4-hydroxy-2-nonenal and Lupus-Erythematosus--Systemic

Non-enzymatic lipid peroxidation of cellular membranes occurs during periods of sustained oxidative stress. 4-Hydroxynonenal (HNE), the most reactive lipid peroxidation product, is capable of modifying and/or cross-linking proteins leading to impaired physiological functions. The formation of protein adducts produce structural modifications which generate neo-antigens and induce auto-antibodies. Enhanced oxidative stress and accumulation of HNE-modified proteins are associated with systemic lupus erythematosus (SLE) and other autoimmune diseases. This study has probed the role of lipid peroxidation derived aldehydes in SLE. We report the structural perturbations in human serum albumin (HSA) upon modification with HNE and the consequential enhanced immunogenicity. The induced antibodies were found to be highly specific for the immunogen and exhibited cross-reactivity with HNE-modified epitopes on proteins, amino acids and nucleic acid. The experimentally induced anti-HNE-HSA antibodies appreciably recognized HNE modified epitopes on the HSA obtained from SLE patients. These antibodies, therefore, form a good immunochemical probe to detect such damages in lupus patients. Possible role of anti-HNE-HSA antibodies as a marker for detection/progression of SLE has been discussed.

Topics: Adolescent; Adult; Aged; Aldehydes; Antibodies; Case-Control Studies; Female; Humans; Immunochemistry; Lupus Erythematosus, Systemic; Middle Aged; Oxidative Stress; Serum Albumin; Young Adult

Even though systemic lupus erythematosus (SLE) is associated with high morbidity and mortality rates among young and middle-aged women, the molecular mechanisms of disease pathogenesis are not fully understood. Previous studies from our laboratory suggested an association between oxidative stress and SLE disease activity (SLEDAI). To further assess the role of reactive oxygen species (ROS) in SLE, we examined the contribution of lipid-derived reactive aldehydes (LDRAs)-specific immune complexes in SLE. Sera from 60 SLE patients with varying SLEDAI and 32 age- and gender- matched healthy controls were analyzed for oxidative stress and related markers. Patients were divided into two groups based on their SLEDAI scores (<6 and ≥ 6). Both SLEDAI groups showed higher serum 4-hydroxynonenal (HNE)-/malondialdehyde (MDA)-protein adducts and their specific immune complexes (HNE-/MDA-specific ICs) together with IL-17 than the controls, but the levels were significantly greater in the high SLEDAI (≥ 6) group. Moreover, the serum levels of anti-oxidant enzymes Cu/Zn superoxide dismutase (SOD) and catalase (CAT) were significantly reduced in both patient groups compared to controls. Remarkably, for the first time, our data show that increased HNE-/MDA-specific ICs are positively associated with SLEDAI and elevated circulating immune complexes (CICs), suggesting a possible causal relationship among oxidative stress, LDRA-specific ICs and the development of SLE. Our findings, apart from providing firm support to an association between oxidative stress and SLE, also suggest that these oxidative stress markers, especially the HNE-/MDA-specific ICs, may be useful in evaluating the prognosis of SLE as well as in elucidating the mechanisms of disease pathogenesis.

Topics: Adult; Aged; Aldehydes; Antigen-Antibody Complex; Blood Proteins; Case-Control Studies; Catalase; Enzyme-Linked Immunosorbent Assay; Female; Humans; Interleukin-17; Lipid Peroxidation; Lupus Erythematosus, Systemic; Male; Malondialdehyde; Middle Aged; Oxidative Stress; Reactive Oxygen Species; Severity of Illness Index; Superoxide Dismutase

