Antibodies to malondialdehyde-acetaldehyde modified low-density lipoprotein in patients with newly diagnosed inflammatory joint disease.

Objective:To assess antibodies to malondialdehyde-acetaldehyde-modified low-density lipoprotein (MAA-LDL) in patients with newly diagnosed inflammatory joint disease.Method: Patients with rheumatoid arthritis (RA), spondyloarthritis (SpA), and undifferentiated arthritis (UA), participating in the Northern Savo 2010 Study, were evaluated for metabolic syndrome (MetS), metabolic and inflammatory markers, antibodies to MAA-LDL, Aggregatibacter actinomycetemcomitans, and Porphyromonas gingivalis.Results: Among 135 newly diagnosed untreated patients, of whom 53 (39%) were diagnosed to have RA, 44 (33%) SpA, and 38 (28%) UA, 49%, 30%, and 47%, respectively, had MetS. After adjusting for age and gender, anti-MAA-LDL immunoglobulin (Ig)A (p = 0.009), IgG (p = 0.031), and IgM (p = 0.001) levels differed between the diagnostic categories, but not in patients with MetS present or absent. All antibody classes to MAA-LDL correlated with erythrocyte sedimentation rate (ESR), and IgA and IgG antibodies with high-sensitivity C-reactive protein (hs-CRP). IgA antibodies to MAA-LDL correlated with rheumatoid factor (RF), anti-citrullinated protein antibodies (ACPAs), fasting plasma glucose, IgA antibodies to A. actinomycetemcomitans, and in IgA and IgG antibodies to P. gingivalis.Conclusion: Among various arthritis groups, antibodies to MAA-LDL were most common in RA. Antibodies to modified lipoproteins were associated with inflammation measured by ESR and hs-CRP. IgA antibodies to MAA-LDL correlated with age, antibodies to periodontal bacteria, RF, ACPA, and fasting glucose. Associations between antibodies to MAA-LDL and antibodies to periodontal bacteria, RA-associated antibodies, inflammatory parameters, and plasma glucose already reflect cardiovascular burden in inflammatory joint diseases at diagnosis.

Carotid artery calcification in panoramic radiographs associates with oral infections and mortality.

AIM: To study the prevalence of carotid artery calcification (CAC) in relation to apical and marginal periodontitis, subgingival dysbiotic bacterial species and serum and saliva immune responses against them. In addition, the aim was to analyse the association of CAC with angiographically verified coronary artery disease (CAD) and mortality. METHODOLOGY: In the present random Parogene cohort, the patients had an indication for coronary angiography. Apical and marginal periodontitis were diagnosed during clinical and radiographic oral examinations, and CAC on panoramic radiographs (n = 492). Presence and severity of CAD were registered from angiography. Subgingival dysbiotic bacterial species were quantitated using checkerboard DNA-DNA-hybridization, and serum and saliva antibody levels were determined by immunoassays. The cohort was followed-up for 10 years or until death (median 9.9, range 0.21-10.4) via linkage to the national death register. The statistical models were adjusted for age, gender, smoking, hypertension, diabetes and dyslipidemia. RESULTS: A total of 102 (20.7%) patients had detectable CAC, which was moderate in 81 (16.4%) and severe in 21 (4.3%). CAC was associated (OR, 95% CI) with severe apical periodontitis (2.25, 1.15-4.41), root canal fillings (1.15, 1.04-1.26), alveolar bone loss (2.66, 1.21-5.84), severe periodontal inflammation (2.23, 1.11-4.47), high level of gram-negative subgingival species (2.73, 1.34-5.50), saliva IgG against dysbiotic species (1.05, 1.01-1.10/unit) and severe (2.58, 1.36-4.90) and chronic (2.13, 1.15-3.93) CAD. A total of 105 (20.7%) patients died during the follow-up and 53 (10.4%) deaths were because of cardiovascular diseases (CVD). Severe CAC predicted worse survival with HRs (95% CI) of 3.08 (1.58-6.06) for all-cause and 3.43 (1.42-8.25) for CVD death. CONCLUSIONS: CAC on panoramic tomography was associated with (i) apical and marginal periodontitis and dysbiotic bacterial species giving rise to an immunological response, and with (ii) severe, chronic CAD and increased mortality. The results further emphasize the role of oral infections in CAD and the importance of referring a patient with CAC for a cardiovascular evaluation.

