Immunological responses to oxidized LDL

Free Radical Biology & Medicine, Vol. 28, No. 12, pp. 1771–1779, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/00/$–see front matter

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Forum: Role of Oxidation in Atherosclerosis IMMUNOLOGICAL RESPONSES TO OXIDIZED LDL SOHVI H¨ORKKO¨ , CHRISTOPH J. BINDER, PETER X. SHAW, MI-KYUNG CHANG, GREGG SILVERMAN, WULF PALINSKI, and JOSEPH L. WITZTUM Department of Medicine, University of California, San Diego, La Jolla, CA, USA (Received 2 May 2000; Accepted 5 May 2000)

Abstract—Considerable evidence now points to an important role for the immune system in experimental models of atherosclerosis. We have reviewed the growing body of evidence that oxidation of LDL generates a wide variety of neoself determinants that lead to cellular and humoral immune responses. In particular, we have demonstrated that at least some of the oxidation-specific epitopes generated on the oxidized LDL particle, such as oxidized phospholipid epitopes, are also generated on apoptotic cells and are also present on the surface of some bacteria. Many of these same epitopes serve as important ligands mediating the binding and clearance of oxidatively damaged lipoprotein particles and apoptotic cells, and the innate immune response to these epitopes can be seen as a conserted response to effect their removal. In addition, other epitopes of OxLDL also undoubtedly play a role in the immune activation that characterizes the progressive atherosclerotic plaque. It will be of great importance to define the importance of the role of these responses and to understand which are beneficial and which deleterious. Such information could lead one day to novel therapeutic approaches to inhibit atherogenesis that take advantage of the ability to manipulate the immune response. © 2000 Elsevier Science Inc. Keywords—Oxidized LDL, Atherosclerosis, Immunity, Free radical, Oxidized phospholipids, T cells, Immunoglobulins, Autoantibodies

INTRODUCTION

formation of fatty streaks to transitional lesions and complex plaques that converts relatively benign lesions into ones that have the potential to alter artery wall function, as well as plaque rupture, thrombosis, and clinical events [2]. Once initiated, this progressive atherogenic process has many characteristics of a chronic inflammatory process [1,3,4]. Indeed, there is now much experimental evidence in animal models that the immune system plays an important role and, under certain circumstances, may even substantially alter the course of the atherosclerotic process [3,5,6]. Although a review of the extensive experimental evidence documenting a role of immune mediated mechanisms is beyond the scope and intent of this chapter, a brief summary is in order. Immunocompetent cells, including monocyte/macrophages and T cells, are prominent components of nearly all stages of lesions. Data support a central role for monocyte/macrophages in atherogenesis. While these cells play many roles, their ability to serve as professional antigen presenting cells (APC) undoubtedly is related to the variety of autoantibodies generated to oxidatively modified lipids and pro-

Hypercholesterolemia is undoubtedly the major factor involved in the initiation of fatty streaks, the initial lesion of the atherogenic process [1]. However, it is the transSohvi Ho¨rkko¨, MD, PhD, Assistant Research Biochemist, Department of Medicine, University of California, San Diego, CA, USA. Christoph J. Binder, MD, Postdoctoral Fellow, Department of Medicine, University of California, San Diego, CA, USA. Peter X. Shaw, PhD, Research Project Scientist, Department of Medicine, University of California, San Diego, CA, USA. Mikyung Chang, MD, Postdoctoral Fellow, Department of Medicine, University of California, San Diego, CA, USA. Gregg Silverman, MD, Assoc. Professor of Medicine, Division of Rheumatology, Department of Medicine, University of California, San Diego, CA, USA. Wulf Palinski, MD, Professor of Medicine, Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego, CA, USA. Joseph L. Witztum, MD, Professor of Medicine, Division of Endocrinology and Metabolism, Department of Medicine, and Director of SCOR in Molecular Medicine and Atherosclerosis, University of California, San Diego, CA, USA. Address correspondence to: Joseph L. Witztum, MD, Dept. of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0682, USA; Tel: (858) 534-4347; Fax: (858) 5342005; E-Mail: [email protected]. 1771