Oxidative stress is elevated in systemic lupus erythematosus (SLE) patients, and associated extensively with SLE pathogenesis. However, no common indicators of oxidative stress are yet in routine clinical use because of their instability, nonspecificity, and non-representation of all SLE symptoms. Moreover, the method for reproducible analysis of reactive oxygen species is still lacking. Lipids and their metabolites are essential components of biological systems, many of which serve as molecular targets of oxidative stress and play crucial roles in signaling, inflammation, and immune responses. Thus, determining the changed levels of lipids and their metabolites may serve the needs for SLE research. In the pilot study, shotgun lipidomics of sera from 30 SLE patients and 30 controls was performed and revealed a marked reduction of ethanolamine plasmalogen (pPE) species from 85.03±3.06 to 62.39±4.34 nmol/mL serum in controls and patients, respectively, accompanying significant increases in lysoPE (LPE) content (~46mol%) and 4-hydroxynonenal (an indictor of oxidative stress) in patients. Representative proinflammatory cytokines were also determined, revealing significant elevation of IL-6, IL-10, and TNF-α in SLE patients. Multivariate and multiple regression analyses showed for the first time that significant correlation among the SLE disease activity index, IL-10 levels, and pPE content exists, providing insights into SLE pathogenesis. The study also indicates that the changes of pPE (molecular targets of oxidative stress) and their peroxidation products may serve as novel biomarkers for diagnosis of SLE.

Topics: Adult; Aldehydes; Biomarkers; Case-Control Studies; Female; Humans; Interleukin-10; Interleukin-6; Lupus Erythematosus, Systemic; Lysophospholipids; Middle Aged; Oxidative Stress; Pilot Projects; Plasmalogens; Severity of Illness Index; Tumor Necrosis Factor-alpha

We have previously reported accelerated acquisition of new autoreactivity upon immunization with 4-hydroxy-2-nonenal (HNE)-modified Ro60, as well as differential induction of lupus or Sjögren's syndrome by immunization with Ro60 containing varying amounts of HNE. Since the number of HNE molecules on Ro60 appears to be important, we hypothesized that specific sequences on Ro60 are targets for HNE-modification. Using surface plasmon resonance (SPR) we have also shown intramolecular protein-protein interaction between Ro60 and Ro multiple antigenic peptides (MAPs). We also hypothesized that intramolecular protein-protein interaction would be abolished by HNE-modification. To test this hypotheses we investigated (a) the epitopes of Ro60, using 19 Ro MAPs in an in vitro assay (involving HNE-modification of MAPs following immobilization on ELISA plates) to identify targets of HNE modification on Ro60 and (b) the protein-protein interaction between unmodified Ro60 MAPs, immobilized on the sensor surface of BIAcore, and unmodified Ro60 or HNE-modified Ro60 using SPR. New data obtained with SPR strengthens our earlier observation that immunization with HNE-Ro60 induces a stronger response. Unmodified Ro60 bound to several Ro60 MAPs through protein-protein interaction analyzed using SPR. This interaction was totally abrogated using HNE-modified Ro60 suggesting that sequences on Ro had become modified with HNE. When 19 Ro60 MAPs were modified in vitro with HNE, it was found that 10/19 MAPs significantly bound HNE covalently (p<0.001 compared to MAPs binding HNE poorly). The amino acid sequences 126-137, 166-272 and 401-495 on Ro60 were strongly HNE modified. Using computational model system based on the recently published crystal structure for Ro60 enabled us to identify regions on the Ro60 molecule represented by the HNE-modified Ro MAPs, which are part of the exposed tertiary structure of the Ro60 protein.

Topics: Aldehydes; Amino Acid Sequence; Animals; Autoantibodies; Autoantigens; Cattle; Cross-Linking Reagents; Epitopes, B-Lymphocyte; Lupus Erythematosus, Systemic; Molecular Sequence Data; Protein Structure, Secondary; Rabbits; Ribonucleoproteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Surface Plasmon Resonance

