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Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:864-870
Published online before print February 2, 2006, doi: 10.1161/01.ATV.0000206122.61591.ff
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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:864.)
© 2006 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Adoptive Transfer of CD4+ T Cells Reactive to Modified Low-Density Lipoprotein Aggravates Atherosclerosis

Xinghua Zhou; Anna-Karin L. Robertson; Charlotta Hjerpe; Göran K. Hansson

From the Center for Molecular Medicine and Department of Medicine, Karolinska Institutet, Stockholm, Sweden. A.-K.L.R.’s current address: Section of Immunobiology, Yale University School of Medicine, New Haven, Conn.

Correspondence to Xinghua Zhou, MD, PhD, FESC, Center for Molecular Medicine L8:03, Karolinska Hospital, SE-17176 Stockholm, Sweden. E-mail Xinghua.Zhou{at}ki.se


*    Abstract
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 *Abstract
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Objective— Atherosclerosis is associated with immune responses to oxidized low-density lipoprotein (oxLDL). The presence of activated macrophages and T cells in lesions suggests that cell-mediated immune reactions are taking place during the disease process. However, the role of specific immune responses has remained unclear. We have previously shown that transfer of CD4+ T cells from apolipoprotein E knockout mice (apoE–/–) into immunodeficient apoE–/– scid/scid mice accelerates disease.

Methods and Results— To test whether this effect is dependent on specific disease-associated antigens, purified CD4+ T cells from oxLDL-immunized mice were transferred into apoE–/– scid/scid mice. CD4+ T cells from mice immunized with a nonrelevant antigen, keyhole limpet hemocyanin (KLH), and naïve CD4+ T cells were used as controls. After 12 weeks, all mice that received T cells had larger lesions than untouched apoE–/– scid/scid controls. However, mice receiving CD4+ T cells from oxLDL immunized mice had substantially accelerated lesion progression compared with those receiving naive or KLH-primed T cells. Circulating levels of interferon-{gamma} were increased in proportion to the acceleration of atherosclerosis.

Conclusion— These data show that adoptive transfer of purified CD4+ T cells from oxLDL-immunized mice accelerates atherosclerosis. They support the notion that Th1 cellular immunity is proatherogenic and identify oxLDL as a culprit autoantigen.

To test whether atherogenesis depends on specific disease-associated antigens, we transferred CD4+ T-cells from immunized donors into apoE–/– scid/scid mice. Mice receiving T cells from oxLDL-immunized mice had accelerated lesion progression compared with those receiving naive or KLH-primed T cells. These findings demonstrate that T-cell response to oxLDL is critical in atherogenesis.


Key Words: atherosclerosis • lymphocytes • low-density lipoprotein • immune system • mice, knockout–/–


*    Introduction
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Research over the last decades has identified immune mechanisms as pathogenically important in atherosclerosis.1,2 T lymphocytes and macrophages are present in lesions throughout the development of disease, both in man and experimental models.3–6 Most of the T cells in the lesions are CD4+ TCRab+ T cells expressing activation markers such as human leukocyte antigen-DR (HLA-DR) and interferon-{gamma} (IFN-{gamma}).3,4,7–9 The expression of major histocompatibility complex (MHC) class II molecules such as HLA-DR on activated macrophages and activated T cells adjacent to these macrophages in the lesions strongly suggests that cell-mediated immune reactions are taking place in atherosclerosis.

A series of investigations have suggested modified low density lipoproteins (LDLs) as autoantigens in atherosclerosis.10 LDL accumulates in lesions, where it is modified by oxidative and enzymatic processes.11–15 Circulating autoantibodies to epitopes of oxidized LDL (oxLDL) have been detected in patients and experimental animals with atherosclerosis,13,16 and antibodies isolated from atherosclerotic lesions recognize oxLDL.17 Macrophages can take up, process, and present neoantigens such as oxLDL to CD4+ T cells, eliciting cellular immune responses.18 Indeed, &10% of the CD4+ T cells cloned from human lesions recognize oxLDL in an MHC class II-dependent manner.9

The apolipoprotein E knockout (apoE–/–) mouse is a useful model of human disease because it develops severe hypercholesterolemia and spontaneous atherosclerosis.19,20 In this model, a local immune response dominated by CD4+ T cells occurs in all phases of atherosclerosis.5,21 T cells from immune organs of apoE–/– mice exhibit strong reactivity toward oxLDL.22 In addition, high titers of circulating autoantibodies to oxLDL are detected in this model.23 However, the role of the T-cell immune response to modified LDL in the disease process has remained unclear.