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teins as discussed below. Indeed, the effectiveness of the ability of macrophages to induce T cell stimulation after taking up modified proteins via “scavenger-receptors” has been demonstrated [7,8]. T cells are also prominent components of early and late lesions. They appear to be activated, including the expression of Th1 cytokines such as INF␥. In turn, most lesional vascular cells express MHC Class II molecules. It is likely that there is “cross talk” between T cells and macrophages, for example, INF␥ secreted by T cells activates macrophages and leads to IL-12 release, which in turn increases INF␥ release [5]. In addition, CD40 and CD40L are widely expressed on vascular cells in lesions [9], and of particular relevance for the ability of APCs to present lipids, including oxidized lipids, is the observation that CD-1 molecules are prominent in lesions [10]. It should be noted that while immunoglobulins are prominent components of atherosclerotic lesions, there are only rare reports of B cells in lesions [11]. Similarly, while plasma cells have been observed in inflammatory infiltrates in the adventitia surrounding aneurysmal formation, they have not been reported in lesions. An exception was the recent report of immunoglobulin-secreting plasma cells in lesions of hypercholesterolemic rabbits [12]. Evidence that the immune system modulates atherogenesis comes from experimental studies demonstrating that specific immune-based interventions can influence lesion progression. These data have been reviewed elsewhere [3,5,6]. In brief, elimination of T cells by cyclosporine treatment of hypercholesterolemic rabbits accelerated atherosclerosis [13]. MHC-Class I deficient mice on a high fat diet have been reported to have increased aortic valve lesions [14]. Administration of an antiCD40L antibody to cholesterol-fed LDLR-negative mice reduced lesion formation by 60% [15]. In addition, it was recently reported that intravenous administration of polyspecific IgG decreased lesion formation [16]. Both of these latter two interventions have been used for treatment of experimental and/or clinical autoimmune disorders. Furthermore, as described below, immunization of a number of hypercholesterolemic animal models with models of OxLDL has led to the amelioration of atherosclerotic lesion progression [17–21]. In other cases, immunization with antigens that are highly homologous to endogenous proteins present in lesions, such as with heat shock proteins, can accelerate atherogenesis [22,23]. In most cases it is likely that immune mechanisms are modulating events with regard to atherogenesis rather than a primary initiating event. For example, in the presence of extreme hypercholesterolemia, such as when plasma cholesterol levels of 1600 mg/dl or greater are achieved in cholesterol fed apoE-deficient mice, the absence of both T and B cell compartments (as achieved by

crossing apoE-deficient mice with RAG-1 KO mice) did not impact the extent of lesion generation [24,25]. However, in the same model, if the double KO animals were fed chow, so that cholesterol levels of only 600 – 800 mg/dl were reached, lesion formation was reduced by 42% [24]. These data demonstrate that initiation of lesion formation, and even progression does not require immune function. It is more likely that once early lesion formation has begun, a variety of antigens are generated within the atherosclerotic lesion, or conceivably even elsewhere, that lead to immune activation, and consequent cellular and humoral responses. As noted above, the presence of activated T cells and extensive expression of MHC-Class II, as well as CD1 molecules, suggest antigen-mediated activation. Emerging evidence suggests that a number of potential antigen classes are present in lesions that could be responsible for this immune activation. These include bacterial and viral antigens [26], heat shock proteins [27], modifications of arterial wall components and lipoproteins, (as occurs with nonenzymatic glycation secondary to hyperglycemia [28,29]), and, importantly, oxidatively modified lipoproteins and their products. The full extent and nature of the likely multiple antigens within the atherosclerotic lesion actually responsible for immune activation are not yet known. However, a recent report suggests that there is an oligoclonal T cell expansion within atherosclerotic lesions in apoE-deficient mice, suggesting a restricted antigen-driven T cell proliferation in lesions, at least under conditions of marked hypercholesterolemia [30]. There is now considerable evidence that neoself epitopes generated on oxidized LDL (and potentially other oxidatively modified lipids and proteins) are important and even dominant antigens that drive this immune response. Indeed, it has been demonstrated that up to 10% of CD4⫹ T cells cloned from human atherosclerotic lesions proliferated specifically to OxLDL in an HLA-DR dependent manner [31]. In this chapter we will review the evidence that there is a significant immune response against a variety of epitopes of OxLDL. IMMUNOGENICITY OF MODIFIED LDL