4-Hydroxynonenal (HNE) is the most abundant and toxic aldehyde generated by the oxidation of plasma membrane polyunsaturated fatty acids. Systemic lupus erythematosus (SLE), a chronic autoimmune disease, is primarily characterized by increased levels of autoantibodies, predominantly against ds-DNA. However, the initial antigenic stimulus for the disease etiopathogenesis has remained elusive. HNE has been extensively used as a biomarker of oxidative stress. It can form adduct with proteins, making them highly immunogenic. Increased levels of such aldehyde-protein adducts have been reported in various pathological states, including autoimmune disorders like SLE and arthritis. In the present study, HNE-mediated structural changes in human serum albumin (HSA) were characterized by UV, fluorescence, CD and FT-IR spectroscopy as well as by polyacrylamide gel electrophoresis. Furthermore, immunogenicity of native and HNE-modified HSA was probed in female rabbits. The HNE-modified HSA was highly immunogenic eliciting high titre immunogen specific antibodies. Binding of SLE anti-DNA antibodies was analyzed by direct binding and competition ELISA. The data show preferential binding of SLE autoantibodies to HNE-modified HSA as compared to native HSA or native DNA. Our results suggest that HNE modification generates neoepitopes on HSA causing enhanced autoantibodies production. The results point towards the possible role of HNE-modified HSA in SLE etiopathogenesis.

Topics: Aldehydes; Antibody Affinity; Autoantibodies; Humans; Immunoglobulin G; Lipid Peroxidation; Lupus Erythematosus, Systemic; Protein Binding; Serum Albumin

Even though reactive oxygen species (ROS) have been implicated in SLE pathogenesis, the contributory role of ROS, especially the consequences of oxidative modification of proteins by lipid peroxidation-derived aldehydes (LPDAs) such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE) in eliciting an autoimmune response and disease pathogenesis remains largely unexplored. MRL/lpr mice, a widely used model for SLE, spontaneously develop a condition similar to human SLE, whereas MRL+/+ mice with the same MRL background, show much slower onset of SLE. To assess if the differences in the onset of SLE in the two substrains could partly be due to differential expression of LPDAs and to provide evidence for the role of LPDA-modified proteins in SLE pathogenesis, we determined the serum levels of MDA-/HNE-protein adducts, anti-MDA-/HNE-protein adduct antibodies, MDA-/HNE-protein adduct specific immune complexes, and various autoantibodies in 6-, 12- and 18-week old mice of both substrains. The results show age-related increases in the formation of MDA-/HNE-protein adducts, their corresponding antibodies and MDA-/HNE-specific immune complexes, but MRL/lpr mice showed greater and more accelerated response. Interestingly, a highly positive correlation between increased anti-MDA-/HNE-protein adduct antibodies and autoantibodies was observed. More importantly, we further observed that HNE-MSA caused significant inhibition in antinuclear antibodies (ANA) binding to nuclear antigens. These findings suggest that LPDA-modified proteins could be important sources of autoantibodies and CICs in these mice, and thus contribute to autoimmune disease pathogenesis. The observed differential responses to LPDAs in MRL/lpr and MRL+/+ mice may, in part, be responsible for accelerated and delayed onset of the disease, respectively.

Topics: Aldehydes; Animals; Autoantibodies; Autoimmunity; Enzyme-Linked Immunosorbent Assay; Female; Lipid Peroxidation; Lupus Erythematosus, Systemic; Malondialdehyde; Mice; Mice, Inbred MRL lpr; Oxidation-Reduction; Oxidative Stress; Proteins

