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Identification of Three New Autoantibodies Associated with Systemic Lupus Erythematosus Using Two Proteomic Approaches

2011; Elsevier BV; Volume: 10; Issue: 6 Linguagem: Inglês

10.1074/mcp.m110.005330

1535-9484

Yasuhiro Katsumata, Yasushi Kawaguchi, Sayumi Baba, Seisuke Hattori, Koji Tahara, Kaori Ito, Tadao Iwasaki, Nozomi Yamaguchi, Masaaki Oyama, Hiroko Kozuka‐Hata, Hiroaki Hattori, Kinya Nagata, Hisashi Yamanaka, Masako Hara,

T-cell and B-cell Immunology

Our objective was to identify new serum autoantibodies associated with systemic lupus erythematosus (SLE), focusing on those found in patients with central nervous system (CNS) syndromes. Autoantigens in human brain proteins were screened by multiple proteomic analyses: two-dimensional polyacrylamide gel electrophoresis/Western blots followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis and immunoprecipitation followed by liquid chromatography-tandem mass spectrometry shotgun analysis. The presence of serum IgG autoantibodies against 11 selected recombinant antigens was assessed by Western blot and enzyme-linked immunosorbent assay (ELISA) in the sera of 106 SLE patients and 100 normal healthy controls. The O.D. values in sera from SLE patients were significantly higher than those of controls for the antigens crystallin αB (p = 0.0002), esterase D (p = 0.0002), APEX nuclease 1 (p < 0.0001), ribosomal protein P0 (p < 0.0001), and PA28γ (p = 0.0005); the first three are newly reported. The anti-esterase D antibody levels were significantly higher in the CNS group than in the non-CNS group (p = 0.016). Moreover, when the SLE patients were categorized using CNS manifestations indicating neurologic or psychiatric disorders, the anti-APEX nuclease 1 antibody levels were significantly elevated in SLE patients with psychiatric disorders (p = 0.037). In conclusion, the association of SLE with several new and previously reported autoantibodies has been demonstrated. Statistically significant associations between anti-esterase D antibodies and CNS syndromes as well as between anti-APEX nuclease 1 antibodies and psychiatric disorders in SLE were also demonstrated. The combined immunoproteomic approaches used in this study are reliable and effective methods for identifying SLE autoantigens. Our objective was to identify new serum autoantibodies associated with systemic lupus erythematosus (SLE), focusing on those found in patients with central nervous system (CNS) syndromes. Autoantigens in human brain proteins were screened by multiple proteomic analyses: two-dimensional polyacrylamide gel electrophoresis/Western blots followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis and immunoprecipitation followed by liquid chromatography-tandem mass spectrometry shotgun analysis. The presence of serum IgG autoantibodies against 11 selected recombinant antigens was assessed by Western blot and enzyme-linked immunosorbent assay (ELISA) in the sera of 106 SLE patients and 100 normal healthy controls. The O.D. values in sera from SLE patients were significantly higher than those of controls for the antigens crystallin αB (p = 0.0002), esterase D (p = 0.0002), APEX nuclease 1 (p < 0.0001), ribosomal protein P0 (p < 0.0001), and PA28γ (p = 0.0005); the first three are newly reported. The anti-esterase D antibody levels were significantly higher in the CNS group than in the non-CNS group (p = 0.016). Moreover, when the SLE patients were categorized using CNS manifestations indicating neurologic or psychiatric disorders, the anti-APEX nuclease 1 antibody levels were significantly elevated in SLE patients with psychiatric disorders (p = 0.037). In conclusion, the association of SLE with several new and previously reported autoantibodies has been demonstrated. Statistically significant associations between anti-esterase D antibodies and CNS syndromes as well as between anti-APEX nuclease 1 antibodies and psychiatric disorders in SLE were also demonstrated. The combined immunoproteomic approaches used in this study are reliable and effective methods for identifying SLE autoantigens. Systemic lupus erythematosus (SLE) 1The abbreviations used are:SLEsystemic lupus erythematosusCNScentral nervous systemNPneuropsychiatricACRAmerican College of RheumatologyRArheumatoid arthritisSScsystemic sclerosisSSSjögren syndromeMSmultiple sclerosisWBwestern blotHRPhorseradish peroxidaseNHCnormal healthy controlPBSphosphate buffered saline. is an autoimmune disease that usually develops in women aged 18–50 years and is characterized by the presence of autoantibodies. Diagnosis is difficult because SLE is a great imitator of other diseases (1Kurien B.T. Scofield R.H. Autoantibody determination in the diagnosis of systemic lupus erythematosus.Scand. J. Immunol. 