The original observation that even very subtle modifications of LDL render it immunogenic came from the serendipitous observation that nonenzymatic glycation of autologous LDL led to its rapid plasma clearance when it was injected intravenously into diabetic and some euglycemic subjects [32]. It was subsequently shown that this was due to immune recognition of the modified LDL. Indeed, a variety of subtle modifications of homologous LDL were also shown to render the modified LDL immunogenic. For example, carbamylation, acetylation, and even methylation of lysine residues of apoB were

Immunity to OxLDL

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Fig. 1. Scheme of potential products resulting from lipid peroxidation of LDL that could give rise to immunogenic epitopes. (Reprinted with modifications from Ho¨rkko¨ et al. [36].)

sufficient to generate neoself determinants of homologous LDL that led to antibody formation [33]. Of considerable interest was the observation that in most cases the resulting antisera generated (e.g., by immunizing guinea pigs with guinea pig methylated-LDL) recognized not only the immunizing agent, e.g., methyl-LDL, but also recognized a variety of similarly methylated proteins, including methyl-lysine itself [33]. Subsequently, when we wished to generate antibodies that could be used to identify the presence of OxLDL in vivo, we used the same principle. We reasoned that breakdown products of oxidatively modified polyunsaturated fatty acids, such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) could form covalent adducts with lysine residues of apoB (see Pathway 1 of Fig. 1). Thus, we prepared MDA-LDL and 4-HNE-LDL with guinea pig and murine LDL and generated monospecific antisera and monoclonal antibodies, respectively [34,35]. These antibodies recognized their respective oxidationderived epitopes not only on OxLDL, but also on a variety of other similarly derived peptides and proteins. We termed these “oxidation-specific” epitopes to emphasize that while occurring on OxLDL, under conditions of lipid peroxidation, these epitopes could also be generated in vivo on other structures, such as proteins and aminocontaining headgroups of phospholipids as well (see “lipid” component of Pathway 1 of Fig. 1). In addition, the residual phospholipid backbone, containing the remaining decomposed fatty acid, which can also contain

reactive groups, such as aldehydes terminating the esterified fatty acid in the sn2 position, could also form a Schiff base with the apoB, yielding in this case an entire oxidized phospholipid covalently attached to the apoB of OxLDL (see Pathway 2 in Fig. 1). In a similar manner, lipid-lipid adducts could also occur (“lipid” adducts in Pathway 2). Any phospholipid containing an unsaturated fatty acid would be susceptible to oxidation and could potentially generate an antigenic neo-epitope. Indeed, as discussed below, it is likely that many of the so-called antiphospholipid antibodies that are associated with rheumatological diseases, or with the primary Antiphospholipid Antibody Syndrome (APS), which is associated with thrombotic disease, are actually antibodies directed against oxidized phospholipids [36,37]. It should also be appreciated that many other oxidative products could occur as well, including modified triglycerides, oxidized cholesterol derivatives, and oxidized cholesteryl esters. A wide variety of these reactive products could also form covalent adducts with residues of apoB, which in addition is subjected to nonenzymatic cleavage [38]. As will be discussed below, the formation of oxidized phospholipid adducts on apoB turned out to be of major biological significance and generated highly immunogenic epitopes. The antibodies originally prepared against the model epitopes of MDA-LDL and 4-HNE-LDL were subsequently used to demonstrate the prominence of such “oxidation-specific” epitopes, both intracellular as well

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as extracellular, in atherosclerotic lesions of animal models and humans [34,39,40]. Frequently these colocalized with apoB as well. To confirm that at least some of the immunostaining in lesions was due to binding to OxLDL, we eluted LDL from lesions of rabbits and humans and demonstrated that it had all of the biological properties of LDL oxidized in vitro [41]. In particular we demonstrated that when tested by ELISA and Western blot techniques, the eluted LDL contained a variety of oxidation-specific epitopes [34,41,42]. AUTOANTIBODIES TO OXLDL