Our previous work showed that immunization of rabbits with 4-hydroxy-2-nonenal-modified Ro60 (HNE-Ro60) accelerates autoimmunity. We extended this model into mice, hypothesizing that the severity of autoimmunity would be dependent on the degree of HNE modification of Ro60. Five groups of BALB/c mice (10/group) were used. Group I was immunized with Ro60. Groups II to IV were immunized with Ro60 modified with 0.4 mM (low), 2 mM (medium), and 10 mM (high) HNE, respectively. Group V controls received Freund's adjuvant. A rapid abrogation of tolerance to Ro60/La antigens occurred in mice immunized with HNE-modified Ro60, especially in the low and medium HNE-Ro60 groups. Lymphocytic infiltration and significantly high decrement in salivary flow (37%) compared to controls was observed only in the high HNE-Ro60 group, suggesting induction of a Sjögren syndrome-like condition in this group. Anti-dsDNA occurred only in mice immunized with medium HNE-Ro60. This group did not have a significant decrement in salivary flow, suggesting induction of a systemic lupus erythematosus-like manifestation in this group. Significantly high antibodies to Ro60 were found in saliva of mice in the low and medium HNE-Ro60 and the Ro60 groups, as well as anti-HNE Ro60 in the low and medium HNE-Ro60 groups. Understanding the mechanism of this differential induction may help discriminate between these two autoimmune diseases.

Topics: Aldehydes; Animals; Autoimmunity; Female; Lipid Peroxidation; Lupus Erythematosus, Systemic; Mice; Mice, Inbred BALB C; Ribonucleoproteins; Salivary Glands; Sjogren's Syndrome

Systemic lupus erythematosus (SLE) is a multifactorial disorder characterized by the presence of autoantibodies. We and others have implicated free radical mediated peroxidative damage in the pathogenesis of SLE. Since harmful free radical products are formed during this oxidative process, including 4-hydroxy 2-nonenol (4-HNE) and malondialdehyde (MDA), we hypothesized that specific HNE-protein adducts would be present in SLE red blood cell (RBC) membranes. Catalase is located on chromosome 11p13 where linkage analysis has revealed a marker in the same region of the genome among families with thrombocytopenia, a clinical manifestation associated with severe lupus in SLE affected pedigrees. Moreover, SLE afflicts African-Americans three times more frequently than their European-American counterparts. Hence we investigated the effects of a genetic polymorphism of catalase on risk and severity of SLE in 48 pedigrees with African American ancestry.. Tryptic digestion followed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis was used to identify the protein modified by HNE, following Coomassie staining to visualize the bands on the acrylamide gels. Genotyping analysis for the C --> T, -262 bp polymorphism in the promoter region of catalase was performed by PCR-RFLP and direct PCR-sequencing. We used a "pedigree disequilibrium test" for the family based association analysis, implemented in the PDT program to analyze the genotyping results.. We found two proteins to be HNE-modified, migrating around 80 and 50 kD respectively. Tryptic digestion followed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis of the Coomassie stained 80 kD band revealed that the target of HNE modification was catalase, a protein shown to associate with RBC membrane proteins. All the test statistics carried out on the genotyping analysis for the C --> T, -262 bp polymorphism in the promoter region of catalase were non-significant (p > 0.05) in our data, which suggested that this SNP is not associated with SLE.. Our results indicate that catalase is one of the proteins modified due to oxidative stress. However, catalase may not be a susceptibility gene for SLE. Nonetheless, catalase is oxidatively modified among SLE patients. This suggests a possible role between oxidative modification of catalase and its affects on enzymatic activity in SLE. An oxidatively modified catalase could be one of the reasons for lower enzymatic activity among SLE subjects, which in turn could favor the accumulation of deleterious hydrogen peroxide. Furthermore, HNE-products are potential neoantigens and could be involved in the pathogenesis of SLE. Decrease in catalase activity could affect the oxidant-antioxidant balance. Chronic disturbance of this balance in patients with SLE may work favorably for the premature onset of atherogenesis with severe vascular effect.

