Protein-bound 4-Hydroxy-2-nonenalAN ENDOGENOUS TRIGGERING ANTIGEN OF ANTI-DNA RESPONSE*□S

Received for publication, April 11, 2007, and in revised form, June 1, 2007 Published, JBC Papers in Press, June 22, 2007, DOI 10.1074/jbc.M703039200

Kazuyo Toyoda‡, Ritsuko Nagae‡, Mitsugu Akagawa‡, Kosuke Ishino‡, Takahiro Shibata‡, Sohei Ito§,Noriyuki Shibata¶, Tomoko Yamamoto¶, Makio Kobayashi¶, Yoshinari Takasaki�, Tsukasa Matsuda‡,and Koji Uchida‡1

From the ‡Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan, the §School of Food andNutritional Sciences, University of Shizuoka, Shizuoka 422-8526, Japan, the ¶Departments of Pathology, Tokyo Women’sMedical University, Tokyo 162-8666, Japan, and the �Department of Internal Medicine and Rheumatology,Juntendo University School of Medicine, Tokyo 113-8421, Japan

Several lines of evidence indicate that the nonenzymatic oxi-dative modification of proteins and the subsequent accumula-tion of the modified proteins have been found in cells duringaging and oxidative stress and in various pathological states,including premature diseases, muscular dystrophy, rheumatoidarthritis, and atherosclerosis. Our previous work suggested theexistence of molecular mimicry between antibodies raisedagainst hydroxy-2-nonenal (HNE)-modified protein and anti-DNAautoantibodies, a serologic hallmark of systemic lupus ery-thematosus (SLE). In the present study, we investigated the pos-sible involvement of HNE-modified proteins as the endogenoussource of the anti-DNAantibodies. Accumulation of the antigenrecognized by the antibody against the HNE-modified proteinwas observed in the nucleus of almost all of the epidermal cellsfrom patients with autoimmune diseases, including SLE. TheSLEpatients also showed significantly higher serum levels of theanti-HNE titer than healthy individuals. To determine if a spe-cific anti-DNA response could be initiated by the HNE-derivedepitopes, we immunized BALB/c mice with the HNE-modifiedprotein and observed a progressive increase in the anti-DNAresponse. Moreover, we generated the monoclonal antibodies,showing recognition specificity toward DNA, and found thatthey can bind to two structurally distinct antigens (i.e. the nativeDNA and protein-bound 4-oxo-2-nonenal). The findings in thisstudy provide evidence to suspect an etiologic role for lipid per-oxidation in autoimmune diseases.

Several lines of evidence indicate that the nonenzymatic oxi-dative modification of proteins and the subsequent accumula-tion of the modified proteins have been found in cells during

aging and oxidative stress and in various pathological states,including premature diseases,muscular dystrophy, rheumatoidarthritis, and atherosclerosis (1, 2). It has also been suggestedthat many of the effects of cellular dysfunction under oxidativestress are mediated by the products of nonenzymatic reactions,such as the peroxidative degradation of polyunsaturated fattyacids (3, 4). Lipid peroxidation leads to the formation of a broadarray of different products with diverse and powerful biologicalactivities. Among them are a variety of different aldehydes. Theprimary products of lipid peroxidation, lipid hydroperoxides,can undergo carbon-carbon bond cleavage via alkoxyl radicalsin the presence of transitionmetals, giving rise to the formationof short chain, unesterified aldehydes of 3–9 carbons in length,and a second class of aldehydes still esterified to the parent lipid(5). These aldehydes generated during the lipid peroxidationhave been implicated as causative agents in cytotoxic processesinitiated by the exposure of biological systems to oxidizingagents.Some of the lipid peroxidation products exhibit a facile reac-

tivity with proteins, generating a variety of intra- and intermo-lecular covalent adducts. Such adducts could be the targets of Bcell-mediated immune responses and induce T cell responsesand add the potential of certain aldehydes to induce an autoim-munity by breaking the B cell tolerance to nonmodified pro-teins. It has been shown that the modification of self-proteinsby lipid peroxidation products indeed results in a break of atolerance to self-proteins (6). The fact that post-transla-tional modification of proteins is enhanced in aging andstressed cells and arises under physiological conditions (1, 2)suggests the existence of an association between covalentmodification of protein with lipid peroxidation products andautoimmune diseases.Anti-DNA autoantibodies are a prime feature of human sys-

temic lupus erythematosus (SLE)2 (7). The appearance of theseantibodies in humans and inmurine models of lupus correlateswith the progression of the disease, and by comparison with allof the other lupus autoantibodies, those against the double-

* This work was supported by a research grant from the Ministry of Education,Culture, Sports, Science, and Technology and by the Center of Excellence(COE) Program in the 21st Century in Japan (to K. U.), by a research grantfrom the Institute for Advanced Research, Nagoya University (to K. U.), andby the COE Program in the 21st Century in Japan (to K. U. and T. M.). Thecosts of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked “advertise-ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. S1–S3.

1 To whom correspondence should be addressed: Laboratory of Food andBiodynamics, Graduate School of Bioagricultural Sciences, Nagoya Univer-sity, Nagoya 464-8601, Japan. Tel.: 81-52-789-4127; Fax: 81-52-789-5741;E-mail: uchidak@agr.nagoya-u.ac.jp.