Because OxLDL was present in atherosclerotic lesions and was immunogenic, one would predict the presence of autoantibodies to the oxidation-specific epitopes of OxLDL in plasma. Indeed, we first reported the presence of such autoantibodies in rabbits and humans [34]. Since that original report there have been numerous reports of such autoantibodies to “oxidation-specific” epitopes of OxLDL by our group and by many others, both in animal models of atherosclerosis and in humans. In animal models, autoantibody titers to epitopes of OxLDL show a strong correlation to measures of atherosclerosis. For example, our laboratory reported that such autoantibodies progressively rose over time in cholesterol-fed LDLR-deficient mice and at time of sacrifice the antibody titer correlated with the extent of atherosclerosis present [43]. Such antibody titers also rise in chowfed C57BL/6 mice as they age, though the levels of antibody titers are lower [44]. Extremely high titers of such autoantibodies are also observed in cholesterol-fed apoE-deficient mice [40]. Furthermore, if their atherogenesis is inhibited by deletion of the 12,15-lipoxygenase gene (e.g., by crossing of apoE-deficient x homozygous 12,15-LO KO mice), then the levels of autoantibodies are correspondingly reduced [45]. In addition, we demonstrated that in these animal models of atherosclerosis the autoantibodies were present in lesions, in part as immune complexes with OxLDL [42]. Such autoantibodies are also found in human populations as well, both in patients with clinically evident cardiovascular disease as well as in apparently normal populations. We initially reported that the baseline titer of autoantibodies to MDA-LDL (as a model epitope of OxLDL) was an excellent predictor of progression of carotid intimal-medial thickness over a 2-year period in a group of healthy Finnish males [46]. Since that initial report many, but not all, studies have shown a relationship between elevated autoantibody titers to epitopes of OxLDL and various cardiovascular diseases or risk factors, such as coronary artery disease, myocardial infarction, peripheral vascular disease, hypertension, preeclampsia, or diabetes (see [47,48] for reviews).

However, careful prospective epidemiological studies remain to be done before the clinical utility of such measurements in nonselected populations can be ascertained. PROPERTIES OF MONOCLONAL AUTOANTIBODIES CLONED FROM APOE-DEFICIENT MICE

The observation that apoE-deficient mice had extremely high titers of autoantibodies to a wide variety of oxidation-specific epitopes suggested the possibility that such autoantibodies could be cloned from the spleens of the mice, despite the fact that the animals had not received any exogenous immunization. Indeed, from a single fusion made from the spleens of two cholesterolfed apoE-deficient mice we obtained a large number of viable hybridomas that bound to one or more epitopes of OxLDL, reflecting the exuberant immune response [49]. In fact, of 768 pooled viable hybridomas, 64% showed binding to one or more epitopes of OxLDL. In fact, the spleens of the apoE-deficient mice were nearly 40% heavier than those of C57BL/6 controls, presumably reflecting the exuberant immunological response. From the pooled hybridomas, 13 monoclonals (termed EO antibodies) were eventually cloned that bound to OxLDL or to MDA-LDL. Each of these were shown to be IgM and to immunostain atherosclerotic tissue. In subsequent studies we demonstrated that each of the EO antibodies selected for their ability to bind to OxLDL bound specifically to the isolated lipids of OxLDL as well as to the delipidated apoB, but not to the lipids or protein of native LDL. Specifically, it was found that each of these OxLDL-specific antibodies recognized oxidized phospholipid epitopes [50]. For example, antibody EO6 recognized the oxidized phospholipid POVPC (1-palmitoyl-2(5-oxovaloryl)-3-phosphorylcholine), but not the parent, unoxidized phospholipid, PAPC (1-palmitoyl-2-arachidonyl-3-phosphorylcholine). EO6 specifically was able to bind to an adduct of POVPC with BSA. Of great interest was the observation that these OxLDL-specific autoantibodies had important biological properties. Each of these IgM autoantibodies, such as EO6, was able to nearly fully block the binding and degradation of OxLDL by elicited peritoneal macrophages. Furthermore, Fab fragments of EO6 were also able to block uptake, suggesting that it was not simply the size of the IgM that was responsible for the inhibition of degradation. Indeed, the hypothesis that the epitopes to which these antibodies bind represent important ligands on OxLDL mediating macrophage uptake was strongly supported by the demonstration that POVPCBSA was also able to compete to the same extent for the binding of OxLDL to macrophages [50]. In further studies it was demonstrated that both microemulsions of the lipids of OxLDL, as well as the resolubilized, delipidated