Salivary IgA to MAA-LDL and Oral Pathogens Are Linked to Coronary Disease.

A large body of literature has established the link between periodontal disease and cardiovascular disease. Oxidized low-density lipoproteins (OxLDLs) have a crucial role in atherosclerosis progression through initiation of immunological response. Monoclonal IgM antibodies to malondialdehyde-modified low-density lipoprotein (MDA-LDL) and to malondialdehyde acetaldehyde-modified low-density lipoprotein (MAA-LDL) have been shown to cross-react with the key virulence factors of periodontal pathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. We have previously shown that salivary IgA antibodies to MAA-LDL cross-react with P. gingivalis in healthy humans. In this study, we aim to assess whether oral mucosal immune response represented by salivary IgA to MAA-LDL and oral pathogens is associated with coronary artery disease (CAD). Also, the molecular mimicry through antibody cross-reaction between salivary IgA to MAA-LDL and oral pathogens was evaluated. The study subjects consisted of 451 patients who underwent a coronary angiography with no CAD ( n = 133), stable CAD ( n = 169), and acute coronary syndrome (ACS, n = 149). Elevated salivary IgA antibody levels to MAA-LDL, Rgp44 (gingipain A hemagglutinin domain of P. gingivalis), and Aa-HSP60 (heat shock protein 60 of A. actinomycetemcomitans) were discovered in stable-CAD and ACS patients when compared to no-CAD patients. In a multinomial regression model adjusted for known cardiovascular risk factors, stable CAD and ACS were associated with IgA to MAA-LDL ( P = 0.016, P = 0.043), Rgp44 ( P = 0.012, P = 0.004), Aa-HSP60 ( P = 0.032, P = 0.030), Tannerella forsythia ( P = 0.002, P = 0.004), Porphyromonas endodontalis ( P = 0.016, P = 0.020), Prevotella intermedia ( P = 0.038, P = 0.005), and with total IgA antibody concentration ( P = 0.002, P = 0.016). Salivary IgA to MAA-LDL showed cross-reactivity with the oral pathogens tested in the study patients. The study highlights an association between salivary IgA to MAA-LDL and atherosclerosis. However, whether salivary IgA to MAA-LDL and the related oral humoral responses play a causal role in the development in the CAD should be elucidated in the future.

Intestinal alkaline phosphatase at the crossroad of intestinal health and disease - a putative role in type 1 diabetes.

BACKGROUND: Patients with type 1 diabetes have shown an increase in circulating cytokines, altered lipoprotein metabolism and signs of vascular dysfunction in response to high-fat meals. Intestinal alkaline phosphatase (IAP) regulates lipid transport and inflammatory responses in the gastrointestinal tract. We therefore hypothesized that changes in IAP activity could have profound effects on gut metabolic homeostasis in patients with type 1 diabetes. METHODS: Faecal samples of 41 nondiabetic controls and 46 patients with type 1 diabetes were analysed for IAP activity, calprotectin, immunoglobulins and short-chain fatty acids (SCFAs). The impact of oral IAP supplementation on intestinal immunoglobulin levels was evaluated in C57BL/6 mice exposed to high-fat diet for 11 weeks. RESULTS: Patients with type 1 diabetes exhibited signs of intestinal inflammation. Compared to controls, patients with diabetes had higher faecal calprotectin levels, lower faecal IAP activities accompanied by lower propionate and butyrate concentrations. Moreover, the amount of faecal IgA and the level of antibodies binding to oxidized LDL were decreased in patients with type 1 diabetes. In mice, oral IAP supplementation increased intestinal IgA levels markedly. CONCLUSION: Deprivation of protective intestinal factors may increase the risk of inflammation in the gut - a phenomenon that seems to be present already in patients with uncomplicated type 1 diabetes. Low levels of intestinal IgA and antibodies to oxidized lipid epitopes may predispose such patients to inflammation-driven complications such as cardiovascular disease and diabetic nephropathy. Importantly, oral IAP supplementation could have beneficial therapeutic effects on gut metabolic homeostasis, possibly through stimulation of intestinal IgA secretion.