2 The abbreviations used are: SLE, systemic lupus erythematosus; ELISA,enzyme-linked immunosorbent assay; HNE, 4-hydroxy-2-nonenal; Ab,antibody; mAb, monoclonal antibody; ONE, 4-oxo-2-nonenal; HPLC, highpressure liquid chromatography; BSA, bovine serum albumin; PBS, phos-phate-buffered saline; KLH, keyhole limpet hemocyanin; VH, variable heavychain; VL, variable light chain; CDR, complementarity-determining region.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 35, pp. 25769 –25778, August 31, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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stranded DNA are thought to be the most pathogenic andinvolved in the development of renal pathology (8). However,due to the systemic character and complexity of the disease, itstill remains unclear what exactly are the primary stimuli thatdrive such autoantibody responses and the mechanisms thatregulate the entire pathological process in lupus. Althoughsome bacterial DNAs appear to be immunogenic in normalmouse strains, they do not elicit the production of antibodiesthat cross-react with the eukaryotic DNA (9, 10). Thus, itremains unclear if DNA or nucleosomes induce the anti-DNAantibodies in lupus-prone mice or if the anti-DNA antibodiesare made in response to some other antigen, either itself orforeign.4-Hydroxy-2-nonenal (HNE), one of the most prominent

lipid peroxidation-specific aldehydes, is believed to be largelyresponsible for the cytopathological effects observed duringoxidative stress (4, 5). HNE exerts these effects because of itsfacile reactivity with biological materials, including proteins(Fig. 1) (5). Upon reaction of the protein, HNE specificallyreacts with nucleophilic amino acids, such as cysteine, histi-dine, and lysine, to form stable Michael addition adducts pos-sessing the cyclic hemiacetal structure (5). Previously, we raisedthe anti-HNE monoclonal antibodies (mAbs), which enantios-electively recognized the (R)-HNE-histidine Michael adducts(11), and unexpectedly found that the sequence of an anti-HNEmAb was highly homologous to the anti-DNA autoantibodies(12). In addition, we characterized the ability of the mAb torecognize DNA and identified the 4-Oxo-2-nonenal (ONE)-modified 2�-deoxynucleoside (7-(2-oxo-heptyl)-substituted1,N2-etheno-type 4-oxo-2-nonenal-2�-deoxynucleoside) as analternative epitope. Based on these findings, we proposed the

hypothesis that post-translational protein modification withlipid peroxidation products, such as HNE, could serve as animmunological trigger for the production of anti-DNA autoan-tibodies in autoimmune diseases.

EXPERIMENTAL PROCEDURES

Materials—The stock solutions ofHNEwere prepared by theacid treatment (1 mM HCl) of HNE dimethylacetal, which wassynthesized according to the procedure of De Montarby et al.(13). The (R)- and (S)-HNEs were prepared by the enzymaticresolution of racemic HNE (14) and purified by a chiral phaseHPLC on a ChiralPak AD-RH column (0.46 � 15 cm) (DaicelChemical Industries, Ltd., Osaka, Japan) (11). ONEwas synthe-sized by the oxidation of HNE dimethyl acetal with pyridiniumdichlorochromate, followed byHCl hydrolysis (15). Other alde-hydeswere purchased fromCaymanChemical Co. (AnnArbor,MI). The horseradish peroxidase-linked anti-rabbit IgG immu-noglobulin, horseradish peroxidase-NeutrAvidin, and ECLWestern blotting detection reagentswere obtained fromAmer-sham Biosciences.Animals—Female BALB/cmice were obtained fromChubu

Kagaku Shizai Co., Ltd. (Nagoya, Japan). All animal proto-cols were approved by the Animal Experiment Committee inthe Graduate School of Bioagricultural Sciences, NagoyaUniversity.Modification of Protein and DNA by Reactive Aldehydes—

Modification of the protein by aldehydes was performed byincubating BSA (1.0mg/ml) with 1–10mM aldehydes in 1ml of50 mM sodium phosphate buffer (pH 7.4) at 37 °C for 24 h.Modification of theDNAwas performed by incubating calf thy-mus DNA (1.0 mg/ml) with 10 mM aldehydes in 50 mM sodiumphosphate buffer (pH 7.4) at 37 °C for 24 h.ELISA—A 100-�l aliquot of the antigen solution was added

to each well of a 96-well microtiter plate and incubated for 20 hat 4 °C. The antigen solution was then removed, and the platewas washed with phosphate-buffered saline (PBS) containing0.5%Tween 20 (PBS/Tween). Eachwell was incubatedwith 200�l of 4% Blockace (Yukijirushi, Sapporo, Japan) in PBS/Tweenfor 60min at 37 °C to block the unsaturated plastic surface. Theplate was then washed three times with PBS/Tween. A 100-�laliquot of a 102� dilution of serum was added to each well andincubated for 2 h at 37 °C. After discarding the supernatantsand washing three times with PBS/Tween, 100 �l of a 5 � 103dilution of goat anti-mouse IgG conjugated to horseradish per-oxidase in PBS/Tween was added. After incubation for 1 h at37 °C, the supernatant was discarded, and the plates werewashed three times with PBS/Tween. The enzyme-linked anti-body bound to the well was revealed by adding 100 �l/well of1,2-phenylenediamine (0.5 mg/ml) in a 0.1 M citrate/phosphatebuffer (pH 5.5) containing 0.003% hydrogen peroxide. Thereaction was terminated by the addition of 2 M sulfuric acid (50ml/well), and the absorbance at 492 nmwas read using amicro-ELISAplate reader. In a competitive ELISA, the competitorwasincubatedwith the antibody for 20 h at 4 °C to yield competitor/antibody mixtures containing the antibody at 0.8 �g/ml andvariable concentrations of the competitor. A 100-�l aliquot ofthe competitor/antibody mixture was added to each well andincubated for 2 h at 37 °C. After discarding the supernatants

X

FIGURE 1. Formation of HNE-modified protein. A, formation of reactiveelectrophiles during lipid peroxidation of �6 polyunsaturated fatty acids.B, mechanism of covalent binding of HNE to proteins. X represents the sidechain of nucleophilic amino acids, such as cysteine, histidine, and lysine.