To study the role of adaptive immunity in atherosclerosis, we generated an immunodeficient apoE–/– scid/scid mouse by crossing apoE–/– mice with the scid/scid strain, which lacks T and B cells.24 We reported previously that transfer of CD4+ T cells from aged apoE–/– mice into immunodeficient apoE–/– scid/scid mice accelerates the progression of atherosclerosis.24 To determine whether this effect is dependent on specific disease-associated antigens, CD4+ T cells from mice immunized with oxLDL were transferred into apoE–/– scid/scid mice. Controls included CD4+ T cells from mice immunized with a nonrelevant antigen, keyhole limpet hemocyanin (KLH), or with incomplete Freund’s adjuvant (IFA) alone, as well as untouched mice. Our results demonstrate an important role for the oxLDL-specific T-cell response as an accelerating factor in atherosclerosis.


*    Methods
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 up arrowIntroduction
 *Methods
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Animals, Diets, and Serum Analysis
C57BL/6J mice obtained from Taconic Europe, Ry Denmark, and apoE–/– scid/scid mice on the C57BL/6J background were generated in our laboratory,24 kept under sterile conditions, and fed standard mouse chow. All experiments were approved by the local ethics committee. The apoE–/– scid/scid mice were injected intravenously with CD4+ T cells at 7 weeks of age and euthanized at 16 weeks. To exclude a potential "leakage" (ie, appearance of immune cells), which has been reported in aged scid/scid animals, we assessed the frequency of immunocompetent cells by flow cytometry. Splenocytes were stained with fluorescein isothiocyanate-conjugated rat anti-CD3, phycoerythrin-conjugated rat anti-CD8, phycoerythrin-conjugated rat anti-CD19, and CyChrome conjugated rat anti-CD4. All antibodies were from PharMingen. Blood was obtained by heart puncture at euthanization and allowed to clot. Sera were centrifuged at 14 000g for 20 minutes to remove chylomicrons. The serum IFN-{gamma}, interleukin-4 (IL-4), and IL-5 levels were analyzed by sandwich ELISA with OptELA antibody sets (PharMingen) and serum cholesterol by the cholesterol oxidase method (Boehringer Mannheim).

Homologous LDL Isolation and Modification
Blood was obtained by heart puncture from 8- to 10-week-old apoE–/– mice and pooled into vacuum tubes containing K2EDTA. LDL was isolated from plasma by ultracentrifugation through a discontinuous NaCl gradient of 1.020 to 1.063 mg/mL for 20 hours at 4°C in a Beckman L8-80 ultracentrifuge with a 50.3-Ti Beckman fixed-angle rotor.16,25 In plasma from apoE–/– mouse, this density cutoff may contain chylomicron remnants in addition to LDL.25,26 The protein content was determined by the Lowry method, and the LDL preparation with added Na2EDTA (1 mg/mL) was sterile filtered, kept at 4°C under N2, and used within 2 weeks. Malondialdehyde (MDA) modification of LDL was performed as described previously.23

Immunization Protocol and Analysis of Antigen Specificity
At 6 weeks of age, male C57BL/6J donor mice were randomly divided into 3 groups (n=40 per group). Two groups were injected peritoneally with either homologous MDA-LDL (100 µg protein per mouse) or KLH (Pierce; 100 µg protein per mouse) and boosted once after 2 weeks. MDA-LDL and KLH were emulsified with IFA before immunization. The third group was used as donors of naïve T cells.

Splenocytes were obtained on day 5 after the booster. The splenocytes from individual mice were cultured in duplicate in serum-free medium including 1:100 ITS+Premix (Collaborative Biomedical), 1 mg/mL BSA, 10 mmol/L HEPES, 1 mmol/L Na pyruvate, 1 mmol/L nonessential amino acids, and 50 µg/mL gentamicin sulfate (Sigma) after osmotic lyses of red blood cells. These cells were incubated in the absence of or in the presence of titrated amounts of homologous MDA-LDL or KLH for 72 hours. The incubation was followed by overnight incorporating with 3H-thymidine for the assay of cell proliferation. To quantify the T-cell cytokines secreted by the splenocytes, supernatants harvested 72 hours after incubation were measured using a Th1/Th2 Cytokine CBA Kit (BD Biosciences) according to manufacturer instruction.

Negative Selection and Adoptive Transfer of CD4+ T Cells
Splenocytes from immunized mice were pooled and incubated in plastic dishes for 90 minutes at 37°C after red blood cells were lysed. Nonadherent cells were incubated at 4°C for 30 minutes with biotin-conjugated rat-anti-mouse-CD19/CD22, anti-mouse-CD8, anti-mouse-Mac-1 (CD11b), and anti-mouse-c-kit (CD117) to remove potential peripheral stem cells. This was followed by addition of streptavidin-coated microbeads and elimination on MiniMACS columns. The purified cells were >95% viable and contained >99% CD3+CD4+ T cells and <1% CD22+ B cells as judged by flow cytometry. At 7 weeks of age, female apoE–/– scid/scid mice were randomly divided into 4 groups (n=5 to 9 per group) as follows: (1) transfer of CD4+ T cells from mice immunized with MDA-LDL (MDA-LDLCD4+), (2) transfer of CD4+ T cells from KLH-immunized mice (KLHCD4+), (3) transfer of CD4+ T cells from unimmunized mice (naïveCD4+), and (4) untouched controls. Every mouse received 12x106 CD4+ T cells via the tail vein. All recipients were euthanized at 16 weeks of age (ie, 9 weeks after cell transfer).