Early atherosclerosis and IgG2 to bacteria are associated with FcgammaRIIa genotype in non-smokers.

BACKGROUND: Involvement of low density lipoprotein (LDL) immune complexes (ICs) in atherogenesis has been proposed. Human FcgammaRIIa receptor (CD32) plays a crucial role in the phagocytosis of IgG(2) ICs and a functional point mutation 131His/Arg diminishes IgG(2) binding to the receptor. STUDY DESIGN: We examined FcgammaRIIa-131His/Arg polymorphism, IgG(2) antibody titres to oxidized low-density lipoprotein (OxLDL) and Streptococcus pneumoniae cell wall polysaccharide (CWPS) and subclinical atherosclerosis in a large cohort of Finnish subjects (n = 1041). RESULTS: Non-smoking subjects with homozygous 131His/His genotype had more premature atherosclerosis (P = 0.004) and higher IgG(2) to bacterial CWPS (P = 0.002) compared with other genotypes. Smoking subjects had significantly higher intima-media thickness (IMT) than that of non-smokers (P < 0.001) and genotype-dependent associations were indistinct. There was no association between FcgammaRIIa genotype and antibody titres to OxLDL. CONCLUSIONS: Our data demonstrate that FcgammaRIIa 131His/Arg polymorphism is associated with subclinical atherosclerosis in non-smoking subjects. Furthermore, FcgammaRIIa genotype is associated with IgG(2) titres to bacterial CWPS, but not to OxLDL. These data propose possible involvement of FcgammaRIIa receptor in atherogenesis.

Plasma ghrelin concentrations are positively associated with carotid artery atherosclerosis in males.

BACKGROUND: Ghrelin, a peptide hormone from stomach, stimulates food intake and decreases fat utilization. Ghrelin binds to growth hormone secretagogue receptor (GHSR). GHSR density has been shown to be upregulated in atherosclerotic lesions, but the relationship between ghrelin concentration and atherosclerosis has not yet been studied. We, therefore, characterized the association between ghrelin concentration and carotid artery intima-media thickness (IMT) in a population-based cohort of 1024 middle-aged (40-60 years) men and women. METHODS: Intima-media thickness and the number of atherosclerotic plaques were determined ultrasonographically. Fasting plasma ghrelin concentrations were analysed using RIA-kit (PhoenixPeptide). RESULTS: There was a positive association between mean IMT and ghrelin concentration in the analysis of males before and after adjustments for the traditional risk factors of atherosclerosis [age, systolic blood pressure, LDL cholesterol, body mass index (BMI), and smoking (ancova, P = 0.004 and P = 0.007, respectively)]. However, no such association was found in females (P = 0.985 and P = 0.915). There was no correlation between ghrelin and CRP concentrations or ghrelin and smoking. CONCLUSION: Ghrelin concentrations and carotid artery atherosclerosis are positively associated in males even after adjustment for the commonly recognized risk factors of atherosclerosis. Experimental and prospective studies are warranted to elucidate the role of ghrelin in atherosclerosis.

The negative association between plasma ghrelin and IGF-I is modified by obesity, insulin resistance and type 2 diabetes.

