Anti-Ox-LDL Antibodies and Anti-Ox-LDL-Β 2 GPI Antibodies in Patients with Systemic Lupus Erythematosus

original papers Adv Clin Exp Med 2012, 21, 3, 331 335 ISSN 1899 5276 Copyright by Wroclaw Medical University Beata Nowak 1, 2, Magdalena Szmyrka-Kaczmarek 2, Anna Durazińska 2, Rafał Płaksej 3, Krzysztof Borysewicz 2, Lucyna Korman 2, Piotr Wiland 2 Anti-Ox-LDL Antibodies and Anti-Ox-LDL-Β 2 GPI Antibodies in Patients with Systemic Lupus Erythematosus Przeciwciała przeciwko oksydowanym LDL oraz przeciwko kompleksom oxldl-β2gpi u chorych na tocznia rumieniowatego układowego 1 Department of Pharmacology, Wroclaw Medical University, Poland 2 Department of Rheumatology and Internal Medicine, Wroclaw Medical University, Poland 3 Department of Cardiology, Wroclaw Medical University, Poland Abstract Objectives. The aim of the study was to assess the concentration of anti-oxidized low-density lipoprotein (antioxldl) antibodies and antibodies against oxldl-β 2 GPI (oxldl-beta 2 glycoprotein I) complexes in the serum of patients with systemic lupus erythematosus (SLE). Correlations between clinical and laboratory factors and the intima media thickness (IMT) were also investigated. Material and Methods. The study included 16 patients (14 females, 2 males) with an established diagnosis of SLE. The mean disease duration was 6.3 years (range: 2 23 years). Thirteen age-matched healthy volunteers comprised the control group. IMT, the concentration of anti-oxldl and anti-oxldl-β 2 GPI antibodies and lipid profile were assesed. Data concerning other cardiovascular risk factors were also collected. Results. In the SLE group the intima media was significantly thicker than in control group. In the SLE group a statistically significant positive correlation was noted between age and mean IMT. Immunological assays revealed elevated serum concentration of anti-oxldl antibodies in the SLE group; serum concentration of IgG anti-oxldlβ 2 GPI antibodies and IgM anti-oxldl-β 2 GPI antibodies were also elevated in the SLE group compared to the controls. There was a statistically significant positive correlation between LDL concentration and anti-oxldl antibody concentration in the SLE group. Conclusions. The study findings support the thesis that cardiovasular risk is significantly higher in SLE patients. Elevated concentrations of anti-oxldl antibodies, IgG anti-oxldl-β 2 GPI antibodies and IgM anti-oxldl-β 2 GPI antibodies were detected in the SLE group, which may contribute to the elevated cardiovascular risk in SLE patients (Adv Clin Exp Med 2012, 21, 3, 331 335). Key words: anti-oxidizes-low density lipoprotein antibodies, anti-oxidized-low density lipoprotein beta2-glicoprotein I antibodies, systemic lupus erythematosus. Streszczenie Cel pracy. Ocena stężenia przeciwciał skierowanych przeciwko cząsteczkom oxldl (utlenowanych lipoprotein o niskiej gęstości) oraz przeciwko kompleksom zawierających oxldl i beta2-glikokoproteinę I (oxldl-β 2 GPI) w surowicy chorych na toczeń rumieniowaty układowy (SLE). Ponadto szukano związku między czynnikami klinicznymi i laboratoryjnymi a grubością błony wewnętrznej (IMT). Materiał i metody. Do badania włączono 16 chorych (14 kobiet i 2 mężczyzn) z ustalonym rozpoznaniem SLE oraz 13 zdrowych ochotników jako grupę kontrolną. U wszystkich uczestników badania oceniono IMT, stężenia przeciwciał anty-oxldl oraz anty-oxldl-β 2 GPI i gospodarkę lipidową, a także zebrano dane dotyczące klasycznych czynników ryzyka chorób sercowo-naczyniowych. Wyniki. W grupie chorych na SLE stwierdzono istotne pogrubienie IMT w porównaniu z grupą kontrolną. W grupie chorych na SLE stwierdzono dodatnią korelację między wiekiem a IMT. Badania immunologiczne wykazały zwiększone stężenie przeciwciała anty-oxldl w grupie chorych na SLE. Wykazano także istotnie zwiększone stęże-