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and washing three times with PBS/Tween, the second antibodywas added, and the enzyme-linked antibody bound to the wellwas revealed as already described.Western Blot Analysis—BSA (1 mg/ml) was incubated with

ONE (0–5 mM) in 1 ml of 50 mM sodium phosphate buffer, pH7.4, for 24 h at 37 °C. The protein sampleswere electrophoresedthrough a 10%polyacrylamide gel. After electrophoresis, the gelwas transblotted onto a nitrocellulose or polyvinylidene difluo-ride membrane (Amersham Biosciences), incubated withBlockace for blocking, washed, and then incubated with a pri-mary antibody for detection of theONE-modified protein. Thisprocedure was followed by the addition of horseradish peroxi-dase conjugated to the goat anti-rabbit IgG immunoglobulinandECL reagents. The bandswere visualized using aCool SaverAE-6955 (ATTO, Tokyo, Japan).Preparation of mAbs Using HNE-modified Protein as an

Immunogen—The immunogen was prepared by incubatingkeyhole limpet hemocyanin (KLH) (1.0mg/ml) with 5mMHNEin 10ml of 50mM sodiumphosphate buffer (pH 7.4) at 37 °C for24 h. We immunized the female BALB/c mice on day 1 withcomplete Freund adjuvant and 0.5 mg of immunogen (HNE-modified KLH) and boosted on days 7, 17, and 27 with incom-plete Freund adjuvant by emulsifying and intraperitoneal injec-tion. Spleen cells from the immunized mice were fused withP3/U1 murine myeloma cells and cultured in HAT (hypoxan-tine/aminopterin/thymidine) selection medium. The culturesupernatants of the hybridoma were screened using an ELISA,employing pairs of wells in microtiter plates on which wereabsorbed calf thymus DNA and HNE-treated BSA as antigens(0.5 �g of protein or DNA per well). After incubation with 100�l of the hybridoma supernatants, and with intervening washeswith Tris-buffered saline, pH 7.8, containing 0.05% Tween 20(TBS-Tween), the wells were incubated with alkaline phospha-tase-conjugated goat anti-mouse IgG, followed by a substratesolution containing 1 mg/ml p-nitrophenyl phosphate. Hybri-doma cells, corresponding to the supernatants that were posi-tive on either DNA or HNE-modified BSA and negative onnative BSA, were then cloned by limited dilution. Afterrepeated screenings, three clones showing the most distinctiverecognition of DNA and two clones showing the most distinc-tive recognition of both HNE-modified BSAs were obtained.Identification of Antigenic Adducts Recognized by mAbs

Raised against HNE-modified Protein—ONE modification ofthe amino acid derivatives was performed by incubating 10mM

N�-acetylcysteine, N�-acetylhistidine, or N�-acetyllysine with10 mM ONE in 1.0 ml of 50 mM sodium phosphate buffer (pH7.4) at 37 °C for 24 h. The antigenicONE-cysteine adducts wereanalyzed by a reverse-phase HPLC using a Develosil ODS-MG-5 column (4.6 � 250 mm) (Nomura Chemicals, Aichi,Japan) with a linear gradient from 10% aqueous acetonitrile(0.01% trifluoroacetic acid) to 100% acetonitrile (0.1% trifluoro-acetic acid) for 50 min at a flow rate of 0.8 ml/min. The elutionprofiles were monitored by absorbance at 240 nm. The anti-genic ONE-cysteine adducts were also analyzed by liquid chro-matography-mass spectrometry using a Jasco PlatformII-LCinstrument.

Cloning, Sequencing, and Analysis of Variable Heavy (VH)and Light Chain (VL) Genes—Immunogloblin variable regiongenes were cloned and sequenced following amplification byPCR. Total RNAwas prepared from 5� 106 hybridoma cells bythe phenol-guanidine isothiocyanate method (TRIzol reagent;Invitrogen) according to the manufacturer’s protocol. The firststrand cDNA synthesis was performed with recombinantMoloney murine leukemia virus reverse transcriptase (Super-script II; Invitrogen) using themanufacturer’s protocol. A 5-�gsample of the total RNA was primed with 10 pmol of randomprimers. Variable region geneswere amplified using degeneratesense primers homologous to the mouse heavy and light chainleader sequences and antisense constant primers (Novagen), aspreviously described (16). The amplification products wereligated into the pGEM-TEasyVector (Promega) using standardprotocols, and both strands of inserts were sequenced using anautomated dye-chain termination DNA sequencer. Theobtained sequences were analyzed using the DNASTAR soft-ware (DNASTAR, Madison, WI). The Basic Local AlignmentSearch Tool (BLAST) protocol was used to search the Gen-BankTM data base to determine the homology with the Vregions of other murine Abs that have been sequenced (17).Serum Samples—The serum samples were prepared from 90

patients with SLE, 20 patients with progressive systemic sclero-sis, and 30 patients with rheumatoid arthritis, all of whom metthe criteria for diagnosis proposed by the American College ofRheumatology (18–20); 15 patients with polymyositis/der-matomyositis, who met the criteria proposed by Bohan et al.(21); and 20 patients with Sjogren’s syndrome, who were diag-nosed according to the criteria proposed by the EuropeanCom-munity (22). The anti-DNA and anti-HNE titers in the serumsamples were measured by ELISA using calf thymus DNA andHNE-modified protein, respectively, as the coating antigens.Immunohistochemical Analysis—Skin specimens were ob-

tained by biopsy from eight control subjects (sex: twomales andsix females; age: 33–49 (40.75� 5.57) years), eight contact der-matitis patients (sex: two males and six females; age: 25–74(50.13 � 17.60) years), three pemphigus vulgaris patients (sex:two males and one female; age: 21–55 (36.67 � 17.04) years),and five SLE patients (sex: one male and four females; age:21–37 (26.80� 8.90) years).Multiple 4-�m-thick sectionswerecut from formalin (20%)-fixed, paraffin-embedded skinmateri-als of each case that were used for the hematoxylin-eosin stain-ing and immunohistochemical staining. The sections weredeparaffinized, rehydrated, quenched for 10 min at 4 °C with3% hydrogen peroxide, rinsed in phosphate-buffered saline, pH7.6, pretreated for 20 min at room temperature with 3% skimmilk in PBS, and subsequently incubated overnight at 4 °C withthe primary mAb HNEJ2 (23) at a final concentration of 0.1�g/ml. The antibody binding was visualized by the avidin-biotin-immunoperoxidase complex method using theappropriate Vectastain ABC kit (Vector Laboratories, Burl-ingame, CA) according to the manufacturer’s instructions.The chromogen was 3,3�-diaminobenzidine tetrahydrochlo-ride, and the counterstain was hematoxylin. Sections fromwhich the primary antibody was omitted served as the neg-ative reaction controls. The immunohistochemical localiza-ton of the protein-bound HNE was verified by comparison

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with consecutive sections stained with the hematoxylin-eo-sin-stained consecutive sections.Statistical Analyses—The statistical analysis was performed

with the StatView software (version 4.0; Abacus Concepts,Berkeley, CA). Differences were analyzed by the unpaired two-tailed Student’s t test or Welch’s t test as appropriate, and pvalues of �0.05 were considered significant.