AIMS/HYPOTHESIS: Ghrelin is a natural growth hormone-releasing peptide thought to be involved in the regulation of energy metabolism. The recent studies concerning the association between ghrelin and insulin-like growth factor-I (IGF-I) concentrations have shown either negative correlation or no correlation at all. The aims of this study were to clarify the association between ghrelin and IGF-I concentrations in a large cohort and to characterize whether obesity, insulin resistance and type 2 diabetes affect this association. METHODS: We analysed fasting plasma ghrelin and IGF-I concentrations of 1,004 middle-aged subjects of the population-based OPERA study. Insulin resistance was estimated using QUICKI. RESULTS: IGF-I concentrations were negatively associated with ghrelin concentrations in the analysis of all subjects before (beta=-0.32, p<0.001) and after adjustments for BMI, insulin levels, sex and age (beta=-0.40, p<0.001). The association was particularly strong in males and in the higher BMI tertiles. The degree of association varied in relation to the glycaemic status: no insulin resistance: r(2)=6.5% (p<0.001), insulin resistance without type 2 diabetes: r(2)=21.0% (p<0.001), type 2 diabetes: r(2)=25.4 (p<0.001). IGF-I levels explained larger proportion (r(2)=9.8%) of the variation in ghrelin concentrations compared to fasting insulin concentration (r(2)=3.0%) and BMI (r(2)=1.5%). CONCLUSIONS/INTERPRETATION: There is a negative and independent association between ghrelin and IGF-I concentrations in middle-aged subjects. The interaction between IGF-I and ghrelin is modified by obesity, IR and type 2 diabetes. Further studies are warranted to elucidate the role of ghrelin in the development of these states.

Human-derived anti-oxidized LDL autoantibody blocks uptake of oxidized LDL by macrophages and localizes to atherosclerotic lesions in vivo.

Autoantibodies to oxidation-specific epitopes of low density lipoprotein (LDL), such as malondialdehyde-modified LDL (MDA-LDL), occur in plasma and atherosclerotic lesions of humans and animals. Plasma titers of such antibodies are correlated with atherosclerosis in murine models, and several such autoantibodies have been cloned. However, human-derived monoclonal antibodies to epitopes of oxidized LDL (OxLDL) have not yet been reported. We constructed a phage display antibody library from a patient with high plasma anti-MDA-LDL titers and isolated 3 monoclonal IgG Fab antibodies, which specifically bound to MDA-LDL. One of these, IK17, also bound to intact OxLDL as well as to its lipid and protein moieties but not to those of native LDL. IK17 inhibited the uptake of OxLDL by macrophages and also bound to apoptotic cells and inhibited their phagocytosis by macrophages. IK17 strongly immunostained necrotic cores of human and rabbit atherosclerotic lesions. When (125)I-IK17 was injected intravenously into LDL receptor-deficient mice, its specific uptake was greatly enriched in atherosclerotic plaques versus normal aortic tissue. Human autoantibodies to OxLDL have important biological properties that could influence the natural course of atherogenesis.

Anticardiolipin antibodies from patients with the antiphospholipid antibody syndrome recognize epitopes in both beta(2)-glycoprotein 1 and oxidized low-density lipoprotein.

BACKGROUND: We recently suggested that many anticardiolipin antibodies bind only to oxidized cardiolipin (OxCL) and/or to OxCL-beta(2)-glycoprotein 1 (beta(2)GP1) adducts but not to a "reduced" cardiolipin that is unable to undergo oxidation. To test this hypothesis, we investigated 24 sera, 4 protein A-purified IgG fractions, and 3 human monoclonal antibodies that were all isolated from patients with antiphospholipid antibody syndrome (APS); testing was also performed in 7 controls. Two monoclonal antibodies (IS3 and IS4) were selected for binding to CL and one was selected for binding to beta(2)GP1 (LJB8). METHODS AND RESULTS: By chemiluminescent immunoassay, all APS sera samples bound only to OxCL and not to reduced CL, and the binding was inhibited >95% by OxCL but not reduced CL. All purified IgG fractions bound to beta(2)GP1 but only when the beta(2)GP1 was plated on microtiter wells coated with OxCL. All 3 monoclonal antibodies bound only to OxCL. On Western blots, IS4 and LJB8 bound to beta(2)GP1 as well as to delipidated apoB of oxidized LDL but not to native apoB. IS3 also bound to oxidized apoB on Western blot. Covalent modification of beta(2)GP1 with oxidation products of CL made it more antigenic for APS serum samples, for purified IgG fractions, and for the monoclonal antibodies. CONCLUSIONS: These data support the hypothesis that oxidation of CL is needed to generate epitopes for many anticardiolipin antibodies and that some of these epitopes are covalent adducts of OxCL with beta(2)GP1 or apoB.