332 B. Nowak et al. nie przeciwciał anty-oxldl-β 2 GPI w klasie IgG oraz w klasie IgM w grupie chorych na SLE. W grupie chorych na SLE stwierdzono także istotną statystycznie dodatnią korelację między stężeniami LDL i przeciwciał anty-oxldl. Wnioski. Uzyskane wyniki potwierdzają tezę o istotnie zwiększonym ryzyku sercowo-naczyniowym u chorych na toczeń rumieniowaty układowy. Stwierdzone zwiększone stężenie przeciwciał anty-oxldl oraz anty-oxldl-β 2 GPI w klasie IgG i w klasie IgM w grupie chorych na SLE może być jednym z czynników zwiększającym to ryzyko (Adv Clin Exp Med 2012, 21, 3, 331 335). Słowa kluczowe: przeciwciała przeciwko utlenowanym lipoproteinom o niskiej gęstości, przeciwciała przeciwko kompleksom utlenowanych lipoprotein o niskiej gęstości i beta2-glikoproteiny I, toczeń rumieniowaty układowy. Premature atherosclerosis in patients with systemic lupus erythematosus (SLE) was described for the first time more than thirty years ago by Bulkley and Roberts [1]. Since then many authors have reported symptomatic or asymptomatic premature atherosclerosis in SLE patients. As the traditional Framingham risk factors cannot fully explain the reasons for accelerated atherosclerosis in systemic lupus erythematosus [2], other risk factors need to be identified and interactions between traditional and immunological risk factors are under investigation. The aim of the current study was to assess the concentration of anti-oxldl-antibodies and antibodies against oxldl-β 2 GPI complexes in the serum of SLE patients. A correlation was also sought between clinical and laboratory factors and the intima media thickness (IMT), which is one of the markers of the presence and extent of atherosclerosis [3 5]. Material and Methods The study involved 16 patients (14 females, 2 males) aged 27 60 (mean age 44.4) with an established diagnosis of SLE. The inclusion criteria were an established diagnosis of SLE and age above 18 years; exclusion criteria for the SLE group included overlap syndromes, chronic and acute infections, and a history of malignant neoplasm. The mean disease duration was 6.3 years (range: 2 23 years). The characteristics of the SLE patients are shown in Table 1. Thirteen age-matched healthy volunteers made up the control group. The exclusion criteria for the control group included SLE or any other connective tissue disease, chronic and acute infections, and history of malignant neoplasm. Volunteers were also excluded if their medical history revealed any cardio-vascular disease. People with elevated blood pressure, any abnormalities in ECGs or significant abnormalities in physical examinations were excluded from the study. The patients and the healthy volunteers gave their informed consent prior to their inclusion in the study. Table 1. Characteristics of the SLE group Tabela 1. Charakterystyka grupy chorych na SLE SLEDAI# 0 34 (10) SLICC/ACR# 0 7 (2.87) Concomitant diseases (Choroby współistniejące) antiphospholipid syndrome* 5 (31.25%) diabetes type II* 1 (6.25%) hyperlipidemia* 10 (62.5%) hypertension * 7 (43.75%) Manifestations of atherosclerosis (Objawy miażdżycy) coronary heart disease* 1 (6.25%) peripheral vascular disease* 0 (0%) stroke* 1 (6.25%) Treatment for SLE (Leczenie SLE) number of patients receiving GCs dose of GCs (counted as dose of prednisone# number of patients receiving immunosuppressive drugs* 14 (87.5%) 5 30 (15.5%) 9 (7 AZA, 1 Cy, 1 other) (56%) SLE systemic lupus erythematosus. SLEDAI SLE Disease Activity Index. SLICC/ACR SLE Collaborating Clinics/American College of Rheumatology Damage Index. GCs glicocorticosteroids. AZA azathioprine. Cy cyclophosphamide. # results presented as range (mean). * results presented as number (%). SLE toczeń rumieniowaty układowy. SLEDAI wskaźnik aktywności SLE. SLICC/ACR wskaźnik uszkodzenia narządów w przebiegu SLE. GCs glikokortykosteroidy. AZA azatiopryna. Cy cyklofosfamid. # wyniki przedstawione w postaci: zakres (średnia). * wyniki przedstawione w postaci: liczba (%).

