COMMENTARIES

ANTIBODIES AGAINST OXIDIZED LDL AND ATHEROSCLEROSIS

Alexandru Schiopu and Gunilla Nordin Fredrikson
Department of Medicine, Lund University, Sweden
Experimental Cardiovascular Research Lab
Wallenberg Laboratory
Entrance 46, Floor 1
Malmö University Hospital
20502 Malmö, Sweden
E-mail:
Alexandru.Schiopu@medforsk.mas.lu.se
Gunilla.Nordin_Fredrikson@medforsk.mas.lu.se

 

Atherosclerosis is considered to be the result of a chronic inflammatory process in the tunica intima of large and medium sized arteries. Oxidized LDL plays a major role both in disease initiation and progression. Native LDL enters the vessel wall by diffusion, binds to matrix proteoglycans and suffers enzymatic and non-enzymatic modifications (aggregation, oxidation, lipolysis, proteolysis). During oxidation, the constitutive protein of LDL, apoB-100, is fragmented and the lysine and histidine residues on the peptide fragments bind malondialdehyde (MDA) and other reactive aldehydes. MDA is a result of the oxidation process of the polyunsaturated fatty acids of the LDL phospholipids. Oxidation of LDL phospholipids also generates phophorylcholine (PC) containing compounds, such as 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine, known as POVPC. PC is also common on the surface of apoptotic cells and encapsulated pathogens (e.g. Streptococcus Pneumoniae). The new formed structures, denominated oxidation-specific epitopes, are recognized by the immune system and have been shown to activate T cells and trigger immune responses, leading to generation of antibodies.
Numerous studies have indicated the presence of both IgM and IgG antibodies against these neo-epitopes in human plasma. Nevertheless, correlations between the amounts of antibodies and presence or progression of carotid artery disease, coronary artery disease or risk for developing acute events (myocardial infarction, stroke) have been contradictory so far [1]. One possible explanation is that these immune responses are differently modulated at different stages of disease development and also at different ages and are strongly influenced by other risk factors, for example concomitant pathologic conditions, sex, genetic background as well as the general state of the immune system.
In animal studies, immunization with oxLDL reduced atherosclerosis in rabbits [2,3] and apoE-/- mice [4-6] and reduced intimal plaque development after vascular injury in rabbits [7]. Moreover, passive treatment with polyclonal immunoglobulins inhibited atherosclerosis development in apoE-/- mice [8] and B cell substitution reduced atherosclerosis development in splenectomized apoE-/- mice [9] and the injury-induced plaque formation in Rag-1 mice [10]. Taken together, these results indicated the existence of an atheroprotective antibody-mediated adaptive immune response against oxLDL epitopes, and opened up the tempting perspective of using this effect in active and passive immunization strategies for protection against atherosclerosis.
Nevertheless, there are important differences between immune responses involving antibodies against the two major types of oxLDL epitopes, the oxidized PC-containing phospholipids and the MDA-modified peptide fragments of apoB-100, and these effects need to be thoroughly characterized before being used for therapeutic purposes.

Antibodies against Oxidized Phospholipids

Palinski et al. isolated monoclonal antibodies specific for copper-oxidized LDL (Cu-LDL) from spleens of hypercholesterolemic apoE-/- mice. These antibodies, which received the name of EO-antibodies, proved all to be of IgM isotype and specific for the PC-containing epitopes of Cu-LDL, such as POVPC [11].
The representative prototype antibody, EO6, was intensively studied and characterized. It has been shown that EO6 binds to the PC epitopes on both oxLDL and apoptotic cells, blocks their binding to macrophage scavenger receptors (SR) CD36 and SR-B1 and inhibits cellular uptake. The same effects were obtained by using BSA-bound POVPC. In contrast, EO antibodies selected for binding to MDA-LDL did not have the same effect, suggesting that binding and uptake of oxLDL and apoptotic cells in macrophages via the scavenger receptors are mediated by the PC containing oxidized phospholipids and that there are natural antibodies in plasma that can block this effect [12,13].
Furthermore, the antigen-binding domains of the EO6 antibody (and of all the other EO antibodies as well) proved to be genetically, structurally and functionally identical to those of T15, a natural IgA antibody secreted by innate B1 cells and functioning as a first line of defense against microbial pathogens, such as Streptococcus Pneumoniae [14,15]. Immunization of LDLR-/- mice with Streptococcus Pneumoniae lead to expansion of activated B1 cells in the spleen, associated with strongly enhanced secretion of the IgM EO6 antibodies and significant inhibition of atherosclerosis development in the aortic sinus [16]. These results might be explained by cross reactivity between PC epitopes on oxLDL and those on the polysaccharide capsule of the bacteria.
Interestingly, immunization with MDA-LDL, which does not contain the PC headgroup lead to increased secretion of IgM EO6 antibodies and an atheroprotective effect. In this case the results are believed to depend on activation of IL-5 secreting Th2 cells specific for MDA-LDL. In turn, IL-5 may provide stimulation of B1 cells, enhancing production of the EO6/T15 antibodies. The levels of EO6 antibodies reached in this study were approximately 35% of those obtained by immunization with Streptococcus pneumoniae [17].