Circulating autoantibodies to oxidized cardiolipin correlate with isoprostane F(2alpha)-VI levels and the extent of atherosclerosis in ApoE-deficient mice: modulation by vitamin E.

Lipid peroxidation plays an important role in atherogenesis. Previous studies suggested that autoantibodies against epitopes of oxidized low-density lipoprotein may indicate the extent or rate of progression of atherosclerosis. The aim of this study was to investigate whether autoantibodies to oxidized phospholipids, such as oxidized cardiolipin (OxCL), correlate with levels of isoprostane F(2alpha)-VI, a sensitive marker of in vivo lipid peroxidation, as well as with the extent of atherosclerosis. Two groups of apolipoprotein E-deficient mice were fed chow with or without vitamin E (2000 IU/kg diet) for 16 weeks. In untreated animals, autoantibodies against OxCL and urinary, plasma, and aortic isoprostane F(2alpha)-VI levels increased significantly. Vitamin E treatment significantly reduced antibody titers, isoprostane levels, and atherosclerosis at the end of the study, compared with untreated mice. Autoantibodies to OxCL correlated with aortic isoprostane F(2alpha)-VI levels (r(2) = 0.42, P =.001 for IgG and r(2) = 0.63, P <.001 for IgM). Both aortic isoprostane F(2alpha)-VI levels (r(2) = 0.59, P <.001) and titers of OxCL antibodies (r(2) = 0.70, P <.001 for IgG and r(2) = 0.68, P <.001 for IgM) correlated with the extent of aortic atherosclerosis. The fact that the levels of autoantibodies to OxCL correlated with a sensitive direct measure of lipid peroxidation in vivo and that both autoantibodies and aortic isoprostane F(2alpha)-VI levels correlated with the extent of atherosclerosis suggests that antibodies to OxCL are a sensitive indicator of in vivo lipid peroxidation and atherosclerosis.

Scavenger receptors, oxidized LDL, and atherosclerosis.

Oxidized LDL (OxLDL) competes with oxidatively damaged and apoptotic cells for binding to mouse peritoneal macrophages, implying the presence of one or more common domains. However, the nature of the ligands involved has not been determined. Studies in this laboratory over the last several years provide evidence that oxidized phospholipids, present in OxLDL and also in the membrane of apoptotic cells, represent one such ligand. These oxidized phospholipids, either in the lipid phase of OxLDL or becoming attached covalently to apoprotein B during LDL oxidation, have been shown to play a major role in the binding of OxLDL to CD36 and to SR-B1 expressed in transfected cells. The lipid and protein moieties compete with each other to some extent, indicating that they are binding to at least one common site. A monoclonal antibody selected because of its reactivity with OxLDL proved to be an antibody against oxidized phospholipids (but not native phospholipids). This antibody (EO6) blocked the uptake of OxLDL by CD36 and by SR-B1 in transfected cells by as much as 80%; it also inhibited macrophage phagocytosis of apoptotic cells by about 40%. Thus, the persistence of receptors for OxLDL during evolution is probably accounted for by their role in recognition of ligands on the surfaces of oxidatively damaged or apoptotic cells. This has important implications in biology generally and specifically in atherogenesis, because apoptosis is a prominent feature of late lesions.

