© 2001 American Heart Association, Inc.
Editorials |
The First Human Monoclonal Antibody to Oxidized LDL
From the Department of Pathology, University of Chicago, Chicago, Ill.
Correspondence to Dr Godfrey S. Getz, Department of Pathology, University of Chicago, BH-329, MC 3083, 5841 S Mayfield Ave, Chicago, IL 60637-1470. E-mail g-getz{at}uchicago.edu
Largely as a result of the seminal observations of Steinberg and colleagues1 on oxidized lipoproteins and the work of Witztum and his colleagues on the immunological response to these modified lipoproteins, there has been much recent interest in the significance and role of antibodies to oxidized LDL (oxLDL) in the plasma of humans and animals with evolving atherosclerosis. This interest has focused on the value of these antibodies for the diagnosis of atherosclerosis and, more important, in its pathogenesis. This story is now greatly advanced by the article from Witztum and colleagues (Shaw et al2) in this issue of the Journal, in which they report on the isolation and characterization of the first human monoclonal antibody that recognizes epitopes of oxLDL. These authors started with RNA extracted from peripheral blood mononuclear cells of a patient with coronary artery disease that were expressing high titers of antibody to malondialdehyde LDL (MDA-LDL). They used this RNA to prepare cDNA, from which they generated 2 phage (filamentous) display libraries expressing the variable chains of heavy and light immunoglobulins with both the k and l light-chain families. These libraries were screened against MDA-LDL through several rounds. Three specific IgG antibodies were isolated that bind MDA-LDL. One of these antibodies, 1K 17, was characterized in some detail with respect to its immunological and biological properties. This antibody bound to the lipid and protein moieties of oxLDL but not to native LDL. 1K 17 also recognized apoptotic cells. It inhibited the uptake of both oxLDL and apoptotic cells by macrophages in culture. It was used to immunostain human and animal atherosclerotic lesions, where it localized most strongly to the necrotic cores. When this antibody was iodinated and injected into LDL receptor-deficient mice that had advanced plaques, the antibody was highly enriched in the plaque area in contrast to the normal aortic tissue.
See page 1333
This article highlights a number of important issues. It is the first description of a human monoclonal antibody directed against specific epitopes of oxLDL. It further emphasizes the great value and utility of DNA libraries from either spleen or circulating lymphocytes in characterizing individual antibody species. This particular antibody exhibits properties that are unique, not only in its human origin but also in its recognition characteristics. Although the antibody is directed at MDA-LDL, it is significantly different in its immunochemical properties than the previously described murine antibody MDA2.3 The latter stains atherosclerotic lesion areas rich in macrophage foam cells, which areas are poorly stained by 1K 17. Also unlike MDA2, 1K 17 does not recognize MDA-albumin. It is important to obtain more precise information on the nature of the epitope it recognizes to more easily interpret its tendency to localize to the necrotic cores of lesions. By using further refinements of the techniques and the antigens used for screening, this technology may yet uncover a range of antibodies with differing specificities and so allow for a more detailed immunochemical description of the composition of atherosclerotic lesions.
The differences in the properties of this antibody from those previously described highlight the diversity of monoclonal antibodies to epitopes within oxLDL. One set of antibodies isolated from the B cells of apo E–ldeficient mice has been carefully characterized.4 Several of these, designated EO3, EO4, EO6, and EO7, are reactive against oxidized phospholipids, especially the oxidation product of palmitoyl arachidonoyl phosphatidylcholine (PAPC), namely, palmitoyl oxovaleroyl glycerophosphorylcholine. The latter oxidation product has been shown to be highly bioactive in promoting proatherogenic changes in gene expression in the cells of the vessel wall, which changes have been implicated in the early stages of atherogenesis.5 These antibodies, which are all IgM antibodies, are identical to the germ line natural antibody that recognizes phosphorylcholine,6 a major determinant on the teichoic acid of Streptococcus pneumoniae. They do not recognize unoxidized PAPC. Interestingly, they also recognize oxidized but not unoxidized cardiolipin,7 which contains no choline. So even in this highly characterized antibody present in the plasma of some humans and in the plasma and lesions of the apo E–deficient mouse, the precise nature of the cognate epitope is not fully defined. Perhaps it is the phosphodiester bond conformation that is altered by an oxidation-induced change in the hydrophilicity of the nonpolar portion of the phospholipids that is the epitope recognized by these antibodies. The fact that the EO panel of antibodies and 1K 17 described in this current study both recognize apoptotic cells and inhibit their phagocytosis by macrophages raises the question about the precise role of oxLDL in promoting their production. It is possible that apoptotic cells play an important role in this regard. Apoptosis is an integral physiological process involved in tissue development and differentiation. The oxidative stress associated with either apoptosis or hyperlipidemia could promote the increased production of these antibodies.