Quantitation of Plaque Size, Cellular Components, and Markers of Immune/Inflammatory Activation
Recipient apoE–/– scid/scid mice were euthanized in a CO2 chamber at 16 weeks of age and perfused transcardially with PBS. The heart was dissected out and the tissue segment from sinus aorticus to the lower tips of the right and left atria was isolated, snap-frozen in liquid nitrogen, and embedded with O.C.T. (Tissue-Tek) compound. The tissue was sectioned at a thickness of 10 µm. For morphometric analysis, 5 sections starting at the level of the aortic valves were chosen at 100-µm intervals. Sections were stained with Oil Red O and counterstained with hematoxylin. The size of the plaque was measured with Leica Q500MC image analysis software. The mean value of plaque cross-section areas from 5 sections was used to estimate the extent of atherosclerosis.24

To evaluate T-cell infiltration and inflammatory molecule expression in the plaques, immunohistochemistry staining was performed by using monoclonal biotin-conjugated rat anti-CD4, anti-I-Ab (MHC class II) and anti-vascular cell adhesion molecule-1 (VCAM-1) followed by avidin-horseradish peroxidase.24

Statistics
Results are expressed as mean±SEM. Data were analyzed by the Mann-Whitney test. The significance level was set at P<0.05.


*    Results
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 *Results
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Induction of Antigen-Specific T Cells
The adoptive transfer experiment was performed to assess the role of specific antigen in the disease-aggravating action of CD4+ T cells on atherosclerosis. Six-week-old C57BL/6J (denoted B6) rather than apoE–/– mice were chosen as donors because their T cells are largely näive, whereas the autoimmune process starts early in apoE–/– mice. The experimental design is outlined in Figure 1. Donor B6 mice were immunized twice with MDA-LDL or the control antigen KLH. To avoid inducing a T-cell response to other antigens (eg, heat shock proteins), IFA rather than complete Freund’s adjuvant was used in our immunization protocol. The splenocytes of MDA-LDL-immunized mice showed a dose-dependent proliferation in response to in vitro stimulation with MDA-LDL but not to KLH (Figure 2A and 2B). Strong systemic IgG responses were registered to the respective antigens (Figure I, available online at http://atvb.ahajournals.org). T-cell proliferation was accompanied by increased expression of the T-cell cytokines IFN-{gamma} and IL-2 (Figure 2C and 2D). Splenocytes collected from KLH-primed mice exhibited significant proliferation when exposed to KLH but not to MDA-LDL in vitro and was accompanied by increased expression of the T-cell cytokines IFN-{gamma}, IL-2, and IL-5 (Figure 2B, 2F through 2H). The results of the proliferation assay and the expression pattern of the T-cell cytokines therefore indicated the induction of antigen-specific T cells to the respective antigens used for immunization. No IL-4 production was detected in any of the groups, as is usually the case with B6 mice (data not shown). Robust IL-5 secretion was registered from the KLH-primed T cells when challenged in vitro with KLH (Figure 2I). In contrast, IL-5 level did not increase when T cells from MDA-LDL-immunized mice were exposed to their cognate antigen (ie, MDA-LDL; Figure 2I). Expression of tumor necrosis factor-{alpha} (TNF-{alpha}) increased in T cells from MDA-LDL-immunized mice in response to in vitro MDA-LDL stimulation (Figure 2E), whereas TNF-{alpha} levels were not significantly changed in KLH-primed group after stimulation with KLH (data not shown). In conclusion, a Th1 response was observed when splenocytes from mice immunized with MDA-LDL were challenged with MDA-LDL in vitro. In contrast, both Th1 and Th2 responses took place in the splenocytes primed with KLH and stimulated in vitro with KLH (Figure 2I).


Figure 1
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  		Figure 1. Illustration of experimental design.At 6 weeks of age, C57BL/6J mice were injected peritoneally with either homologous MDA-LDL or KLH in IFA and boosted after 2 weeks. An untreated group served as donors of naïve T cells. Purified spleenic CD4+ T cells were transferred intravenously into the apoE–/– scid/scid mice.


Figure 2
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  		Figure 2. Induction of antigen-specific T cells in immunized donor mice and effects of CD4+ T cell transfer on circulating IFN-{gamma} levels. A and B, Splenocyte proliferation assay. The cells from individual mouse after lyses of red blood cells were cultured in serum-free medium with titrated amounts of homologues MDA-LDL or KLH and the antigen-specific proliferation was measured by 3(H)-thymidin incorporation. C through I, Antigen-specific T-cell cytokine secretion. Analysis of the cytokines in cultures of the cells from individual mouse was performed after 72-hour incubation with different amounts of antigens. J, IFN-{gamma} concentration (pg/mL) in peripheral blood of the T-cell receivers at 16 weeks old. Mean±SD; n=6; *P<0.05 vs control.

