti-influenza virus control mAb 51A9. Figure 2C shows that FB1 treatment reduced the lipid lesions from 23% in control mAb–treated mice to 13% in FB1-treated mice (P ⫽ 0.007), representing a 43% reduction in atherosclerosis. Again, this reduction in atherosclerosis in FB1-treated mice was independent of alterations in total serum cholesterol levels (Figure 2D). The results of both of these experiments indicated that treatment with FB1 significantly reduces the extent of aortic lesions in LDLR⫺/⫺ mice. We compared the morphology and architecture of the lesions between the 51A9- and FB1-treated mice with the use of a light microscope to examine the oil red O–stained cryosections. Figure 3 shows representative sections taken from the aortic sinuses of both groups in experiment 2. The extent and severity of the lesions in the FB1-treated mice were qualitatively reduced compared with those in the 51A9-treated mice (Figures 3A and C). These histochemical staining results are consistent with those obtained using en face staining of the aortas from these 2 groups of mice (Figure 2C). In addition, we observed differences in the aortic medial layer between the 2 groups of mice. The aortic medial layer of the FB1-treated mice tended to be thicker than that of the 51A9-treated mice (Figures 3B and D). Moreover, lipid-containing cells were present in the aortic medial layer of the FB1-treated mice, whereas ROLE OF aPL IN THE PREVENTION OF ATHEROSCLEROSIS Figure 3. Effect of antiphospholipid monoclonal antibody (mAb) FB1 treatment on atherosclerosis in LDLR⫺/⫺ mice. Transverse cryosections were cut from aortic roots and stained with oil red O and hematoxylin. A, Representative section of aortic root from a control LDLR⫺/⫺ mouse receiving irrelevant isotype-matched control antibody. B, High-power photomicrograph of the area of lesion indicated by the asterisk in A, showing intimal atherosclerotic plaque composed of extracellular matrix with numerous lipid-containing cells (arrows) and small lipid droplets, which occupy most of the luminal surface. Aortic media (M) shows essentially no lipid deposits. C, Representative section from aortic root of FB1-treated LDLR⫺/⫺ mouse. The extent of luminal involvement by atherosclerosis is reduced in comparison with control mAb 51A9–treated mice, with reduction comparable with that seen in en face studies. D, High-power photomicrograph of the area of lesion indicated by the asterisk in C, showing relatively reduced intimal plaque thickness and lipid-containing cells (arrow) in the media. (Original magnification ⫻ 40 in A and C; ⫻ 400 in B and D.) none were seen in the aortic medial layer of the 51A9treated mice (Figures 3B and D). DISCUSSION Various studies suggest that autoantibodies to lipid antigens are present during both APS and atherosclerosis, and several mechanisms have been invoked to explain the epitope specificities common to both aPL and anti–ox-LDL antibodies. The molecular basis for this cross-reactivity may be related to the presence of phospholipids, such as CL, in the LDL particle (11). Some studies have suggested that the recognition of oxidation-specific epitopes is the explanation for this cross-reactivity (2). Those investigators generated a panel of mAb obtained from apolipoprotein E–deficient (Apo E⫺/⫺) mice (3). The mAb display preferential 2977 binding to oxidation-specific epitopes since they bind ox-LDL, but not native LDL. Likewise, the mAb from Apo E⫺/⫺ mice react with spontaneously oxidized CL, but not with reduced CL. FB1, the mAb used in the present study, displayed a different pattern of crossreactivity. FB1 reacted with both native LDL and oxLDL. In fact, FB1 binding to ox-LDL was decreased relative to native LDL. Likewise, FB1 reacted with both naturally oxidized and hydrogenated CL (data not shown). The differences in binding specificities between the mAb FB1 and Apo E⫺/⫺ may be related to their different origins. Consistent with our current findings, a recent study showed that human aPL react equally well with reduced and oxidized forms of CL (12). A remarkable property of FB1 is its ability to decrease atherosclerosis in LDLR⫺/⫺ mice. This effect may seem paradoxical since this mAb comes from diseased animals. The role of antibodies during atherosclerosis remains unclear and has led to contradictory suggestions. Autoantibodies could play a role in the disease by forming immune complexes and aggravating the inflammatory process. Alternatively, antibodies may not participate in the pathogenesis and could merely be an epiphenomenon resulting from the hyperlipidemia during the disease. More recently, several authors have suggested that some antibodies may in fact improve atherosclerotic manifestations, although their mechanisms of action have not been elucidated. Some have suggested that autoantibodies to ox-LDL may act as scavengers and clear ox-LDL from the circulation or prevent their uptake by macrophages (6,7). Because FB1 can bind to both native LDL and ox-LDL, it is likely that FB1 has additional effects besides preventing ox-LDL internalization. The finding of increased medial foam cells in the aortas of FB1-treated mice was unexpected, and the mechanism underlying this effect is not currently obvious. Potentially involved mechanisms could include altered permeability characteristics and/or ligand– receptor interactions of FB1-modified lipoproteins, or FB1-related changes in adhesion molecule expression in the aortic vasa vasorum of treated animals. Elucidation of the underlying mechanism will require identification of the cell type(s) of the medial foam cells, which is beyond the scope of current studies. Studies in atherosclerosis-prone mice suggest that humoral immunity can be beneficial, since immunization with ox-LDL can reduce the severity of disease (13,14). However, it is likely that within the same atherosclerotic individual, several antibody populations coexist that can have beneficial or detrimental effects 2978 NICOLO ET AL upon the disease. Harnessing humoral immunity to reduce plaque formation in human atherosclerosis will depend upon identifying the fine specificities of protective antibodies. 7. ACKNOWLEDGMENTS We thank M. Fowler, Z. Khan, H. Bradford, and Drs. A. 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