Neo-self antigens and the expansion of B-1 cells: lessons from atherosclerosis-prone mice.

The pathogenesis of atherosclerosis involves an inflammatory process that is modulated by the immune system, and within these complex responses we have discerned a possible role for an archetypic B-1 clone. We speculate that due to their immunogenicity and in vivo distribution the "neo"-self determinants created in oxidatively modified LDL are highly stimulatory for certain B-1 cell clones. These neo-self determinants, which can be created chemically, by somatic processes, may in fact represent the molecular analogues of somatic maturation, or even aging. These changes, including those on non-protein antigens induced by oxidative metabolism, amongst others, create neo-determinants against which the host no doubt can not develop rigorous B-cell tolerance. The onset of expression of these oxidative neo-determinants relatively late in development may well serve a useful function for the highly evolved mammalian immune system, as targeting by evolutionarily selected B-1 clones may facilitate the amplification of other useful antibody-mediated physiologic functions. As in the case of the T15 clone, these antibodies may aid in protection against common microbial pathogens. Hence we postulate that during the evolution of the adaptive immune system the neo-self antigenic milieu may have been exploited for the natural selection of primordial clonal specificities. The T15 B-1 clone may then illustrate a common paradigm in which there has been natural selection based on utility for the defense of the individual from environmental threats, as well as for possible "housekeeping" role(s) and the maintenance of cellular homeostasis.

Immunological responses to oxidized LDL.

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 concerted 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.

Natural antibodies with the T15 idiotype may act in atherosclerosis, apoptotic clearance, and protective immunity.

The immune response to oxidized LDL (OxLDL) may play an important role in atherogenesis. Working with apoE-deficient mice, we isolated a panel of OxLDL-specific B-cell lines that secrete IgM Abs that specifically bind to oxidized phospholipids such as 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC). These Abs block uptake of OxLDL by macrophages, recognize similar oxidation-specific epitopes on apoptotic cells, and are deposited in atherosclerotic lesions. The Abs were found to be structurally and functionally identical to classic "natural" T15 anti-PC Abs that are of B-1 cell origin and are reported to provide optimal protection from virulent pneumococcal infection. These findings suggest that there has been natural selection for B-1 cells secreting oxidation-specific/T15 antibodies, both for their role in natural immune defense and for housekeeping roles against oxidation-dependent neodeterminants in health and disease.

Oxidized phospholipids, linked to apolipoprotein B of oxidized LDL, are ligands for macrophage scavenger receptors.

Previous studies have shown that macrophage receptors for oxidized LDL (OxLDL) recognize both the lipid and protein moieties, and that a monoclonal antibody against OxLDL, EO6, also recognizes both species. The present studies show directly that during LDL oxidation phospholipids become covalently attached to apolipoprotein B (apoB). After exhaustive extraction of lipids, apoB of native LDL contained 4 +/- 3 moles of phosphorus/mole protein. In contrast, apoB of OxLDL contained approximately 75 moles of phosphorus/mole protein. Saponification of this apoB released phosphorus, choline, and saturated fatty acids in a molar ratio of 1.0:0.98:0.84. When LDL was reductively methylated prior to oxidation, the amount of phospholipid covalently bound was reduced by about 80%, indicating that the phospholipids attach at lysine epsilon amino groups. Progressive decreases in the phospholipid associated with apoB of OxLDL decreased the ability of the protein to compete for binding to macrophage scavenger receptors and decreased its reactivity with antibody EO6. We postulate that some oxidized phospholipids containing fatty acid aldehydes at the sn-2 position bind to lysine residues of apoB while others remain unreacted within the lipid phase. This would account for the interchangeability of lipid and apolipoprotein of OxLDL with respect to receptor binding and antibody recognition.

The binding of oxidized low density lipoprotein to mouse CD36 is mediated in part by oxidized phospholipids that are associated with both the lipid and protein moieties of the lipoprotein.