Anti-Ox-LDL Antibodies and Anti-Ox-LDL-Β2GPI in SLE 333 The data for analysis were obtained from medical histories, physical examinations, laboratory tests and ultrasound examinations of the carotid arteries. In the SLE group the disease activity was measured with the Systemic Lupus Erythematosous Disease Activity Index (SLEDAI) and organ damage was assessed using the SLE Collaborating Clinics/ American College of Rheumatology Damage Index (SLICC/ACR). The ultrasound examinations of the carotid arteries in both groups were carried out using a high frequency linear transducer (10 MHz, Vingmed System 5). Two measurements of the IMT of the common carotid artery (CCA), internal carotid artery (ICA) and the bulb of common carotid artery (BCCA) were performed bilaterally. The mean IMT was calculated as mean value of all these measurements. Serum concentrations of anti-oxldl antibodies and anti-oxldl-β 2 GPI IgG and IgM antibodies were assessed with ELISA tests (Oxidized LDL, Mercodia and Anti-AtherOx Test Kit IgG, and Anti-AtherOx Test Kit IgM, both from Corgenix Medical Corporation, USA). The statistical analysis was performed using Statistica Software. The differences in mean IMT, anti-oxldl antibodies, anti-oxldl-β 2 GPI IgG and IgM antibodies were compared using a U-Mann Whitney test. Correlations between the assessed parameters were tested using non-parametrical tests. The study protocol was reviewed in November 2005 (KB923/2005) by the Ethics Committee of Wroclaw Medical University, and the study was been performed in accordance with the ethical standards of the1964 Declaration of Helsinki. Results The results of IMT measurements and immunological assays are shown in Table 2. In the SLE group the intima media was significantly thicker than in the control group (the mean IMT in the SLE group was 0.71 ± 0.21 mm, as opposed to the mean IMT in the control group, which was 0.50 ± 0.04 mm; p = 0.00015). This proved that arteriosclerosis was more advanced in the SLE patients. In the SLE group a statistically significant positive correlation was found between age and mean IMT (R S = 0.68; p = 0.004). In the SLE group there was also a statistically significant negative correlation between mean IMT and dose of glicocorticosteroids (R S = 0.64; p = 0.013). There was no significant correlation between SLICC/ACR and IMT. In the control group a statistically significant positive correlation was detected between mean IMT and serum concentration of IgG anti-oxldlβ 2 GPI antibodies (R S = 0.64; p = 0.019) The immunological assays revealed elevated serum concentration of anti-oxldl antibodies in the SLE group (97.8 ± 22.6 U/L vs. 70.2 ± 19.0 U/L; p = 0.0023). The serum concentrations of IgG antioxldl-β2gpi antibodies and IgM anti-oxldl- Table 2. IMT and immunological assays in the SLE group and the control group Tabela 2. IMT I wyniki badań immunologicznych w grupie chorych na SLE I w grupie kontrolnej SLE group (Grupa SLE) n = 16 Control group (Grupa kontrolna) n = 13 p * IMT mm 0.71 ± 0.21 0.50 ± 0.04 0.00015 Anti-oxLDL antibodies (Przeciwciała anty-oxldl) U/l Anti-oxLDL-β2GPI IgG antibodies G Units number of positive sera (Przeciwciała anty-oxldl-β2gpi klasy IgG jednostki G] liczba surowic z wynikiem dodatnim) 97.8 ± 22.6 70.2 ± 19.0 0.0023 38.7 ± 41.0 (10) 11.9 ± 5.6 (1) 0.0015 Anti-oxLDL-β2GPI IgM antibodies M Units number of positive sera (Przeciwciała anty-oxldl-β2gpi klasy IgM jednostki M liczba surowic z wynikiem dodatnim) Results presented as mean ± SD. SD standard deviation. IMT intima media thickness. SLE: systemic lupus erythematosus. oxldl: oxidized LDL cholesterol. oxldl-β2gpi complex of oxldl and β2-glycoprotein I. * p values concern the comparison of levels in the two groups. 42.1 ± 63.7 (7) 11.7 ± 6.7 (2) 0.008 Wyniki przedstawiono w postaci: średnia ± SD. SD odchylenie standardowe. IMT grubość błony wewnętrznej. SLE toczeń rumieniowaty układowy. oxldl utlenowany cholesterol frakcji LDL. oxldl-β 2 GPI kompleks oxldl i β 2 -glikoproteiny I. * wartości p dotyczą porównywanych stężeń.