RESULTS

Presence of HNE-specific Epitopes in Human AutoimmuneDiseases—To establish the association between the HNEmod-ification of protein and autoimmune diseases, we first deter-mined the presence of theHNE-specific epitopes in the contactdermatitis, pemphigus vulgaris, and SLE using themAb againstthe HNE-modified proteins. Contact dermatitis is known to bea T cell-mediated immune reaction, whereas pemphigus vul-garis and SLE are based on T cell- and B cell-mediated autoim-mune mechanisms with the appearance of antiepidermal cellmembrane antibodies and antinuclear component antibodies,respectively. The hematoxylin-eosin-stained skin sectionsdemonstrated the characteristic features of these diseases asfollows. Briefly, (i) the contact dermatitis patients showed focalinfiltration of the lymphocytes in the epidermis associated withspongiosis and in the dermis surrounding the superficial ves-sels, (ii) the pemphigus vulgaris patients exhibited suprabasilaracantholytic blister formation, resulting in bullous cavity for-mation, and (iii) the SLE patients indicated keratinization andliquefaction degeneration of the epidermis and fibrinoid degen-eration of the dermis. No immunoreaction product depositswere detectable on sections with the omission of the primaryantibody (data not shown). In the control subjects, the HNE-specific epitopes were obscure and weakly detectable in thesuperficial layer of the epidermis (Fig. 2A). In the chronic der-matitis patients, the immunoreactivity was distinct and focallyseen in the nucleus of the epidermal cells restricted to regionsadjacent to the lymphocytic infiltrates (Fig. 2B). In the patients

with pemphigus vulgaris (Fig. 2C) and SLE (Fig. 2D), the immu-noreactivity was distinct and diffusely seen in the nucleus ofalmost all of the epidermal cells. In addition, the HNE-specificepitopes were detectable in the cytoplasm of the epidermalcells in the examined cases, and the staining intensities var-ied from case to case, as shown in Fig. 2D. The HNE-specificepitopes were also localized in the nucleus of the endothelialcells of the capillary vessels surrounded by lymphocytic infil-trates in the dermis, whereas the epitopes were undetectablein the lymphocytes.SLE Patients Have Higher Titers of Anti-HNE Antibodies—

Based on the detection of the HNE-specific epitopes in thepatients with the autoimmune diseases, we speculated that theproduction of specific antibodies against the epitopes mightalso be accelerated. Hence, to evaluate the presence of theseantibodies in the autoimmune diseases, wemeasured the serumantibody titers directed against the nativeDNAandHNE-mod-ified serum albumin. Although it was not statistically signifi-cant, we observed a modest increase in the anti-DNA antibod-ies in the SLE patients (Fig. 3A). In contrast, the SLE patientsexhibited significant increases in the anti-HNE antibodiescompared with the controls (Fig. 3B). We also measured theserum anti-DNA and anti-HNE antibody titers in otherautoimmune diseases, including progressive systemic scle-rosis, rheumatoid arthritis, polymyositis/dermatomyositis,and Sjogren syndrome. Although there was no significantincrease in the serum anti-DNA levels, we observed signifi-cant increases in the anti-HNE antibodies compared withthe controls (Fig. S1). In addition, in our preliminary exper-iments, we have observed pronounced increases in anti-DNA and anti-HNE titers in MRL-lprmice, a representativemurine model of SLE.3 Thus, the antibody response againstthe HNE-specific epitopes may be an immunological char-acteristic common to various types of autoimmune diseases,and the serum anti-HNE titer has a potentially importantdiagnostic value in human autoimmune diseases. These clin-

3 K. Toyoda and K. Uchida, unpublished observation.

FIGURE 2. Immunohistochemical detection of HNE-modified protein inskin sections from patients with autoimmune diseases. We determinedthe presence of HNE-modified protein in the skin specimens obtained bybiopsy from eight control subjects, eight contact dermatitis patients, threepemphigus vulgaris patients, and five SLE patients. The most representativedata in each group are shown. A, control; B, contact dermatitis; C, pemphigusvulgaris; D, SLE. Original magnification was �40.

FIGURE 3. Cross-reactivity of serum from SLE patients with native DNAand HNE-modified protein. A, cross-reactivity of serum from SLE patientswith native DNA. B, cross-reactivity of serum from SLE patients with HNE-modified protein. The serum samples were prepared from 14 healthy individ-uals and 90 patients with SLE. The anti-DNA and anti-HNE titers in the serumsamples were measured by ELISA using calf thymus DNA and HNE-modifiedprotein, respectively, as the coating antigens. The HNE-modified protein wasprepared by incubating BSA (1.0 mg/ml) with 1 mM HNE in 1 ml of 50 mM

sodium phosphate buffer (pH 7.4) at 37 °C for 24 h. Five micrograms of anti-gen (calf thymus DNA or HNE-modified protein) was coated per well on poly-styrene plates, and the antibody binding was detected. Shown are themeans � S.E. The calculated p values were obtained by Welch’s t test analysis.