Phenotype of Recipient ApoE–/– scid/scid Mice
At 16 weeks of age, no differences were registered in body weight or serum cholesterol levels between treatment groups (Table). No T cells, B cells, or immunoglobulins could be detected in the untouched apoE–/– scid/scid mice. As expected, CD4+ T cells but not CD8+ T cells or B cells were found in mice that had received CD4+ T cell transfer. There was no significant difference in the number of CD4+ T cells in spleens between the groups that received T cells from different donor groups (Table). Interestingly, serum concentrations of the Th1 cytokine IFN-{gamma} were very low both in untouched apoE–/– scid/scid mice and in mice receiving either naïveCD4+ or KLHCD4+ T cells but increased significantly in the group transferred with MDA-LDLCD4+ T cells (Figure 2J). This may reflect an antigen-specific activation of MDA-LDL specific CD4+ cells in vivo. Serum levels of the Th2 cytokines IL-4 and IL-5 were below the detection limit for all groups (data not shown).


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  		Bodyweight, Serum Lipids, and Cellular Composition in the Spleens

Lesion Development in ApoE–/– scid/scid Mice After Adoptive Transfer
Compared with untreated apoE–/– scid/scid mice, fatty streak lesions in the aortic root were on average 227% larger in apoE–/– scid/scid mice that received MDA-LDLCD4+ T cells, 112% larger in the KLHCD4+ T cell transfer group, and 127% larger in the group receiving naïveCD4+ T cells from untouched B6 mice (Figure 3). Lesion development was significantly increased in apoE–/– scid/scid mice that received MDA-LDLCD4+ T cells compared with those receiving either KLHCD4+ or naïveCD4+ T cells (P=0.008 and 0.019; Figure 3). The latter 2 treatment groups did not differ significantly from each other with regard to lesion size but differed from lesion size in untreated mice (Figure 3).


Figure 3
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  		Figure 3. Effects of CD4+ T cell transfer on atherosclerotic lesion development. Morphometric quantitation of lesion size (µm2) in aortic root from 16-week-old untouched apoE–/– scid/scid and from apoE–/– scid/scid, which received naïveCD4+ T cells, KLHCD4+ T cells, or MDA-LDLCD4+ T cells. Mean±SD; *P<0.05 vs the group received CD4+ T cells from untreated group; #P<0.05 vs the groups received CD4+ T cells from either naïve or KLH groups.

T-Cell Homing and Inflammatory Molecule Expression in the Lesions After Specific T-Cell Transfer
Immunohistochemical analysis showed a strikingly increased infiltration of CD4+ cells into the lesions after MDA-LDLCD4+ T cell transfer (Figure 4A andB) compared with other treatments. Furthermore, expression of the MHC class II molecule I-Ab was increased several-fold in mice receiving MDA-LDLCD4+ T cells (Figure 4A and 4C). VCAM-1 expression was significantly increased in the lesions of all apoE–/– scid/scid mice that had received CD4+ T cells, irrespective of the specificity of the transferred cells (Figure 4A and 4D).


Figure 4
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  		Figure 4. Effects of CD4+ T-cell transfer on lipid accumulation and inflammatory gene expression in aortic lesions. A, The micrographs show lesion lipid deposition by oil red O (ORO) staining (original magnification x50), CD4+ T-cell infiltration (x400), I-Ab, and VCAM-1 expression (x400) from 16-week-old untouched apoE–/– scid/scid and from apoE–/– scid/scid, which received naïveCD4+ T cells, KLHCD4+ T cells, or MDA-LDLCD4+ T cells. C and D, Data were obtained by counting lesions at the aortic root at x400 magnification from 4 to 6 mice of each group. Values represent CD4 or I-Ab or VCAM-1–positive cells divided by the lesion area and are given as mean±SD. *P<0.05 vs other groups.