334 B. Nowak et al. β2gpi antibodies were also elevated in the SLE group as compared to the controls (38.7 ± 41.0 G Units vs. 11.9 ± 5.6 G Units; p = 0.0015 and 42.1 ± 63.7 M Units vs. 11.7 ± 6.7 M Units; p = 0.0080, respectively). No correlations between the levels of IgG and IgM anti-oxldl-β 2 GPI antibodies were found. In the SLE group there were statistically significant correlations between the concentration of anti-oxldl antibodies and the concentrations of LDL, triglycerides and total cholesterol (R S = 0.74, p = 0.0027; R S = 0.57, p = 0.032; and R S = 0.53, p = 0.047, respectively). No correlations between the SLEDAI values and the concentrations of anti-oxldl and anti-oxldl-β 2 GPI antibodies were detected. However, there was a statistically significant negative correlation between erythrocyte sedimentation rate (ESR) and high density lipoprotein (HDL) concentration (R S = 0.60; p < 0.05). No correlation between SLICC/ ACR scores and concentrations of anti-oxldl and anti-oxldl-β₂gpi antibodies were detected. Discussion Thicker intima media were found in the SLE patients than in the healthy volunteers. As prospective studies have revealed a strong correlation between carotid atherosclerosis and the risk of myocardial infarction [6] and the Carotid Atherosclerosis Progression Study (CAPS) proved that carotid IMT independently predicted future vascular events and that its predictive value was high in younger and older subjects [7], the current findings support the thesis that cardiovasular risk is significantly higher in SLE patients. Elevated concentrations of anti-oxldl antibodies were found in the SLE group. Acting as autoantigens, oxldl particles stimulate the synthesis of anti-oxldl-antibodies, which are elevated in patients with unstable angina and myocardial infarction [8, 9]. The findings of the current study indicate that anti-oxldl antibodies may also contribute to the elevated cardiovascular risk in SLE patients. Although in most studies, a positive correlation between anti-oxldl antibodies and IMT was found, suggesting that these antibodies might be markers of progression of atherosclerosis, some experimental data indicate that anti-oxldl antibodies may play a protective role [10, 11]. β 2 glycoprotein I (β 2 GPI), which is one of the main antigens for antiphospholipids antibodies, binds to oxldl, forming stable complexes. These complexes increase the uptake of oxldl by macrophage scavenger receptors and accelerate foam cell formation. The presence of antibodies against oxldl-β 2 GPI-complexes accelerates their internalization and strongly correlates with the risk of arterial thrombosis [12]. The in vitro macrophage uptake of oxldl-β 2 -GPI-complexes increases in the presence of autoantibodies against those complexes [13]. Like Lopez et al. [14], the current authors detected significantly elevated level of IgG anti-oxldl-β 2 GPI antibodies in the serum of SLE patients. In contrast to Lopez s study, the current study revealed a correlation between mean IMT and serum concentration of IgG anti-oxldl-β 2 GPI antibodies. This supports the hypothesis that those antibodies contribute not only to thrombosis formation, but also to the growth of atherosclerotic plaque. It has been suggested by other authors that IgG antibodies are proatherogenic, while IgM antibodies may be protective, but the role of antioxldl-β 2 GPI antibodies in atherogenesis remains controversial [14, 15]. Some authors suggest that anti-oxldl-β 2 GPI antibodies may be involved in organ damage in SLE patients [16], but the current study revealed no correlation between IgG or IgM antibodies and SLICC/ACR scores. The authors concluded that in SLE patients, anti-oxldl, IgG and IgM anti-oxldl-β 2 GPI antibodies may contribute to premature atherogenesis. References [1] Bulkley B, Roberts W: The heart in SLE and changes induced in it by corticosteroid therapy: a study of 36 necroscopy cases. Am J Med 1975, 53, 243 264. [2] Esdaile J, Abrahamowicz M, Grodzicky T, Li Y, Panaritis C, du Berger R: Traditional Framingham risk factors fail to fully account for accelerated atherosclerosis in systemic lupus erythematosous. Arthritis Rheum 2001, 44, 2231 2237. [3] Haller C, Schulz J, Schmidt-Trucksäss A, Burkardt H, Schmitz D, Dickhuth H, Sandrock M: Sequential based analysis of intima-media thickness (IMT) in common carotid artery studies. Atherosclerosis 2007, 195, e203 e209. [4] Rosvall M, Janzon L, Berglund G, Engström G, Hedblad B: Incidence of stroke is related to carotid imt even in the absence of plaque. Atherosclerosis 2005, 179, 325 331. [5] Lee E, Emoto M, Teramura M, Tsuchikura S, Ueno H, Shinohara K, Morioka T, Mori K, Koyama H, Shoji T, Okuno Y, Inaba M, Nishizawa Y: The combination of IMT and stiffness parameter beta is highly associated with concurrent coronary artery disease in type 2 diabetes. J Atheroscler Thromb 2009, 16, 33 39.