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ical and animal data offer an attractive hypothesis that theHNE-specific epitopes may represent immunologic trigger-ing antigens for the human autoimmune diseases.HNE-specific Epitopes Elicit the Anti-DNA Response in

BALB/c Mice—Since HNE physiologically arises in cells, wedetermined if the anti-DNA response could be initiated by theHNE-derived epitopes in vivo. To this end, female BALB/cmicewere immunized every 2 weeks with the HNE-modified KLHemulsified with complete Freund’s adjuvant, and both the anti-HNE and anti-DNA responses were examined. Not only anti-HNE response but also anti-DNA response began to appearafter the third immunization, during week 8 (Fig. 4A), althoughBALB/c mice are not the strain with spontaneous autoimmunedisease. All of the HNE-modified protein-immunized micedeveloped an IgG anti-DNA response, which was comparablewith the anti-HNE response. BALB/c mice immunized withKLH alone did not induce any significant anti-DNA response(data not shown). Four hybridoma clones (D1H3, D2A3, D3A8,and D3A11) producing the antibodies, showing recognitionspecificity toward native DNA, and two hybridoma clones(P2G11 and P4E6) producing the anti-HNE mAbs were pre-pared from mice immunized with the HNE-modified proteinafter screening based on specific binding to each corresponding

antigen, DNA, or HNE-modified protein. As shown in Fig. 4B,the antibodies, showing recognition specificity toward nativeDNA, did not react with the HNE-modified BSA at all, whereasthe anti-HNE mAbs did not show any cross-reactivity towardthe native DNA. Thus, it appeared that animals immunizedwith the HNE-modified protein progressively developed twodistinct populations of B cell clones, which are specific to eitherthe HNE-modified proteins or the native DNA.Sequence Analysis of the mAbs Raised against HNE-specific

Epitopes—To further confirm that the mAbs raised from ani-mals immunized with HNE-specific epitopes represent theanti-DNA antibodies, the identity of their V region genes wasdetermined for homologies to the known V region genes usingthe BLAST protocol (17). A homology search of the Gen-BankTM revealed that D2A3 shared a high degree of sequenceidentity with the anti-DNAand anti-polysaccharide antibodies.The VH domain of D2A3 was highly homologous (85.0–91.6%identity) to the anti-DNAmAbs (Jel466, VH30, and BW2 18-2)and to the anti-polysaccharide mAbs (HmenB3 and mAb 735)(Fig. 5A). In addition, the sequence identity of greater than 93%for the VL domain of D2A3 was shared with the antibodies,anti-CD3 (HuM291 and OKT3) and the anti-�(1–6) dextranantibodies (Fig. 5B). The V genes of D3A8 and D3A11 alsorevealed a sequence similarity with the anti-DNA mAbs.Clonally related VH genes of D3A8 and D3A11 were weaklyhomologous to D2A3 and its homologous anti-DNA and anti-polysaccharide antibodies: Jel466, VH30, BW2 18-2, HmenB3,and mAb 735 (78.5–85.0%). The VL domains of D3A8 andD3A11 were homologous to the anti-DNA mAbs, ZB2G10(95.4%) and DP7VK (94.4%), respectively.On the other hand, theVH genes of the anti-HNEmAbs P4E6

and P2G11 revealed a weak sequence similarity with the anti-DNA mAb ZB2F12 (78.5–81.3%) and anti-HNE mAb RS17(78.5–79.4%), whereas their identities were significantly lowerthan the identity of D2A3 with the anti-DNA mAbs (Fig. 5A).TheVL domain of P4E6was homologous to the anti-DNAmAb(5.9-1, ZA1H3) and anti-HNE mAbs R310 and RS17. Nosequence similarity with the anti-DNA mAbs was observed inthe VL genes of P2G11 (Fig. 5B). In addition, the VH domain ofrheumatoid factor RFA28-A showed a high similarity withR310 and DNA-1 (89.7% identity). The VL domain of rheuma-toid factor 6-19 was almost identical with R310 and RS17(96.4–97.3%) and was homologous to P4E6 and the anti-DNAantibodies: 202.s38 and ZA1H3. However, the VH domain of6-19 showed a weak sequence similarity with the anti-DNA,including the anti-polysaccharide mAbs.Dual Specificity of the mAbs Raised against HNE-specific

Epitopes—We have previously shown that the mAb raisedagainst the modified protein with the HNE enantiomer ((R)-HNE) shows only a slight cross-reactivity with the native DNA,whereas the immunoreactivity is dramatically enhanced by themodification of DNAwithONE, an analog of HNE (12). Hence,we examined the immunoreactivity of the mAbs raised fromanimals immunized with the HNE-specific epitopes toward thealdehyde-modified DNA. Consistent with our previous find-ings, the immunoreactivity of the anti-HNEmAbP4E6with thenative DNA was dramatically enhanced by the modification ofthe DNA with ONE. It was also revealed that ONE, among the

FIGURE 4. Immunization of animals with HNE-specific epitopes triggersanti-DNA response. A, anti-HNE and anti-DNA responses in mice immunizedwith HNE-specific epitopes. The immunogen was prepared by incubatingKLH (1.0 mg/ml) with 1 mM HNE in 10 ml of 50 mM sodium phosphate buffer(pH 7.4) at 37 °C for 24 h. Female BALB/c mice were immunized on day 1 withcomplete Freund adjuvant and 0.5 mg of immunogen (HNE-modified KLH)and boosted on days 7, 17, and 27 with incomplete Freund adjuvant by emul-sifying and intraperitoneal injection. Antibody response was examined by anELISA employing pairs of wells in microtiter plates on which were absorbedcalf thymus DNA (red diamond), BSA (black triangle), and HNE-treated BSA(blue square) as antigens. B, cross-reactivity of the mAbs raised against HNE-specific epitopes. Coating antigens were DNA (red bar), HNE-modified BSA(blue bar), and native BSA (black bar). The affinity of the antibodies was deter-mined by a direct antigen ELISA, employing pairs of wells in microtiter plateson which were absorbed calf thymus DNA, BSA, and HNE-treated BSA asantigens.