Topics: Aldehydes; Catalase; Erythrocyte Membrane; Genetic Predisposition to Disease; Humans; Lipid Peroxidation; Lupus Erythematosus, Systemic; Oxidative Stress; Polymorphism, Single Nucleotide; Risk Factors

Topics: Aldehydes; Animals; Autoantibodies; Autoimmune Diseases; Disease Models, Animal; Lupus Erythematosus, Systemic; Oxidation-Reduction; Ribonucleoproteins

Several lines of evidence indicate that the nonenzymatic oxidative modification of proteins and the subsequent accumulation of the modified proteins have been found in cells during aging and oxidative stress and in various pathological states, including premature diseases, muscular dystrophy, rheumatoid arthritis, and atherosclerosis. Our previous work suggested the existence of molecular mimicry between antibodies raised against hydroxy-2-nonenal (HNE)-modified protein and anti-DNA autoantibodies, a serologic hallmark of systemic lupus erythematosus (SLE). In the present study, we investigated the possible involvement of HNE-modified proteins as the endogenous source of the anti-DNA antibodies. Accumulation of the antigen recognized by the antibody against the HNE-modified protein was observed in the nucleus of almost all of the epidermal cells from patients with autoimmune diseases, including SLE. The SLE patients also showed significantly higher serum levels of the anti-HNE titer than healthy individuals. To determine if a specific anti-DNA response could be initiated by the HNE-derived epitopes, we immunized BALB/c mice with the HNE-modified protein and observed a progressive increase in the anti-DNA response. Moreover, we generated the monoclonal antibodies, showing recognition specificity toward DNA, and found that they can bind to two structurally distinct antigens (i.e. the native DNA and protein-bound 4-oxo-2-nonenal). The findings in this study provide evidence to suspect an etiologic role for lipid peroxidation in autoimmune diseases.

Topics: Aldehydes; Animals; Antibodies, Antinuclear; Arthritis, Rheumatoid; Atherosclerosis; Autoantigens; Cattle; Cellular Senescence; Epitopes; Female; Humans; Lipid Peroxidation; Lupus Erythematosus, Systemic; Mice; Mice, Inbred BALB C; Molecular Mimicry; Muscular Dystrophies; Oxidation-Reduction; Oxidative Stress; Protein Processing, Post-Translational; Serum Albumin, Bovine

4-Hydroxy-2-nonenal (HNE), a racemic mixture of 4R- and 4S-enantiomers, is a major product of lipid peroxidation and is believed to be largely responsible for the cytopathological effects observed during oxidative stress. HNE reacts with histidine to form a stable HNE-histidine Michael addition-type adduct possessing three chiral centers in the cyclic hemiacetal structure. We have previously raised the mAbs, anti-R mAb 310 and anti-S mAb S412, that enantioselectively recognized the R-HNE-histidine and R-HNE-histidine adducts, respectively, and demonstrated the presence of both epitopes in vivo. In the present study, to further investigate the anti-HNE immune response, we analyzed the variable genes and primary structure of these Abs and found that the sequence of R310 was highly homologous to anti-DNA autoantibodies, the hallmark of systemic lupus erythematosus. An x-ray crystallographic analysis of the R310 Fab fragment showed that the R-HNE-histidine adduct binds to a hydrophobic pocket in the antigen-binding site. Despite the structural identity to the anti-DNA autoantibodies, however, R310 showed only a slight crossreactivity with the native double-stranded DNA, whereas the Ab immunoreactivity was dramatically enhanced by the treatment of the DNA with 4-oxo-2-nonenal (ONE), an analog of HNE. Moreover, the 7-(2-oxo-heptyl)-substituted 1,N2-etheno-type ONE-2'-deoxynucleoside adducts were identified as alternative epitopes of R310. Molecular mimicry between the R-HNE-histidine configurational isomers and the ONE-DNA base adducts is proposed for the dual crossreactivity.

Topics: Aldehydes; Amino Acid Sequence; Animals; Antibodies, Antinuclear; Antibodies, Bispecific; Antibodies, Monoclonal; Autoantibodies; Cross Reactions; Crystallography, X-Ray; Deoxyribonucleosides; DNA; DNA Adducts; Epitopes; Immunoglobulin Fab Fragments; Lipid Peroxidation; Lipids; Lupus Erythematosus, Systemic; Mice; Molecular Mimicry; Molecular Sequence Data; Oxidation-Reduction; Proteins