Immunity to OxLDL

protein moiety of OxLDL, demonstrated saturable and specific binding to macrophage scavenger receptors. This binding could be competed for by intact OxLDL. Furthermore, monoclonal antibody EO6 was also able to effectively inhibit the binding of both the lipid microemulsions and the protein of OxLDL to macrophages, and so was the POVPC-BSA adduct [50,51]. These data suggest that the oxidized phospholipid epitopes in OxLDL, present either as lipids or as lipid-protein adducts, are dominant ligands for one or more macrophage scavenger receptors. The fact that apoE-deficient mice have such IgM blocking antibodies suggests that they could play a “protective” role in vivo in inhibiting macrophage uptake of OxLDL and thereby prevent foam-cell formation. In addition, because such oxidized phospholipid epitopes exist on circulating LDL, it is possible that such antibodies could effect removal from plasma of such oxidatively modified lipoproteins (presumably into the liver, spleen, and bone marrow [52], thereby preventing their entrance into the arterial wall. Of course, it should be appreciated that other autoantibodies with different isotypes, e.g., IgG, that could also bind to OxLDL and which contain Fc domains capable of binding to macrophage Fc receptors, could actually promote the uptake of OxLDL as well [53]. The surface of an LDL particle in many ways resembles the surface of a cell. It has been previously demonstrated that OxLDL could inhibit the binding and uptake of apoptotic cells by macrophages [54], suggesting the presence of common oxidation-specific epitopes on OxLDL and apoptotic cells. To test this hypothesis we recently demonstrated that several of the oxidation-specific antibodies, such as EO6, specifically bound to apoptotic cells, but not to viable cells. Furthermore, it was also able to block macrophage phagocytosis of apoptotic cells, as was the POVPC-BSA adduct [55]. Once the apoptotic program has been initiated, cells are known to be subjected to “oxidative stress,” and as shown by these studies, this is accompanied by the expression of oxidation-specific epitopes on the cell’s surface. In turn, these epitopes are recognized by the oxidation-specific antibodies and form ligands mediating phagocytosis by macrophage scavenger receptors. Using the hybridomas secreting the EO antibodies, we have recently cloned and sequenced the antibody genes encoding the variable regions of the EO antibodies that bind to OxLDL. Surprisingly, we observed that the DNA encoding the variable regions of the immunoglobulin binding regions consisted of nonmutated, germline recombinations that contained no “N” insertions. Such genetic recombinations of gene usage of the immunoglobulin families suggests that these IgM autoantibodies that bind to OxLDL are primitive antibodies present at birth and are part of what has been termed the innate

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immune system. In fact, the variable regions of these particular antibodies are encoded by genetic recombinations that were first described over 30 years ago. These antibodies have been much studied because they were shown to confer optimal protection to mice from certain lethal bacterial strains. Studies at the protein and functional level have confirmed the identity of the current EO antibodies to OxLDL and the T15 class of natural autoantibodies described many years ago (Shaw et al., unpublished data). It is tempting to speculate that an evolutionary pressure has existed to help select such autoantibodies directed against neoself determinants generated by oxidative processes. Indeed, the presence of such oxidation-specific epitopes are likely to be ubiquitous both during development (as on apoptotic cells) and in response to environmental challenges and inflammation. ANTIPHOSPHOLIPID ANTIBODIES: RELATIONSHIP TO AUTOANTIBODIES TO OXLDL