*    Discussion
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 *Discussion
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The results of this study show, for the first time, that adoptive transfer of cell-mediated immunity to oxLDL accelerates atherosclerosis. Thus, transfer of CD4+ T cells from MDA-LDL-immunized mice caused a 3.4-fold increase in lesion size in apoE–/– scid/scid animals. This was nearly a doubling of lesion size when compared with the situation in mice receiving CD4+ T cells from mice immunized with another antigen: KLH. This increase was accompanied by systemic elevation of the Th1 cytokine IFN-{gamma}, by increased penetration of CD4+ T cells into lesions, and by augmented expression of (the IFN-{gamma}–inducible) I-Ab protein in the lesions, supporting the notion that Th1 cells producing IFN-{gamma} play an important role in the disease process. The fact that transfer of CD4 cells into immunodeficient mice aggravates the disease is in line with our previous finding that absence of CD4 cells in apoE–/– mice leads to reduced atherosclerosis.26 A proposed mechanism by which the transferred MDA-LDL-specific CD4+ T cells promote atherosclerosis was illustrated (Figure 5). It should be noted that the cholesterol levels were not different among the groups, therefore the acceleration of the disease progression was unlikely because of effects on lipid metabolism. In parallel with augmented lesion development, the mice that received MDA-LDLCD4+ T cells showed an increase in the number of CD4+ T cells in lesions. However, the proportion of CD4+ T cells in the spleen did not differ between the recipients of MDA-LDLCD4+ T cells and of KLHCD4+ T cells. These findings suggest that the MDA-LDL-specific CD4+ T cells homed to and expanded in the atherosclerotic lesions, where the MDA-LDL antigen accumulates.


Figure 5
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  		Figure 5. Proposed mechanism by which ox-LDL-specific CD4+ T cells promote atherosclerosis. Hypercholesterolemia leads to the accumulation and oxidative modification of LDL in the intima. Monocytes and T cells adherent to the endothelium are stimulated by chemokines and migrate into the intima, where the monocytes differentiate into macrophages. These macrophages can internalize oxLDL and present processed fragment of it to CD4+ T cells. Among the lesions, those carrying appropriate TCR (dark gray) recognize antigen and are activated, leading to proliferation and cytokine secretion. IFN-{gamma}, a major cytokine of activated CD4+ T cells, acts on lesion cells and initiates a cascade of inflammatory signaling that promotes disease. The CD4+ T cells that recognize nonatherosclerosis-related antigens (light gray) may act as bystanders in lesions or return to the circulation.

A Th1 response to MDA-LDL was registered when splenocytes harvested from the mice immunized with MDA-LDL were challenged with MDA-LDL in vitro. In contrast, Binder et al reported recently that immunization with MDA-LDL induces Th2-biased responses,27 although complete Freund’s adjuvant-containing mycobacteria, which was used in their study, is well known to be more prone to induce Th1 responses than IFA, which we used.28 The discrepancy between 2 studies may be explained by the different types of adjuvant, the dose of antigen used for the immunization, the number of boost injections, the composition of the culture medium, or the concentration of antigen applied in vitro. Serum-free medium was used in our study because sera used in cell culture contain high levels of LDL, which may obscure any T-cell response to exogenous LDL particles. Importantly, the peak response of the T cells in vitro to MDA-LDL was detected when a low concentration of oxLDL was added to the cultures, whereas a concentration of oxLDL >20 µg/mL was found to induce cytotoxicity. The precise T-cell epitopes in the oxLDL preparations remain to be identified.

Interestingly, transfer of naïveCD4+ or KLHCD4+ T cells resulted in modestly increased lesions with signs of increased inflammatory activity when compared with untouched control apoE–/– scid/scid mice. This may reflect the presence in the naïveCD4+ and the KLHCD4+ T cell preparations of a T-cell population with the capacity to recognize modified LDL or other proatherogenic antigens. Such cells would be activated in situ during the course of the disease, respond immunologically, and aggravate disease development. Alternatively, the homeostatic proliferation of cells transferred in an immunodeficient host could accelerate atherosclerosis because of cytokine secretion or other immune interactions.

VCAM-1 is abundantly expressed in atherosclerotic lesions.29–31 It can be induced via the nuclear factor {kappa}B pathway, which is activated by proinflammatory cytokines, endotoxins, and several other stimuli.32 In the current study, VCAM-1 was induced to approx the same extent, irrespective of the antigenic specificity of transferred T cells. Our findings suggest that VCAM-1 expression reflects vascular inflammation but is less dependent on antigenic specificity than I-A.

Several gene products of activated T cells are likely to be important in atherosclerosis. Numerous studies point to a proatherogenic role of Th1 products such as IFN-{gamma} and TNF-{alpha}.1,4,33,34 Not only does IFN-{gamma} promote antigen presentation by upregulating MHC genes, it also controls scavenger receptor expression as well as cholesterol efflux in macrophages, proliferation, differentiation and collagen production in smooth muscle cells, and hemostatic properties of the endothelium.1,35,36 TNF-{alpha} released by activated Th1 cells has profound effects on adhesion molecule expression, lipolytic enzymes, and several other phenomena. The cell surface molecule CD40 ligand (CD154) can activate macrophages and vascular cells, which can express CD40; this leads to secretion of proteolytic enzymes, tissue factor, and other important factors in the atherogenic process.37 Other costimulatory proteins expressed by immune cells include OX40L, a genotype of which was recently found to associate with coronary artery disease in man.38 All these effector molecules may be involved in the proatherogenic activity of CD4+ T cells, but it remains to be determined whether the properties of the activating antigens affect the effector repertoire. At any rate, our results confirm the notion that CD4+ T cells are proatherogenic, support the notion that their disease-aggravating activity may be mediated via Th1 effector molecules, and show that antigenic specificity for the disease-associated antigen oxLDL is associated with dramatic proatherogenic activity. This may be because oxLDL accumulates in the lesions, where it provides a strong local stimulus for T-cell activation.