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with autoantibodies as a near universal feature of the disease. The Ro ribonucleoprotein particle, composed of a 60-kDa protein noncovalently associated with human cytoplasmic RNA, is the target of antibodies in 25-40% of lupus patients. Purified human 60-kDa Ro was found to be oxidatively modified. Earlier investigations from our laboratory revealed increased oxidative damage in SLE patients. Therefore we hypothesized that oxidation by-products, such as 4-hydroxy-2-nonenal (HNE), could lead to neoantigens like HNE-modified 60-kDa Ro, which could in turn initiate autoimmunity or drive epitope spreading. To test this hypothesis we immunized rabbits with either HNE-modified 60-kDa Ro or the unmodified Ro. Intramolecular epitope spreading within the Ro molecule and intermolecular epitope spreading to La, double-stranded DNA, nRNP, and Sm occurred preferentially in HNE-Ro-immunized animals. Nonspecific anti-HNE antibody, generated by immunization with HNE-keyhole limpet hemocyanin conjugate, did not significantly bind to these autoantigens. These data may suggest a hitherto unappreciated mechanism by which oxidative stress facilitates epitope spreading in SLE.

Topics: Aldehydes; Animals; Antigens; Autoantigens; Autoimmunity; Cattle; DNA; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Epitopes; HeLa Cells; Hemocyanins; Humans; Immunoblotting; Lipid Peroxidation; Lupus Erythematosus, Systemic; Microscopy, Fluorescence; Oxidative Stress; Oxygen; Peptides; Plasmids; Proteins; Rabbits; Ribonucleoproteins; RNA, Small Cytoplasmic; Time Factors

To examine the role of oxidative damage in children and adolescents with autoimmune diseases, we compared blood serum levels of the lipid peroxidation (LPO) products 4-hydroxynonenal (HNE) and malondialdehyde (MDA) in 22 children with systemic lupus erythematosus (SLE), 13 children with focal type of scleroderma, and 21 health controls. In order to study the influence of disease activity in SLE on serum LPO product levels, the SLE group was divided into one group with active disease (n = 11) and one group with non-active disease (n = 11) according to SLEDAI-score, 15.1 and 1.8, respectively. SLE patients with active SLE (146 +/- 14 nmol/l, median 145 nmol/l) have significantly higher HNE levels compared to controls (61 +/- 10 nmol/l, median 52 nmol/l), whereas the MDA serum levels are similar to those of the control group, 1.94 +/- 0.18 mumol/l (median: 2.02 mumol/l) and 1.58 +/- 0.11 mumol/l (median: 1.52 mumol/l), respectively. Children with SCL had HNE and MDA levels similar to the control group.

Topics: Adolescent; Adult; Aldehydes; Autoimmunity; Child; Female; Humans; Lipid Peroxidation; Lupus Erythematosus, Systemic; Male; Malondialdehyde; Scleroderma, Localized

Analysis of serum samples of healthy children (n = 11) and children with Systemic Lupus Erythematosus (SLE), (n = 21) was performed by SDS-PAGE and immunoblot with an antibody directed against proteins modified by lipid peroxidation (LPO) product 4-hydroxynonenal (HNE). A single major stained protein band was detected. By comparison of the molecular weights in nonreducing and reducing SDS-PAGE was found that the main protein modified by HNE is immunoglobulin G. Significantly higher concentrations of the aldehyde modified protein were found in children with high disease activity of SLE measured by SLE disease activity index (SLEDAI). Lipid peroxidation measured by malondialdehyde and 4-hydroxynonenal concentrations show an enhanced level of both compounds also in patients with the active autoimmune disease. Therefore, it can be assumed that free radical mediated processes play a pathophysiological role in the active phase of SLE and HNE-modified serum proteins are a further parameter for the detection of in vivo LPO.

Topics: Adolescent; Aldehydes; Blood Proteins; Blotting, Western; Child; Child, Preschool; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Humans; Lipid Peroxidation; Lupus Erythematosus, Systemic; Malondialdehyde