Anti-Ox-LDL Antibodies and Anti-Ox-LDL-Β2GPI in SLE 335 [6] Johnsen SH, Mathiesen EB, Joakimsen O, Stensland E, Wilsgaard T, Løchen M, Njølstad I, Arnesen E: Carotid atherosclerosis is a stronger predictor of myocardial infarction in women than in men: a 6-year follow-up study of 6226 persons: the TROMSØ study. Stroke 2007, 38, 2873 2880. [7] Lorenz MW, von Kegler S, Steinmetz H, Markus HS, Sitzer M: Carotid intima-media thickening indicates a higher vascular risk across a wide age range: prospective data from the carotid atherosclerosis progression study (caps). Stroke 2006, 37, 87 92. [8] Salonen J, Yla-Herttuala S, Yamamoto R: Autoantibody against oxidized LDL and progression of carotid atherosclerosis. Lancet 1999, 339, 883 887. [9] Inoue T, Uchida T, Kamishirado H: Clinical significance of antibody against oxidized low density lipoprotein in patients with atherosclerotic coronary artery disease. J Am Coll Cardiol 2001, 37, 775 779. [10] Karvonen J, Päivänsalo M, Kesäniemi YA, Hörkkö S: Immunoglobulin m type of autoantibodies to oxidized lowdensity lipoprotein has an inverse relation to carotid artery atherosclerosis. Circulation 2003, 108, 2107 2112. [11] Shoenfeld Y, Wu R, Dearing LD, Matsuura E: Are anti-oxidized low-density lipoprotein antibodies pathogenic or protective? Circulation 2004, 110, 2552 2558. [12] Matsuura E, Lopez L: Are oxidized LDL/beta2-glycoprotein I complexes pathogenic antigens in autoimmunemediated atherosclerosis? Clin Dev Immunol 2004, 11, 103 111. [13] Matsuura E, Kobayashi K, Hurley B, Lopez L: Atherogenic oxidized low-density lipoprotein/beta-2-glycoprotein I (oxldl/beta2gpi) complexes in patients with systemic lupus erythematosus and antiphospholipid syndrome. Lupus 2006, 15, 478 483. [14] Lopez LR, Salazar-Paramo M, Palafox-Sanchez C, Hurley BL, Matsuura E, Garcia-De La Torre I: Oxidized low-density lipoprotein and beta2-glycoprotein I in patients with systemic lupus erythematosus and increased carotid intima-media thickness: implications in autoimmune-mediated atherosclerosis. Lupus 2006, 15, 80 86. [15] Bassi N, Ghirardello A, Iaccarino L, Zampieri S, Rampudda ME, Atzeni F, Sarzi-Puttini P, Shoenfeld Y, Doria A: OxLDL/beta2GPI-anti-oxLDL/beta2GPI complex and atherosclerosis in SLE patients. Autoimmun Rev 2007, 7, 52 58. [16] Bassi N, Zampieri S, Ghirardello A, Tonon M, Zen M, Beggio S, Matsuura E, Doria A: OxLDL/beta2GPI complex and anti-oxldl/beta2gpi in SLE: prevalence and correlates. Autoimmunity 2009, 42, 289 291. Address for correspondence: Beata Nowak Department of Pharmacology Wroclaw Medical University Mikulicza-Radeckiego 2 50-345 Wrocław Poland Tel.: +48 71 784 1442 Mobile: +48 607 924 471 E-mail: betanowak@interia.pl Conflict of interest: none declared Received: 2.06.2011 Revised: 28.12.2011 Accepted: 6.06.2012