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tested lipid peroxidation-derived reactive aldehydes, was theonly source of the immunoreactive structures recognized bythe anti-HNEmAb (Fig. S2A). Of interest, the immunoreactiv-ity ofmAbD2A3with the nativeDNAwas also enhanced by themodification of the DNA with ONE (Fig. S2B).On the other hand, the immunoreactivity of the mAbs with

the aldehyde-modified proteins was also examined. As shownin Fig. 6A, the anti-HNE mAb P4E6 cross-reacted with themodified proteins with HNE,regardless of its chirality. Strikingly,mAb D2A3, showing specificitytoward native DNA, was found tocross-react with the ONE-treatedprotein (Fig. 6B). The other mAbs,D1H3, D3A8, and D3A11, showingspecificity toward native DNA, alsocross-reacted with the modifiedprotein (data not shown). Polyacryl-amide gel electrophoresis undernonreducing and nondenaturingconditions followed by immunoblotanalysis revealed that the ONE-modified protein electrophoresedas the multiple protein bands wasdetected by mAb D2A3 (Fig. 6C).However, no immunoreactive pro-tein bands were detected by theSDS-polyacrylamide gel electro-phoresis/immunoblot analysis (datanot shown), suggesting the fragilityof the epitope structure(s). Thesedata suggest that the HNE-specificepitopes may serve as an earlyimmunologic stimulus that givesrise to the production of dual spe-cific antibodies against the nativeDNA and ONE-modified proteins.Identification of an Epitope Rec-

ognized by the mAb D2A3—ONE isa lipid peroxidation product, whichhas been recently established to beformed by the free radical-initiateddegradation of �6-polyunsaturatedfatty acids (15, 24). It has been sug-gested that, upon reaction with pro-tein, ONE covalently modifies thearginine, cysteine, histidine, andlysine residues (25). However, dueto the instability of the adducts, thestructures of the ONE-modifiedamino acid adducts have not beendetermined except for the ONE-ar-ginine adduct (26). We character-ized the specificity of the mAbD2A3using the reactionmixtures ofthe ONE/amino acids. As shown inFig. 7A, binding of the ONE-modi-fied protein to the antibody was

hardly inhibited by the reaction mixtures of ONE/arginine,ONE/histidine, and ONE/lysine, but significantly inhibited bythe reactionmixture of ONE/cysteine, suggesting that themAbmight recognize an ONE-cysteine adduct as the epitope. Toidentify the ONE-cysteine adduct recognized by the mAbD2A3, the immunoreactivity with the reaction products ofONE with N�-acetylcysteine was characterized. As shown inFig. 7B, the reaction ofN�-acetylcysteinewithONEmainly pro-

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duced four peaks, among which the antibody predominantlyreacted with the peak 1. The liquid chromatography-massspectrometry analysis of this peak showed a pseudomolecularion peak at m/z 318 (M � H)� (Fig. S3), suggesting that it wascomposed of one molecule ofN�-acetylcysteine and one mole-cule of ONE (Fig. 7C).

DISCUSSION

SLE is a potentially fatal systemic autoimmune disease, char-acterized by the increased production of autoantibodies,immune complex deposition in the microvasculature, leuko-cyte infiltration, and, ultimately, tissue damage in a range oforgans. Of the multiple autoantibodies described in this dis-ease, antibodies against the native DNA are among the mostcharacteristic, yet the triggering antigen in the disease is stillunknown. We have shown here that the antigen recognized bythe antibody against HNE-modified protein was accumulatedin the nucleus of almost all of the epidermal cells from patientswith SLE and other autoimmune diseases. The increase in thelevels of the anti-HNE antibodies was also observed in SLEpatients. Based on these observations, we havemade the follow-ing two major discoveries in this study (Fig. 8): (i) the HNE-specific epitopes can be a triggering antigen of anti-DNAresponse, and (ii) the mAbs raised against HNE-specificepitopes can bind to two structurally distinct antigens (i.e.native DNA and ONE-modified proteins). These findings pro-vide evidence to suspect an etiologic role for the lipid peroxida-tion in autoimmune diseases.There is increasing evidence that lipid peroxidation plays a

role in SLE. (i) SLE patients have an enhanced urinary excretionof isoprostanes, the well established biomarkers of lipid peroxi-dation (27), (ii) the levels of the lipid peroxidation-derived shortchain aldehydes are significantly elevated in children with ahigh disease activity of SLE (28), and (iii) there are elevatedlevels of the oxidized low density lipoprotein together with ele-vated levels of autoantibodies related to the oxidized low den-sity lipoprotein in female patients with SLE (29). The involve-ment of lipid peroxidation was further suggested by thedetection of the HNE-specific epitopes in tissues from the SLEpatients. The immunohistochemical studies clearly demon-strated the accumulation of the HNE-specific epitopes in thedermis of the patients (Fig. 2). The immunoreactivity was dif-fusely seen in the cytoplasm of the epidermal cells and in thenucleus of the endothelial cells of capillary vessels surroundedby lymphocytic infiltrates in the dermis of the patients. Theseobservations verified for the first time the intracellular accumu-lation of the HNE-specific epitopes in human SLE and raisedthe possibility that the enhanced lipid peroxidation, through itspivotal role in oxidative stress, followed by the generation of