It has been shown that systemic immunization with oxLDL decreases lesion development with significant elevation of IgG antibodies against oxLDL.25,39–41 The finding that immunization with MDA-LDL protects from atherosclerosis in immunocompetent apoE–/– mice25,26,39–41 whereas transfer of CD4+ T cells from mice immunized with MDA-LDL into immunodeficient apoE–/– mice aggravates disease in this study may appear puzzling. However, the apoE–/–scid/scid mice lack both B and T cells. Therefore, any effects of B cells, including antibody-producing plasma cells derived from them as well as effects of CD8+ T cells, were absent in the present transfer study. In addition, the antigen-presenting cells in apoE–/– scid/scid are never directly exposed to the antigens in the preparation used for immunization, including adjuvant, precluding a possible development of tolerogenic antigen-presenting cells. Furthermore, any early protective mechanisms induced by the immunization may not be transferred if the transferred cells are isolated as late as 20 days after the immunization. The fact that the immunization-induced atheroprotection remains in the mice lacking CD4 suggests that other cells than CD4+ T cells may play an atheroprotective role after immunization.26 Recent reports also suggest that humoral immune responses may have protective effects on disease development.10,42 Because immunization with oxLDL enhances both cellular and humoral immune responses to the atherosclerosis-related antigen, it is possible that some of the mechanisms activated by immunization are deleterious, whereas others are protective. The timing for the induction of such reaction may also relevant. Further studies are necessary to explore how the balance between such protection and aggravation induced by immunization can be controlled.

To summarize, the present data identify by adoptive transfer methodology that oxidatively modified LDL is an autoantigen that promotes atherosclerosis in hypercholesterolemic mice.


*    Acknowledgments
 
Our work was supported by the Swedish Research Council (projects 6816 and 14245), Heart Lung Foundation and Söderberg Foundation, AFA, Gun och Bertil Stohnes Foundation, Loo och Hans Ostermans Foundation, Gamla Tjänarinnor Foundation, and Nanna Swartz Foundation. X.Z. is a recipient of a career investigator award from the Swedish Research Council. We thank Ingrid Törnberg and Kristina Edwardsson for excellent technical assistance.


*    Footnotes
 
Consulting editor for this article was Peter Libby, MD, Brigham and Women’s Hospital, Boston, Mass.

Received July 7, 2005; accepted January 11, 2006.


*    References
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 *References
 