As noted above, a number of the autoantibodies cloned from the apoE-deficient mice bound to epitopes of oxidized phospholipids. In parallel studies that were ongoing in our laboratory we were also studying antibodies from patients with the APS. Such patients have a high frequency of fetal death when pregnant, have increased risk for arterial or venous thrombosis, and increased risk for stroke, myocardial infarction, and pulmonary embolism. We became interested in this problem after we found that women with pre-eclampsia had elevated titers of autoantibodies to epitopes of OxLDL [56]. These women also frequently had elevated titers of anticardiolipin antibodies. Because of our interest in autoantibodies to epitopes of OxLDL, we examined the possibility that a similar phenomenon also was occurring in the APS. Indeed, cardiolipin is the standard phospholipid usually used to test for the presence of antiphospholipid antibodies. It is a phospholipid that contains four polyunsaturated fatty acids and is highly susceptible to lipid peroxidation under conditions of solid-phase immunoassay, which requires the lipid to be dried onto the microtiter well and therefore exposed to air. Indeed, we demonstrated that both sera and affinity purified anticardiolipin IgG from APS patients bound only to oxidized cardiolipin, and did not bind at all to a cardiolipin analogue unable to undergo oxidation [36]. We also showed that ␤2GP1, a cofactor for binding of anticardiolipin antibodies, was recognized by the APS sera only when bound to oxidized cardiolipin, but not when bound to the reduced cardiolipin analog unable to undergo oxidation [37]. ␤2GP1 is an avid phospholipid binding protein present in high concentration in plasma, for the most part associated with lipoproteins. We also showed

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that other proteins with high phospholipid affinity could also serve in a similar manner as a cofactor, as for example apoA-I and apoB [37]. We believe that this indicates that many antiphospholipid antibodies are directed against epitopes of oxidized phospholipids or neoepitopes generated after adduct formation with associated proteins. This suggests that OxLDL could give rise to neoantigens that would generate antiphospholipid antibodies, and in turn, oxidation of a variety of phospholipid structures could give rise to antiOxLDL antibodies. The recent demonstration that circulating LDL contains significant amounts of cardiolipin [57] (previously thought to be present only intracellularly) suggests that many anticardiolipin antibodies and antiOxLDL antibodies may be directed at similar epitopes. Indeed, we have observed that several human monoclonal antibodies selected for binding to cardiolipin bind also to ␤2GP1 and to apoB of OxLDL on Western blots (Ho¨rkko¨ et al., unpublished data). This observation is most consistent with the hypothesis that the epitope to which they bind is an adduct of oxidized cardiolipin with the two lipid avid proteins, ␤2GP1 and apoB. These antibodies may possess properties that interfere with the normal coagulation cascade and thereby influence clinical events. Alternatively, or in addition, we have suggested that such antibodies may be a reflection of the underlying state of lipid peroxidation. Indeed, in an explicit test of this hypothesis, it was recently demonstrated in patients with Lupus that there was a good correlation between the titer of anticardiolipin antibody and the excretion of F2 isoprostanes, a measure of in vivo lipid peroxidation [58,59]. This might also explain the epidemiological observations that antibodies to cardiolipin are independent predictors of cardiovascular events [60,61]. CELLULAR IMMUNITY AND OXIDIZED LDL

In general, cytokines secreted by activated T cells may control macrophage activation, such as scavenger receptor expression, cytokine expression, metalloproteinase secretion, and MHC-Class II expression among other effects. In addition, cytokines undoubtedly modulate many other processes in the vascular wall that can affect atherogenesis [3]. Furthermore, an active “cross talk” between T cells and macrophages as well as helper T cells of different subtypes could well influence the inflammatory state within the plaque and subsequently influence the fate of the disease. To date, it is not known whether differences in the T cell populations, such as CD4⫹ T helper (Th) 1 or Th2 cells, or CD8⫹ cytotoxic T cells, or possibly even natural killer cells, influence atherogenesis, although to date most data have emphasized the importance of helper T cells. The ability of plaque-macrophages to present anti-