FIGURE 5. Sequence alignment of mAbs raised against HNE-specific epitopes and homologous antibodies. A, sequence alignment of the VH regions ofanti-DNA mAbs and homologous antibodies. H1 (H31–H35), H2 (H50 –H65), and H3 (H95–H102) above the alignment represent the Kabat definition comple-mentarity-determining regions (CDRs) based on sequence variability and the most commonly used one. HC1 (H30 –H35), HC2 (H47–H58), and HC3 (H93–H101)represent the contact definition CDRs by Macallum et al. (50). Identities are indicated by dots. Several types of structural/functional characteristics are indicatedby color coding as follows. Red, hydrophobic characteristics; green, hydrophilic characteristics; purple, positive charge; blue, negative charge. Sequences werealigned by using the program ClustalW version 1.82 and were manually modified. Accession numbers for the sequences are as follows: H_R310, AB248082;H_S412, AB248084 (DNA Data Bank of Japan); H_CD3_FN18 (anti-monkey CD3 antibody), AAB71638; H_AN_ZB2F12 (IgM, anti-single-stranded DNA antibody),AAR90967; H_10F4, AAC05423; H_RFA28-A, AAB32543; H_202.s38, CAA80108; H_RF_6-19 (IgG3), AAA63337; H_AN_ZB5F3, AAR90979; H_AN_BW2 18-2,AAL92931; H_AN_VH30, 1612332J; H_PS_HmenB3, AAL96661; H_AN_Jel466 (anti-triplex DNA antibody), AAB30095 (GenBankTM); H_CD3_OKT3 (anti-CD3antibody), 1SY6; H_AN_DNA-1, 1XF2; H_PS_mAb735 (anti-polysialic acid antibody), 1PLG (Protein Data Bank). RS17 is previously constructed monoclonal antibodiesfrom an HNE-KLH-immunized mouse (M. Hashimoto and K. Uchida, unpublished data). An antibody sequence test was performed using the AbCheck program (51).B, sequence alignment of the VL regions of anti-DNA mAbs and homologous antibodies. L1 (L24–L34), L2 (L50–L56), and L3 (L89–L97) represent the Kabat definitionCDRs based on sequence variability and the most commonly used one. LC1 (L30–L36), LC2 (L46–L55), and LC3 (L89–L96) represent the contact definition CDRs.Identities are indicated by dots. Accession numbers for the sequences are as follows: L_R310, AB248083 (DNA Data Bank of Japan); L_CD3_HuM291 (humanizedanti-CD3 antibody), AAB00850; L_PS_dextran, A30562; L_AN_ZA7C10, AAS01791; L_AN_ZB2G10 (anti-single-stranded DNA), AAS01809; L_AN_ZB9C3, AAS01837;L_AN_DP11VK, PL0259; L_AN_DP7VK, PL0260; L_RF 6-19, AAA63385; L_202.s38, PH1042; L_AN_ZA1H3, AAS01776 (GenBankTM); L_CD3_OKT3, 1SY6; L_AN_DNA-1,1XF2; L_PS_mAb735 (anti-polysialic acid antibody), 1PLG (Protein Data Bank). H, heavy chain; L, light chain; AN, anti-nucleotide antibody; PS, anti-polysaccharideantibody; CD, anti-CD3 antibody; RF, rheumatoid factor; R, anti-(R)-HNE antibody; S, anti-S-HNE antibody; RS, anti-(R,S)-HNE antibody.

FIGURE 6. Immunoreactivity of the mAbs P4E6 and D2A3 with modifiedproteins. A, immunoreactivity of the anti-HNE mAb P4E6 with aldehyde-treated proteins. B, immunoreactivity of the mAb D2A3, showing specificitytoward native DNA, with aldehyde-treated proteins. In A and B, affinity of theantibodies was determined by a direct antigen ELISA using aldehyde-treatedBSA as the absorbed antigens. A coating antigen was prepared by incubatingBSA (1 mg/ml) with 1 mM aldehyde in 1 ml of 50 mM sodium phosphate buffer,pH 7.4, for 24 h at 37 °C. One microgram of antigen was coated per well onpolystyrene plates, and antibody binding was detected. C, polyacrylamide gelelectrophoresis under nonreducing and nondenaturing conditions followedby immunoblot analysis of ONE-modified protein. BSA (1 mg/ml) was incu-bated with ONE (0 –5 mM) in 1 ml of 50 mM sodium phosphate buffer, pH 7.4,for 24 h at 37 °C. The protein samples were electrophoresed through 10%polyacrylamide gel and subjected to immunoblot analysis with the mAbD2A3. Left, polyacrylamide gel electrophoresis. Right, immunoblot analysis.

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specific epitopes, may be involved in the pathogenesis of auto-immune disorders.One recent study demonstrated that elevated oxidative stress

in erythrocytes due to a superoxide dismutase 1 deficiency trig-gered autoantibody production and that an antioxidant,N-ace-tylcysteine, significantly suppressed the inflammatory response(30). More intriguingly, our preliminary study demonstrated asignificant increase in the levels of the anti-HNE titer in the

superoxide dismutase 1-deficientmice. These observations also sug-gest that, although the potential rolein pathogenesis needs to be furtherexplored, lipid peroxidation maynot simply be an associated sideeffect of disease progression but apossible etiology of SLE and otherautoimmune diseases.It is well known that the immuno-

reactivity for anti-DNA antibodiesis the most characteristic serologi-cal finding in SLE (31). At the clini-cal level, however, we showed thatthe anti-HNE antibodies could bean important serological marker forSLE, showing a higher specificitythan the anti-DNA antibodies (Fig.3). In addition, a similar increase inthe levels of anti-HNE titer has alsobeen observed in the SLE-proneMRL-lprmice. These data, showingthe existence of a close associationbetween the anti-DNA and anti-HNE antibody titers, significantlyemphasized the relevance of HNEand its epitopes for the anti-DNAresponse in SLE.Moreover, since allof these patients had an active dis-

ease and somepatients receiving no steroids hadhigh anti-HNElevels, the increased anti-HNE antibodies may reflect someform of manifestation of SLE. Therefore, the HNE-specificepitopes may be used as the antigenic probe for detecting spe-cific autoantibodies that can serve as reliable biomarkers for thepractical evaluation of the disease activity in a subpopulation ofSLE patients.Although the anti-DNAantibodies are the hallmark of SLE, it

has been difficult to identify an antigen that will elicit this spec-ificity upon immunization. Based on the findings that (i) thesequence of an anti-HNE mAb was highly homologous to theanti-DNA autoantibodies (12), (ii) the HNE-specific epitopeswere detected in tissues from patients with human SLE (Fig. 2),and (iii) the SLE patients with elevated serum levels of the anti-DNA titer were also positive for anti-HNE antibodies (Fig. 3),which is a significantly higher frequency than in patients with-out the elevated serum anti-HNE antibodies, we speculatedthat the HNE-specific epitopes could be an endogenous sourceof the anti-DNA antibodies. Hence, to determine if the HNE-specific epitopes could induce an anti-DNA response, weattempted to raise mAbs from BALB/c mice immunized withthe HNE-modified KLH and successfully prepared the mAbsD1H3, D2A3, D3A8, andD3A11, showing recognition specific-ity toward native DNA, in addition to the anti-HNE mAbs,P2G11 and P4E6 (Fig. 4). Specificity studies demonstrated thatthe mAbs D1H3, D2A3, D3A8, and D3A11 strongly cross-re-actedwithDNAbut did not cross-reactwith theHNE-modifiedBSA, whereas the anti-HNE mAbs did not show any cross-re-activity toward native DNA, which revealed that animals