2006; 64: 227-235Crossref PubMed Scopus (84) Google Scholar). Autoantibodies are clearly central to the pathogenesis of SLE, and different autoantibodies are associated with different clinical features (2Giles I. Putterman C. Autoantibodies and other biomarkers - pathological consequences (1).Lupus. 2008; 17: 241-246Crossref PubMed Scopus (8) Google Scholar). Several of the more than 100 autoantibodies identified to date have been associated with disease activity (1Kurien B.T. Scofield R.H. Autoantibody determination in the diagnosis of systemic lupus erythematosus.Scand. J. Immunol. 2006; 64: 227-235Crossref PubMed Scopus (84) Google Scholar). Although anti-double-stranded DNA antibodies are the most extensively studied autoantibodies in SLE, others play roles in clinical manifestations, particularly in autoimmune hemolytic anemia, thrombocytopenia, skin disease, and neonatal lupus (3Rahman A. Isenberg D.A. Systemic lupus erythematosus.N. Engl. J. 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Delunardo F. Margutti P. Vacirca D. Piro E. Siracusano A. Ortona E. Autoantibodies involved in neuropsychiatric manifestations associated with systemic lupus erythematosus.J. Neuroimmunol. 2009; 212: 3-9Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The identification and characterization of new, specific autoantibodies could help elucidate the etiology of the NP manifestations that accompany SLE, opening new perspectives for more effective diagnostic and therapeutic strategies. Conventionally, study of the autoimmune response has been conducted by analyzing the presence and/or concentration of individual antibodies in biological fluids. Proteomic techniques allow the simultaneous identification and measurement of different autoantibodies in the sera of patients suffering from autoimmune diseases (8Plebani M. Pittoni M. Celadin M. Bernardi D. Mion M.M. Recent advances in diagnostic technologies for autoimmune diseases.Autoimmun. 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MALDI-TOF MS combined with magnetic beads for detecting serum protein biomarkers and establishment of boosting decision tree model for diagnosis of systemic lupus erythematosus.Rheumatology. 2009; 48: 626-631Crossref PubMed Scopus (33) Google Scholar, 15Dai Y. Hu C. Huang Y. Huang H. Liu J. Lv T. A proteomic study of peripheral blood mononuclear cells in systemic lupus erythematosus.Lupus. 2008; 17: 799-804Crossref PubMed Scopus (27) Google Scholar). The possibility of simultaneously measuring a number of correlated analytes is interesting for analytical reasons (e.g. reduced biological sample and reagent volumes and lower costs), logistical and managerial reasons, and pathophysiological reasons (i.e. identifying combinations of markers for use in disease-oriented or organ-oriented profiling) (8Plebani M. Pittoni M. Celadin M. Bernardi D. Mion M.M. Recent advances in diagnostic technologies for autoimmune diseases.Autoimmun. Rev. 2009; 8: 238-243Crossref PubMed Scopus (30) Google Scholar). However, much work remains unfinished in developing, refining, validating, and applying proteomics technologies to identify biomarkers in autoimmune diseases (9Hueber W. Robinson W.H. Proteomic biomarkers for autoimmune disease.Proteomics. 2006; 6: 4100-4105Crossref PubMed Scopus (52) Google Scholar). By using both conventional and newer proteomic approaches, our aim was to find novel serum autoantibodies associated with SLE, focusing on those found in patients with CNS syndromes. Sera from 106 patients with active SLE from 1994 through 2007 were obtained using the Tokyo Women's Medical University SLE Database. These sera were originally collected from the patients' whole blood using standard tubes with a polyester gel separator. Immediately following clotting at room temperature and following centrifugation, the separated sera were aliquoted and stored at −80 °C. All patients had four or more revised ACR (formerly the American Rheumatism Association) criteria for SLE (16Tan E.M. Cohen A.S. Fries J.F. Masi A.T. McShane D.J. Rothfield N.F. Schaller J.G. Talal N. Winchester R.J. The 1982 revised criteria for the classification of systemic lupus erythematosus.Arthritis Rheum. 