gens, and the presence of T cells and OxLDL in plaques presumably allows the initiation of local cellular immune responses of OxLDL-specific T cells. The occurrence of such T cells is supported by the development of IgG antibodies with specificity for epitopes of OxLDL. Such antibody isotypes are usually dependent on T cell help. Furthermore, the biological significance of OxLDL as a T cell antigen is also strengthened by the observation that 10% of CD4⫹ T cells cloned from human carotid atherosclerotic plaques specifically proliferated in response to OxLDL in a MHC class II restricted manner [31]. Functionally polarized responses by the CD4⫹ T helper cell subsets, Th1 and Th2, are critical in the pathogenesis of several diseases [62,63]. It is therefore tempting to speculate that a Th1/Th2 balance of immune responses to OxLDL plays a similar role in atherosclerosis. Recently, it has been reported that in apoE-deficient mice there is a shift from an initial Th1 type response to a Th2 type response during plaque growth, as judged by the development of MDA-LDL-specific IgG1 and IgG2a antibody populations and the parallel appearance of the Th2 cytokine IL-4 mRNA in the plaques of mice [64,65]. Both subsets of Th cells secrete specific T cell cytokines: Th2 cells make IL-4, IL-5, IL-13, and others, whereas Th1 cells secrete IL-2 and INF␥. Th1 and Th2 cells are thought to play different roles in promoting inflammatory conditions, as well as in crossregulation of each other. Therefore, the dominance of one subset of Th cells could well influence the course of lesion progression. For example, studies in genetically engineered mice have indicated that INF␥ responses accelerate atherosclerosis [66,67], suggesting that the Th1 subset of T cells is proatherogenic. In addition, daily administration of IL-12 accelerated atherosclerosis in apoE-deficient mice and, at the same time, led to a modulation of OxLDL specific Th cell responses [65]. On the other hand, mice that lack the anti-inflammatory monocytic/Th2-cytokine IL-10 exhibited increased atherosclerotic lesions, whereas mice with a murine IL-10 transgene under the control of the human IL-2 promoter had decreased lesions [68,69]. IL-10 particularly suppresses IL-12 and INF␥ secretion [70] and has been shown to inhibit the OxLDL induced production of IL-12 in human monocytes [71]. As stated before, immunization with homologous MDA-LDL led to a significant reduction in atherosclerosis in LDLR-negative rabbits and mice, respectively [17,18]. However, in the latter study we observed that immunization with homologous native LDL also had an atheroprotective effect, although the humoral response to epitopes of OxLDL was far less pronounced than in the animals immunized with the MDA-modified LDL. Although we cannot rule out the possibility that humoral responses played a role, these observations suggest that

Immunity to OxLDL

the major beneficial effect of immunization was not due simply to antigen-specific antibody production. It is more likely that immunization with native LDL led to a localized inflammatory condition that resulted in the localized generation of OxLDL, which would be avidly taken up by macrophages and trigger activation of cellular immune responses. It is conceivable that the specific character of this cellular immune response led to the protective outcome of the immunization. The observations that the endogenous immune response to OxLDL in apoEdeficient mice shifts from an initial Th1 type to a Th2 type response and that Th1 and Th2 cytokines play different roles in the progression of atherosclerosis, clearly point toward a difference of functionally polarized Th responses to OxLDL. Only further studies in mice will determine if this hypothesis is correct. However, it should be noted that the rather clear distinction between Th1 and Th2 cell populations in mice is not so well established in humans.

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ABBREVIATIONS

APC—antigen presenting cell apoA1—apolipoprotein A1 apoB—apolipoprotein B apoE—apolipoprotein E APS—antiphospholipid antibody syndrome ␤2GP1—␤2 glycoprotein 1 BSA— bovine serum albumin 4-HNE— 4-hydroxynonenal KO— knock-out LDL—low density lipoprotein MDA—malondialdehyde MHC—major histocompatibility complex OxLDL— oxidized low density lipoprotein PAPC—1-palmitoyl-2-arachidonyl-phosphatidylcholine POVPC—1-palmitoyl-2-(5-oxovaleroyl)-phosphatidylcholine RAG-1—recombination activating gene 1