FIGURE 7. Identification of an epitope recognized by the mAb D2A3. A, competitive ELISA analysis with thereaction mixtures of amino acid derivatives and ONE. Competitors were prepared by incubating 10 mM aminoacid derivatives, N�-acetylcysteine, N�-acetylhistidine, or N�-acetyllysine, in the presence or absence of ONE(10 mM), in 50 mM sodium phosphate buffer (pH 7.2) for 24 h at 37 °C. Competitors were as follows: ONE/N�-arginine (�), ONE/N�-acetyllysine (‚), ONE/N�-acetylhistidine (E), and ONE/N�-acetylcysteine (F). B, compet-itive ELISA analysis of HPLC fractions for immunoreactivity with mAb D2A3. The reaction was performed byincubating 10 mM N�-acetylcysteine with 10 mM ONE in 10 ml of 50 mM sodium phosphate buffer (pH 7.4) at37 °C for 24 h. Solid line, profile of UV absorbance at 220 nm. Bar, competitive ELISA analysis. C, a proposedstructure of antigenic ONE-cysteine adduct.

FIGURE 8. The HNE-specific epitopes as an endogenous triggering anti-gen of anti-DNA response. Immunization with HNE-modified protein resultsin the production of monoclonal antibodies to both HNE-specific epitopesand native DNA.

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immunized with the HNE-specific epitopes progressivelydeveloped two distinct populations of mAbs that cross-reactedwith either the HNE-modified proteins or native DNA. Anti-bodies cross-reacting with both the HNE epitopes and nativeDNAwere not raised. Specificity studies also revealed that, con-sistent with our previous findings (12), the anti-HNE mAbP4E6 cross-reactedwith theONE-modifiedDNA (Fig. S2). Thisdual cross-reactivity of the anti-HNEmAb was previously pro-posed to arise through molecular mimicry between the HNE-histidine and the 1,N2-etheno-type ONE-2�-deoxyribonucleo-side adducts (12).One unanticipated result of this study is the finding that

the mAb D2A3, showing recognition specificity towardDNA, significantly cross-reacted with the ONE-modifiedprotein (Fig. 6). Our inhibition studies also demonstratedthat the mAb D2A3 recognized the ONE-cysteine Michaeladduct as the major epitope (Fig. 7). Of interest, this dualspecificity toward DNA and the ONE-modified protein isnot unique to this antibody raised from BALB/c mice immu-nized with the HNE-specific epitopes. We have indeedobserved that the anti-DNA mAb BV 16-13, whose specifi-city toward DNA has been well characterized (32), alsocross-reacts with the ONE-modified protein.4 In addition,we have examined the affinity of the anti-DNA mAbs D431and D466 obtained from the 56R and CD40L double trans-genic mice (33, 34), a spontaneous murine model of SLE,with the ONE-modified protein and have found that, of thetwo anti-DNA mAbs, mAb D466 cross-reacted with theONE-modified BSA.4Several different antigenic cross-reactivities have been iden-

tified for the anti-DNA antibodies (35). These antibodies sharestructural similarities with antibodies against bacterial polysac-charide, and some cross-react with the bacterial polysaccharideand protect mice against a lethal bacterial infection (36, 37).Other studies have also demonstrated a cross-reactivity of theanti-DNA antibodies with microbial protein antigens, non-nucleic acid autoantigens, cell membranes, and extracellularmatrix components (38–42). Thus, at least some of the anti-DNA antibodies seen in autoimmune disease are likely to arisefrom antigens associated with modification with HNE andONE, whereas some related adducts arising from other lipidoxidation products or possibly some epitopes that are of quitedistinct origin are also likely to be involved. Although thedetailed mechanisms for the dual cross-reactivity of the mAbD2A3 toward the native DNA and the ONE-cysteine adductremain unclear, there are several possible explanations for theantibody multispecificity: (i) the antibody-combining site hasmore than one contact region for unrelated epitopes (43); (ii)the antigen is a molecular mimic of DNA (commonly referredto as molecular mimicry, which is characterized by an immuneresponse to an environmental agent that cross-reacts with ahost antigen, resulting in disease) (44, 45); and (iii) finally, anintriguing possibility is an antibody conformational isomerism,in which the antibody has two structurally dissimilar binding

site conformations and can bind to two structurally distinctantigens (one site has a deep hole that binds to aromatic hap-tens, and the other binds to an unrelated protein or DNA anti-gen) (46, 47). Among these possibilities, we speculate that theONE-associated dual cross-reactivity of the antibodies may beascribed, at least in part, to the highest reactivity of this majorlipid hydroperoxide-derived bifunctional electrophile towardbiomacromolecules, such as protein and DNA (24–26). Uponreaction with protein and DNA, ONE generates a variety ofdifferent types of adducts on protein andDNAmolecules, someof which are known to have the same or very similar chemicalstructures as those originated from other aldehydes (4, 48, 49).It is likely that some of the adducts may serve as epitopes ofanti-HNE and anti-DNA antibodies through molecular mim-icry. A further study is required to establish the mechanism forthe unexpected cross-reactivities of the anti-DNA mAbstoward covalentlymodified proteinswith the lipid peroxidationproducts.In conclusion, our data demonstrated that the HNE-specific

epitopes could be an endogenous triggering antigen for the pro-duction of the antibodies specific to DNA. In addition, a subsetof the antibodies, showing recognition specificity toward DNA,cross-reacted with the alternative protein-bound epitope.These findings contribute to a paradigm of lipid peroxidationproducts causing at least some autoimmune diseases.

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Takasaki, Tsukasa Matsuda and Koji UchidaSohei Ito, Noriyuki Shibata, Tomoko Yamamoto, Makio Kobayashi, Yoshinari

Kazuyo Toyoda, Ritsuko Nagae, Mitsugu Akagawa, Kosuke Ishino, Takahiro Shibata,ANTIGEN OF ANTI-DNA RESPONSE

Protein-bound 4-Hydroxy-2-nonenal: AN ENDOGENOUS TRIGGERING

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