  1. Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med. 2005; 352: 1685–1695.[Free Full Text]
  2. Zhou X, Hansson GK. Immunomodulation and vaccination for atherosclerosis. Expert Opin Biol Ther. 2004; 4: 599–612.[CrossRef][Medline] [Order article via Infotrieve]
  3. Jonasson L, Holm J, Skalli O, Bondjers G, Hansson GK. Regional accumulations of T cells, macrophages, and smooth muscle cells in the human atherosclerotic plaque. Arteriosclerosis. 1986; 6: 131–138.[Medline] [Order article via Infotrieve]
  4. Hansson GK, Holm J, Jonasson L. Detection of activated T lymphocytes in the human atherosclerotic plaque. Am J Pathol. 1989; 135: 169–175.[Abstract]
  5. Zhou X, Stemme S, Hansson GK. Evidence for a local immune response in atherosclerosis. CD4+ T cells infiltrate lesions of apolipoprotein-E-deficient mice. Am J Pathol. 1996; 149: 359–366.[Abstract]
  6. Llodra J, Angeli V, Liu J, Trogan E, Fisher EA, Randolph GJ. Emigration of monocyte-derived cells from atherosclerotic lesions characterizes regressive, but not progressive, plaques. Proc Natl Acad Sci U S A. 2004; 101: 11779–11784.[Abstract/Free Full Text]
  7. Jonasson L, Holm J, Skalli O, Gabbiani G, Hansson GK. Expression of class II transplantation antigen on vascular smooth muscle cells in human atherosclerosis. J Clin Invest. 1985; 76: 125–131.[Medline] [Order article via Infotrieve]
  8. van der Wal AC, Das PK, Bentz van de Berg D, van der Loos CM, Becker AE. Atherosclerotic lesions in humans. In situ immunophenotypic analysis suggesting an immune mediated response. Lab Invest. 1989; 61: 166–170.[Medline] [Order article via Infotrieve]
  9. Stemme S, Faber B, Holm J, Wiklund O, Witztum JL, Hansson GK. T lymphocytes from human atherosclerotic plaques recognize oxidized low density lipoprotein. Proc Natl Acad Sci U S A. 1995; 92: 3893–3897.[Abstract/Free Full Text]
  10. Binder CJ, Chang MK, Shaw PX, Miller YI, Hartvigsen K, Dewan A, Witztum JL. Innate and acquired immunity in atherogenesis. Nat Med. 2002; 8: 1218–1226.[CrossRef][Medline] [Order article via Infotrieve]
  11. Haberland ME, Fong D, Cheng L. Malondialdehyde-altered protein occurs in atheroma of Watanabe heritable hyperlipidemic rabbits. Science. 1988; 241: 215–218.[Abstract/Free Full Text]
  12. Boyd HC, Gown AM, Wolfbauer G, Chait A. Direct evidence for a protein recognized by a monoclonal antibody against oxidatively modified LDL in atherosclerotic lesions from a Watanabe heritable hyperlipidemic rabbit. Am J Pathol. 1989: 135: 815–825.[Abstract]
  13. Palinski W, Rosenfeld ME, Yla-Herttuala S, Gurtner GC, Socher SS, Butler SW, Parthasarathy S, Carew TE, Steinberg D, Witztum JL. Low density lipoprotein undergoes oxidative modification in vivo. Proc Natl Acad Sci U S A. 1989; 86: 1372–1376.[Abstract/Free Full Text]
  14. Jurgens G, Chen Q, Esterbauer H, Mair S, Ledinski G, Dinges HP. Immunostaining of human autopsy aortas with antibodies to modified apolipoprotein B and apoprotein(a). Arterioscler Thromb. 1993; 13: 1689–1699.[Abstract/Free Full Text]
  15. Palinski W, Ord VA, Plump AS, Breslow JL, Steinberg D, Witztum JL. ApoE-deficient mice are a model of lipoprotein oxidation in atherogenesis. Demonstration of oxidation-specific epitopes in lesions and high titers of autoantibodies to malondialdehyde-lysine in serum. Arterioscler Thromb. 1994; 14: 605–616.[Abstract/Free Full Text]
  16. Yla-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, Witztum JL, Steinberg D. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest. 1989; 84: 1086–1095.[Medline] [Order article via Infotrieve]
  17. Yla-Herttuala S, Palinski W, Butler SW, Picard S, Steinberg D, Witztum JL. Rabbit and human atherosclerotic lesions contain IgG that recognizes epitopes of oxidized LDL. Arterioscler Thromb. 1994; 14: 32–40.[Abstract/Free Full Text]
  18. Nicoletti A, Caligiuri G, Tornberg I, Kodama T, Stemme S, Hansson GK. The macrophage scavenger receptor type A directs modified proteins to antigen presentation. Eur J Immunol. 1999; 29: 512–521.[CrossRef][Medline] [Order article via Infotrieve]
  19. Piedrahita JA, Zhang SH, Hagaman JR, Oliver PM, Maeda N. Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells. Proc Natl Acad Sci U S A. 1992; 89: 4471–4475.[Abstract/Free Full Text]
  20. Plump AS, Smith JD, Hayek T, Aalto-Setala K, Walsh A, Verstuyft JG, Rubin EM, Breslow JL. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells. Cell. 1992; 71: 343–353.[CrossRef][Medline] [Order article via Infotrieve]
  21. Roselaar SE, Kakkanathu PX, Daugherty A. Lymphocyte populations in atherosclerotic lesions of apoE –/– and LDL receptor –/– mice. Decreasing density with disease progression. Arterioscler Thromb Vasc Biol. 1996; 16: 1013–1018.[Abstract/Free Full Text]
  22. Caligiuri G, Nicoletti A, Zhou X, Tornberg I, Hansson GK. Effects of sex and age on atherosclerosis and autoimmunity in apoE-deficient mice. Atherosclerosis. 1999; 145: 301–308.[CrossRef][Medline] [Order article via Infotrieve]