There is growing evidence that CD36 has an important physiological function in the uptake of oxidized low density lipoprotein (OxLDL) by macrophages. However, the ligand specificity and the nature of the ligands on OxLDL that mediate the binding to CD36 remain ill defined. Results from recent studies suggested that some of the macrophage scavenger receptors involved in the uptake of OxLDL recognized both the lipid and the protein moieties of OxLDL, but there was no conclusive direct evidence for this. The present studies were undertaken to test whether a single, well characterized OxLDL receptor, CD36, could bind both the lipid and protein moieties of OxLDL. COS-7 cells transiently transfected with mouse CD36 cDNA bound intact OxLDL with high affinity. This binding was very effectively inhibited ( approximately 50%) both by the reconstituted apoB from OxLDL and by microemulsions prepared from OxLDL lipids. The specific binding of both moieties to CD36 was further confirmed by direct ligand binding analysis and by demonstrating reciprocal inhibition, i.e. apoB from OxLDL inhibited the binding of the OxLDL lipids and vice versa. Furthermore, a monoclonal mouse antibody that recognizes oxidation-specific epitopes in OxLDL inhibited the binding of intact OxLDL and also that of its purified protein and lipid moieties to CD36. This antibody recognizes the phospholipid 1-palmitoyl 2-(5'-oxovaleroyl) phosphatidylcholine. This model of an oxidized phospholipid was also an effective competitor for the CD36 binding of both the resolubilized apoB and the lipid microemulsions from OxLDL. Our results demonstrate that oxidized phospholipids in the lipid phase or covalently attached to apoB serve as ligands for recognition by CD36 and, at least in part, mediate the high affinity binding of OxLDL to macrophages.

All ApoB-containing lipoproteins induce monocyte chemotaxis and adhesion when minimally modified. Modulation of lipoprotein bioactivity by platelet-activating factor acetylhydrolase.

Mildly oxidized LDL has many proinflammatory properties, including the stimulation of monocyte chemotaxis and adhesion, that are important in the development of atherosclerosis. Although ApoB-containing lipoproteins other than LDL may enter the artery wall and undergo oxidation, very little is known regarding their proinflammatory potential. LDL, IDL, VLDL, postprandial remnant particles, and chylomicrons were mildly oxidized by fibroblasts overexpressing 15-lipoxygenase (15-LO) and tested for their ability to stimulate monocyte chemotaxis and adhesion to endothelial cells. When conditioned on 15-LO cells, LDL, IDL, but not VLDL increased monocyte chemotaxis and adhesion approximately 4-fold. Chylomicrons and postprandial remnant particles were also bioactive. Although chylomicrons had a high 18:1/18:2 ratio, similar to that of VLDL, and should presumably be less susceptible to oxidation, they contained (in contrast to VLDL) essentially no platelet-activating factor acetylhydrolase (PAF-AH) activity. Because PAF-AH activity of lipoproteins may be reduced in vivo by oxidation or glycation, LDL, IDL, and VLDL were treated in vitro to reduce PAF-AH activity and then conditioned on 15-lipoxygenase cells. All 3 PAF-AH-depleted lipoproteins, including VLDL, exhibited increased stimulation of monocyte chemotaxis and adhesion. In a similar manner, lipoproteins from Japanese subjects with a deficiency of plasma PAF-AH activity were also markedly more bioactive, and stimulated monocyte adhesion nearly 2-fold compared with lipoproteins from Japanese control subjects with normal plasma PAF-AH. For each lipoprotein, bioactivity resided in the lipid fraction and monocyte adhesion could be blocked by PAF-receptor antagonists. These data suggest that the susceptibility of plasma lipoproteins to develop proinflammatory activity is in part related to their 18:1/18:2 ratio and PAF-AH activity, and that bioactive phospholipids similar to PAF are generated during oxidation of each lipoprotein. Moreover, LDL, IDL, postprandial remnant particles, and chylomicrons and PAF-AH-depleted VLDL all give rise to proinflammatory lipids when mildly oxidized.