Antibodies against MDA-modified Peptides

We have focused our attention on studying the interactions between the immune system and the epitopes generated by modifications of apoB-100 during oxidation, and the effect on atherosclerosis development. Using a library of 302 native and MDA-modified 20 amino acid long peptides, covering the complete sequence of the protein, we have identified several epitopes recognized by IgM and IgG antibodies in human plasma. The IgM antibody amounts inversely correlate with age and oxLDL plasma levels [18].
Immunization of ApoE-/- mice with two of these peptide sequences lead to a significant increase in IgG titers against the MDA-modified peptides, whereas no difference was observed in IgM. These findings contrast with the pneumococcal immunization, which increased IgM, but not IgG. Activation of the immune system with peptides led to a 60% reduction of plaque area in the descending aorta of these mice, and a more stable plaque phenotype, assessed by collagen content of the subvalvular plaques [19].
In order to directly assess antibody effects on plaque development, we have also synthesized recombinant human IgG1 antibodies against two of the MDA-modified peptide sequences. Both the single chain fragment variable (scFv) and the full length IgG1 forms of the antibodies specifically bound to MDA-LDL but not to nLDL. Passive immunization with three doses of one of these antibodies, IEI-E3, reduced plaque area in the descending aorta in a dose-dependent manner up to 50%. The extent of atherosclerosis was determined 4 weeks after the first antibody injection. IEI-E3 also significantly reduced the extent of inflammation in the subvalvular plaques, assessed by immunostainings of oxLDL epitopes and macrophages. Parallel in vitro studies showed that the same antibody induced a 5-fold increase in macrophage binding and uptake of I125-labelled oxLDL [20].
To summarize, active immunization, using Streptococcus Pneumoniae, MDA-LDL or apoB-100 peptides, reduced atherosclerosis development in ApoE-/- or LDLR-/- mice as compared with the respective controls. Nevertheless, what seems to be intriguing is that different antibodies, the IgM EO6 directed against PC epitopes on oxLDL compared to the IgG1 (IEI-E3) directed against MDA-modified apoB-100 peptides, have completely opposite effects on macrophage activity. While EO6 blocks binding of oxLDL to macrophage SR, inhibiting its uptake into the cell, IEI-E3 increases several fold both binding and uptake of oxLDL. This effect of IEI-E3 might be mediated by the fragment crystalline receptors (FcR), considering the ability of the Fc part of IgG antibodies to bind to these receptors. Noticeably, due to their complex structure, IgM do not bind to FcR. The role of the different macrophage receptors in different stages of the atherosclerotic process is not completely understood and needs to be further elucidated.
Since the interaction between macrophages, oxLDL, and antibodies is believed to influence atherosclerosis development, a possible explanation for these inadvertences is that different antibodies act differently at different stages of the disease. Indeed, EO6 successfully reduced plaque area in the subvalvular region, while IEI-E3 was effective only on plaque development in the descending aorta. It is known that atherosclerosis development in hypercholesterolemic mice begins in the aortic sinus and continues distally, whereas plaques in the thoracic and abdominal aorta appear at a later stage.
As shown, different active immunization strategies were proved to be very effective in mice and studies for creating a vaccine against atherosclerosis are currently underway [21-24]. The vaccine could offer durable long time protection by slowing down the atherosclerotic process, leading to smaller, more stable plaques. Nevertheless, some of the very high risk patients would not benefit from active immunization, due to the longer time needed for antibodies to reach effective levels. A direct, immediately effective antibody therapy, adding to current lipid lowering and antiaggregant protocols, could therefore be the answer for the vulnerable patients on the verge of a life-threatening cerebrovascular or cardiac episode. But until then, more studies need to be performed to elucidate the mechanisms behind antibody effects and their efficiency needs to be demonstrated in human studies as well.

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