  23. Zhou X, Paulsson G, Stemme S, Hansson GK. Hypercholesterolemia is associated with a T helper (Th) 1/Th2 switch of the autoimmune response in atherosclerotic apo E-knockout mice. J Clin Invest. 1998; 101: 1717–1725.[Medline] [Order article via Infotrieve]
  24. Zhou X, Nicoletti A, Elhage R, Hansson GK. Transfer of CD4(+) T cells aggravates atherosclerosis in immunodeficient apolipoprotein E knockout mice. Circulation. 2000; 102: 2919–2922.[Abstract/Free Full Text]
  25. Zhou X, Caligiuri G, Hamsten A, Lefvert AK, Hansson GK. LDL immunization induces T-cell-dependent antibody formation and protection against atherosclerosis. Arterioscler Thromb Vasc Biol. 2001; 21: 108–114.[Abstract/Free Full Text]
  26. Zhou X, Robertson AK, Rudling M, Parini P, Hansson GK. Lesion development and response to immunization reveal a complex role for CD4 in atherosclerosis. Circ Res. 2005; 96: 427–434.[Abstract/Free Full Text]
  27. Binder CJ, Hartvigsen K, Chang MK, Miller M, Broide D, Palinski W, Curtiss LK, Corr M, Witztum JL. IL-5 links adaptive and natural immunity specific for epitopes of oxidized LDL and protects from atherosclerosis. J Clin Invest. 2004; 114: 427–437.[CrossRef][Medline] [Order article via Infotrieve]
  28. Billiau A, Matthys P. Modes of action of Freund’s adjuvants in experimental models of autoimmune diseases. J Leukoc Biol. 2001; 70: 849–860.[Abstract/Free Full Text]
  29. Cybulsky MI, Gimbrone MA Jr. Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis. Science. 1991; 251: 788–791.[Abstract/Free Full Text]
  30. Li H, Cybulsky MI, Gimbrone MA Jr, Libby P. An atherogenic diet rapidly induces VCAM-1, a cytokine-regulatable mononuclear leukocyte adhesion molecule, in rabbit aortic endothelium. Arterioscler Thromb. 1993; 13: 197–204.[Abstract/Free Full Text]
  31. Li H, Cybulsky MI, Gimbrone MA Jr, Libby P. Inducible expression of vascular cell adhesion molecule-1 by vascular smooth muscle cells in vitro and within rabbit atheroma. Am J Pathol. 1993; 143: 1551–1559.[Abstract]
  32. Collins T, Read MA, Neish AS, Whitley MZ, Thanos D, Maniatis T. Transcriptional regulation of endothelial cell adhesion molecules: NF-kappa B and cytokine-inducible enhancers. FASEB J. 1995; 9: 899–909.[Abstract]
  33. Gupta S, Pablo AM, Jiang X, Wang N, Tall AR, Schindler C. IFN-gamma potentiates atherosclerosis in ApoE knock-out mice. J Clin Invest. 1997; 99: 2752–2761.[Medline] [Order article via Infotrieve]
  34. Whitman SC, Ravisankar P, Elam H, Daugherty A. Exogenous interferon-gamma enhances atherosclerosis in apolipoprotein E–/– mice. Am J Pathol. 2000; 157: 1819–1824.[Abstract/Free Full Text]
  35. Hansson GK. Immune mechanisms in. Arterioscler Thromb Vasc Biol. 2001; 21: 1876–1890.[Abstract/Free Full Text]
  36. Fazio S, Linton MF. The inflamed plaque: cytokine production and cellular cholesterol balance in the vessel wall. Am J Cardiol. 2001; 88: 12E–15E.[CrossRef][Medline] [Order article via Infotrieve]
  37. Schonbeck U, Libby P. CD40 signaling and plaque instability. Circ Res. 2001; 89: 1092–1093.[Abstract/Free Full Text]
  38. Wang X, Ria M, Kelmenson PM, Eriksson P, Higgins DC, Samnegard A, Petros C, Rollins J, Bennet AM, Wiman B, de Faire U, Wennberg C, Olsson PG, Ishii N, Sugamura K, Hamsten A, Forsman-Semb K, Lagercrantz J, Paigen B. Positional identification of TNFSF4, encoding OX40 ligand, as a gene that influences atherosclerosis susceptibility. Nat Genet. 2005; 37: 365–372.[CrossRef][Medline] [Order article via Infotrieve]
  39. Palinski W, Miller E, Witztum JL. Immunization of low density lipoprotein (LDL) receptor-deficient rabbits with homologous malondialdehyde-modified LDL reduces atherogenesis. Proc Natl Acad Sci U S A. 1995; 92: 821–825.[Abstract/Free Full Text]
  40. Ameli S, Hultgardh-Nilsson A, Regnstrom J, Calara F, Yano J, Cercek B, Shah PK, Nilsson J. Effect of immunization with homologous LDL and oxidized LDL on early atherosclerosis in hypercholesterolemic rabbits. Arterioscler Thromb Vasc Biol. 1996; 16: 1074–1079.[Abstract/Free Full Text]
  41. Freigang S, Horkko S, Miller E, Witztum JL, Palinski W. Immunization of LDL receptor-deficient mice with homologous malondialdehyde-modified and native LDL reduces progression of atherosclerosis by mechanisms other than induction of high titers of antibodies to oxidative neoepitopes. Arterioscler Thromb Vasc Biol. 1998; 18: 1972–1982.[Abstract/Free Full Text]
  42. Schiopu A, Bengtsson J, Soderberg I, Janciauskiene S, Lindgren S, Ares MP, Shah PK, Carlsson R, Nilsson J, Fredrikson GN. Recombinant human antibodies against aldehyde-modified apolipoprotein B-100 peptide sequences inhibit atherosclerosis. Circulation. 2004; 110: 2047–2052.[Abstract/Free Full Text]



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