Receptors for oxidized low-density lipoprotein on elicited mouse peritoneal macrophages can recognize both the modified lipid moieties and the modified protein moieties: implications with respect to macrophage recognition of apoptotic cells.

It has been shown previously that the binding of oxidized low-density lipoprotein (OxLDL) to resident mouse peritoneal macrophages can be inhibited (up to 70%) by the apoprotein B (apoB) isolated from OxLDL, suggesting that macrophage recognition of OxLDL is primarily dependent on its modified protein moiety. However, recent experiments have demonstrated that the lipids isolated from OxLDL and reconstituted into a microemulsion can also strongly inhibit uptake of OxLDL (up to 80%). The present studies show that lipid microemulsions prepared from OxLDL bind to thioglycollate-elicited macrophages at 4 degrees C in a saturable fashion and inhibit the binding of intact OxLDL and also of the apoB from OxLDL. Reciprocally, the binding of the OxLDL-lipid microemulsions was strongly inhibited by intact OxLDL. A conjugate of synthetic 1-palmitoyl 2(5-oxovaleroyl) phosphatidylcholine (an oxidation product of 1-palmitoyl 2-arachidonoyl phosphatidylcholine) with serum albumin, shown previously to inhibit macrophage binding of intact OxLDL, also inhibited the binding of both the apoprotein and the lipid microemulsions prepared from OxLDL. Finally, a monoclonal antibody against oxidized phospholipids, one that inhibits binding of intact OxLDL to macrophages, also inhibited the binding of both the resolubilized apoB and the lipid microemulsions prepared from OxLDL. These studies support the conclusions that: (i) at least some of the macrophage receptors for oxidized LDL can recognize both the lipid and the protein moieties; and (ii) oxidized phospholipids, in the lipid phase of the lipoprotein and/or covalently linked to the apoB of OxLDL, likely play a role in that recognition.

Monoclonal antibodies against oxidized low-density lipoprotein bind to apoptotic cells and inhibit their phagocytosis by elicited macrophages: evidence that oxidation-specific epitopes mediate macrophage recognition.

Apoptosis is recognized as important for normal cellular homeostasis in multicellular organisms. Although there have been great advances in our knowledge of the molecular events regulating apoptosis, much less is known about the receptors on phagocytes responsible for apoptotic cell recognition and phagocytosis or the ligands on apoptotic cells mediating such recognition. The observations that apoptotic cells are under increased oxidative stress and that oxidized low-density lipoprotein (OxLDL) competes with apoptotic cells for macrophage binding suggested the hypothesis that both OxLDL and apoptotic cells share oxidatively modified moieties on their surfaces that serve as ligands for macrophage recognition. To test this hypothesis, we used murine monoclonal autoantibodies that bind to oxidation-specific epitopes on OxLDL. In particular, antibodies EO6 and EO3 recognize oxidized phospholipids, including 1-palmitoyl 2-(5-oxovaleroyl) phosphatidylcholine (POVPC), and antibodies EO12 and EO14 recognize malondialdehyde-lysine, as in malondialdehyde-LDL. Using FACS analysis, we demonstrated that each of these EO antibodies bound to apoptotic cells but not to normal cells, whereas control IgM antibodies did not. Confocal microscopy demonstrated cell-surface expression of the oxidation-specific epitopes on apoptotic cells. Furthermore, each of these antibodies inhibited the phagocytosis of apoptotic cells by elicited peritoneal macrophages, as did OxLDL. In addition, an adduct of POVPC with BSA also effectively prevented phagocytosis. These data demonstrate that apoptotic cells express oxidation-specific epitopes-including oxidized phospholipids-on their cell surface, and that these serve as ligands for recognition and phagocytosis by elicited macrophages.