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. 2010 Oct 29;285(44):33834-42.
doi: 10.1074/jbc.M110.165175. Epub 2010 Aug 24.

Identification of a lipid peroxidation product as the source of oxidation-specific epitopes recognized by anti-DNA autoantibodies

Natsuki Otaki  1 , Miho Chikazawa, Ritsuko Nagae, Yuki Shimozu, Takahiro Shibata, Sohei Ito, Yoshinari Takasaki, Junichi Fujii, Koji Uchida
Affiliations

Identification of a lipid peroxidation product as the source of oxidation-specific epitopes recognized by anti-DNA autoantibodies

Natsuki Otaki et al. J Biol Chem. .

Abstract

Lipid peroxidation in tissue and in tissue fractions represents a degradative process, which is the consequence of the production and the propagation of free radical reactions primarily involving membrane polyunsaturated fatty acids, and has been implicated in the pathogenesis of numerous diseases, including systemic lupus erythematosus (SLE). We have found that bovine serum albumin incubated with peroxidized polyunsaturated fatty acids significantly cross-reacted with the sera from MRL-lpr mice, a representative murine model of SLE. To identify the active substances responsible for the generation of autoantigenic epitopes recognized by the SLE sera, we performed the activity-guiding separation of a principal source from 13-hydroperoxy-9Z,11E-octadecadienoic acid and identified 4-oxo-2-nonenal (ONE), a highly reactive aldehyde originating from the peroxidation of ω6 polyunsaturated fatty acids, as the source of the autoantigenic epitopes. When the age-dependent change in the antibody titer against the ONE-modified protein was measured in the sera from MRL-lpr mice and control MRL-MpJ mice, all of the MRL-lpr mice developed an anti-ONE titer, which was comparable with the anti-DNA titer. Strikingly, a subset of the anti-DNA monoclonal antibodies generated from the SLE mice showing recognition specificity toward DNA cross-reacted with the ONE-specific epitopes. Furthermore, these dual-specific antibodies rapidly bound and internalized into living cells. These findings raised the possibility that the enhanced lipid peroxidation followed by the generation of ONE may be involved in the pathogenesis of autoimmune disorders.

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Figures

FIGURE 1.
FIGURE 1.
Lipid peroxidation modification of serum albumin generates autoantigenic epitopes recognized by the sera from MRL-lpr mice. BSA was incubated with a polyunsaturated fatty acid (arachidonic acid or linoleic acid) in the presence of the Fe2+/ascorbate free radical-generating system. Cross-reactivity of modified proteins with the sera from MRL-lpr and MRL-MpJ mice was examined by ELISA. A, formation of autoantigenic epitopes upon reaction of BSA with peroxidized linoleic acid. Closed square, MRL-lpr mice; open square, MRL-MpJ mice. B, formation of autoantigenic epitopes upon reaction of BSA with peroxidized arachidonic acid. Closed circle, MRL-lpr mice; open circle, MRL-MpJ mice.
FIGURE 2.
FIGURE 2.
Identification of a source of autoantigenic epitopes recognized by the sera from MRL-lpr mice. A, lipid peroxidation products were fractionated by reverse phase HPLC on an ODS column, and the fractions were collected every 2 min. All of the fractions were incubated with serum albumin at 37 °C for 24 h as the coating agent in ELISA. Solid line, profile of UV absorbance at 200–650 nm. Bar, ELISA analysis. B, fraction I was analyzed by reverse phase HPLC on a phenethyl column, and the fractions were collected every 2 min. All of the fractions were incubated with serum albumin at 37 °C for 24 h, as the coating agent in ELISA. Solid line, profile of UV absorbance at 200–650 nm. Bar, ELISA analysis. C, 1H NMR spectrum of the product a. D, chemical structure of ONE. E, cross-reactivity of the MRL-lpr mice sera with ONE-treated proteins. Affinity of the MRL-lpr mice sera was determined by a direct antigen ELISA using ONE-treated BSA as the absorbed antigens. F, cross-reactivity of the MRL-lpr mice sera with aldehydes-treated proteins. Affinity of the MRL-lpr mice sera was determined by a direct antigen ELISA using dsDNA and native and aldehyde-treated BSA as the absorbed antigens.
FIGURE 3.
FIGURE 3.
Anti-ONE response in MRL-lpr mice. A, anti-DNA response in MRL-MpJ (open circle) and MRL-lpr mice (closed circle). The antibody response was examined by an ELISA employing pairs of wells in microtiter plates on which were absorbed calf thymus DNA as the antigen. B, anti-ONE response in MRL-MpJ (open circle) and MRL-lpr mice (closed circle). The antibody response was examined by an ELISA employing pairs of wells in microtiter plates on which were absorbed ONE-treated BSA as the antigen. C, glomerular immunoglobulin deposition in 20-week-old MRL-MpJ and MRL-lpr mice. Kidney sections were stained with Alexa Fluor 488-conjugated antibody against mouse IgG. HE, hematoxylin and eosin. D, immunoreactivity of antibodies eluted from the kidneys of MRL-MpJ and MRL-lpr mice. Affinity of the antibodies was determined by a direct antigen ELISA using ssDNA, dsDNA, BSA, and ONE-treated BSA as the absorbed antigens.
FIGURE 4.
FIGURE 4.
Isolation and specificity of the dual-specific autoantibodies. A, immunoreactivity of the anti-DNA mAbs originating from MRL-lpr mice. The affinity of the antibodies was determined by a direct antigen ELISA using dsDNA, BSA, and ONE-treated BSA as the absorbed antigens. B, specificity of the anti-DNA IgGs originated from MRL-lpr mice. C, sequence alignments of the VH and VL domains of anti-DNA mAbs and homologous antibodies. A dot denotes sequence identity and a space denotes a gap in the sequence. The characters above the alignment represent the IgBLAST complementarity-determined regions (CDR1, CDR2, and CDR3) and framework regions (FWR1, FWR2, and FWR3). The sequences were aligned using the program ClustalW 1.82 and were manually modified. Accession numbers for the sequences are as follows. 202.80, CAA80103; 202.s38, CAA80108; 2-12, AAA97369; F5–58, AAB03597; 1DC7, AAS00741; 1DC10, AAS00737; ZB5G11, AY436953; F4-2 (VH), AAB03592; F4-2 (VL), AAB03601; 3D8, AF232220; 11E6, AAA96774; and BW2 19-19, AAL92956.
FIGURE 5.
FIGURE 5.
Internalization of the dual-specific antibodies into Jurkat cells. A, time-dependent internalization of dual-specific mAbs. Internalization was analyzed by flow cytometry after treating Jurkat cells with the mAbs for different lengths of time. Closed square, DSO; closed circle, A1116; closed diamond, N1131; open circle, normal IgG. B, internalization, not cell surface binding, of dual-specific mAb into the cells. The Jurkat cells were treated with mAb DSO for 1 h. The cells were immediately fixed, extensively washed, and incubated with or without 0.1% Triton X-100. The penetration of the mAb was analyzed by flow cytometry. C, detection of internalized antibodies by confocal microscopy. The Jurkat cells were treated with the mAbs for 6 h. The cells were then stained with propidium iodide (PI) or FITC-labeled Alexa Flour 488-labeled anti-IgG and analyzed by confocal microscopy. Each figure is representative of three independent experiments.
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