Isolation of a variety of antibodies that recognize various epitopes in oxLDL raises the issue of their role in atherogenesis. Despite the chronic inflammatory nature of atherosclerosis, it is clear from mouse models that the capacity to generate antibodies to either oxidized lipoproteins or apoptotic cells is not required for the development of atherosclerotic lesions, even quite complicated ones.8–10 Apo E–deficient mice that are totally unable to generate antibodies develop severe atherosclerosis at the usual sites. Nevertheless, there is considerable evidence that antibodies against oxLDL may modulate atherogenesis. For example, immunization of LDL receptor–deficient rabbits11 and mice12 and of apo E–deficient mice13 with MDA-LDL reduces the extent of aortic root atherosclerosis. However, it is notable that in the latter study, "immunization" with native LDL also reduced the extent of atherosclerosis, and this effect did not seem to be related to an increased titer of antibodies to either LDL or ox LDL.12 It was recently shown that tolerization of neonatal mice with oxLDL reduced the subsequent development of atherosclerosis in these apo E–deficient mice.14 This finding is puzzling in light of the significant presence of oxLDL in fetal arteries,15 which should, in theory, provide an endogenous toleragen. Perhaps each of these observations reflects on the role of different sets of oxLDL antibodies. Bearing in mind the diversity of epitopes on both oxLDL and apoptotic cells that antibodies to oxLDL detect, it is conceivable that each of these specific antibodies may modulate atherogenesis in different if not opposing ways.
The exploration of the role of individual autoantibodies during atherogenesis could be quite informative. The finding of a variety of antibodies to oxLDL has further clinical implications. Several studies have noted the use of these antibodies as risk factors predicting the outcome of atherosclerotic heart16 and cerebral17 disease. The titers of these antibodies in the plasma may reflect the oxidative stress and/or ongoing atherogenesis. The antibodies may also be used as imaging agents to define the composition of atherosclerotic vessels in vivo. These authors have previously used the antibody MDA2 to measure murine atherosclerotic lesions in vivo and even to follow the regression of such lesions.3 In principle, 1K 17 could also be used to image atherosclerotic lesions with prominent necrotic cores, and no doubt this will be exploited by these workers in due course. The principle could be extended by the further characterization of additional monoclonal antibodies yet to be defined that recognize different epitopes than does 1K 17. Such a putative panel of human antibodies directed at cells, lipoproteins, and matrix molecules that have been modified by oxidation or other processes could be of immense value in defining the composition of vulnerable atherosclerotic plaques in vivo. Knowledge about the instability of such vulnerable plaques is critically related to their composition and could be therapeutically important.18 As Shaw and colleagues2 point out, these antibodies could also be useful for targeting the therapeutic reagents to the plaque.
Acknowledgments
Dr Getz was supported by a grant from the National Heart, Lung, and Blood Institute (HL 56827).
The author wishes to thank Dr Catherine Reardon for many valuable discussions.
References
1. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol: modifications of low density lipoprotein that increase its atherogenicity. N Engl J Med. 1989; 320: 915–924.[Medline] [Order article via Infotrieve]
2.
Shaw PX, Horkko S, Tsimikas S, Chang M-K, Palinski W, Silverman GJ, Chen PP, Witztum JL. A human-derived anti-oxidized LDL autoantibody blocks uptake of oxLDL by macrophages and localizes to atherosclerotic lesions in vivo. Arterioscler Thromb Vasc Biol. 2001; 21: 1333–1339.
3.
Tsimikas S, Shortal BP, Witztum JL, Palinski W. In vivo uptake of radiolabeled MDA2, an oxidation-specific monoclonal antibody, provides an accurate measure of atherosclerotic lesions rich in oxidized LDL and is highly sensitive to their regression. Arterioscler Thromb Vasc Biol. 2000; 20: 689–697.
4. Palinski W, Horkko S, Miller E, Steinbrecher UP, Powell HC, Curtiss LK, Witztum JL. Cloning of monoclonal autoantibodies to epitopes of oxidized lipoproteins from apolipoprotein E deficient mice: demonstration of epitopes of oxidized lipoprotein in human plasma. J Clin Invest. 1996; 98: 800–814.[Medline] [Order article via Infotrieve]
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8.
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9.
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12.
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13.
Zhou X, Caliguiri 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.
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