1982; 25: 1271-1277Crossref PubMed Scopus (12580) Google Scholar, 17Hochberg M.C. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus.Arthritis Rheum. 1997; 401725Crossref PubMed Scopus (9116) Google Scholar) and gave informed consent for inclusion in this study. Those who had non-SLE-related NP manifestations arising from infection, uremia, electrolyte imbalance, hypoxia, brain tumor, trauma, primary mental disease, drug use or past histories of NP involvement were excluded. These patients were excluded because we wanted to compare recently diagnosed, active CNS lupus patients to non-NPSLE patients; unrelated conditions could affect current symptoms or laboratory findings. At the time of serum collection, each patient completed a standardized medical history that included medication use and was given a physical examination that included neurologic and rheumatologic assessments. Psychiatric examinations were employed when indicated. Serology profiling for each patient was performed using standard immunoassays. Subjects were classified into the CNS group or the non-CNS group according to the presence or absence of active CNS syndromes. The CNS group was then further classified into the neurologic disorders group consisting of patients with neurologic disorders with or without other NP syndromes, or the psychiatric disorders group comprising patients with psychiatric disorders with or without other NP syndromes (5ACR Ad Hoc Committee on Neuropsychiatric Lupus Nomenclature The American College of Rheumatology nomenclature and case definitions for neuropsychiatric lupus syndromes.Arthritis Rheum. 1999; 42: 599-608Crossref PubMed Scopus (1790) Google Scholar, 18Katsumata Y. Harigai M. Kawaguchi Y. Fukasawa C. Soejima M. Takagi K. Tanaka M. Ichida H. Tochimoto A. Kanno T. Nishimura K. Kamatani N. Hara M. Diagnostic reliability of cerebral spinal fluid tests for acute confusional state (delirium) in patients with systemic lupus erythematosus: interleukin 6 (IL-6), IL-8, interferon-alpha, IgG index, and Q-albumin.J. Rheumatol. 2007; 34: 2010-2017PubMed Google Scholar, 19Katsumata Y. Harigai M. Kawaguchi Y. Fukasawa C. Soejima M. Kanno T. Nishimura K. Yamada T. Yamanaka H. Hara M. Diagnostic reliability of magnetic resonance imaging for central nervous system syndromes in systemic lupus erythematosus: a prospective cohort study.BMC Musculoskelet Disord. 2010; 11: 13Crossref PubMed Scopus (23) Google Scholar). Detailed diagnostic criteria for these groups are described below. Control sera were derived from age- and sex-matched healthy donor subjects and from patients with rheumatoid arthritis (RA), systemic sclerosis (SSc), Sjögren syndrome (SS), and multiple sclerosis (MS) diagnosed using standard criteria (20Arnett F.C. Edworthy S.M. Bloch D.A. McShane D.J. Fries J.F. Cooper N.S. Healey L.A. Kaplan S.R. Liang M.H. Luthra H.S. et al.The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis.Arthritis Rheum. 1988; 31: 315-324Crossref PubMed Scopus (18700) Google Scholar, 21Subcommittee for Scleroderma Criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee Preliminary criteria for the classification of systemic sclerosis (scleroderma). Subcommittee for scleroderma criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee.Arthritis Rheum. 1980; 23: 581-590Crossref PubMed Scopus (4956) Google Scholar, 22Vitali C. Bombardieri S. Moutsopoulos H.M. Coll J. Gerli R. Hatron P.Y. Kater L. Konttinen Y.T. Manthorpe R. Meyer O. Mosca M. Ostuni P. Pellerito R.A. Pennec Y. Porter S.R. Richards A. Sauvezie B. Schiodt M. Sciuto M. Shoenfeld Y. Skopouli F.N. Smolen J.S. Soromenho F. Tishler M. Wattiaux M.J. Assessment of the European classification criteria for Sjogren's syndrome in a series of clinically defined cases: results of a prospective multicentre study. The European Study Group on Diagnostic Criteria for Sjogren's Syndrome.Ann Rheum Dis. 1996; 55: 116-121Crossref PubMed Scopus (474) Google Scholar, 23Polman C.H. Reingold S.C. Edan G. Filippi M. Hartung H.P. Kappos L. Lublin F.D. Metz L.M. McFarland H.F. O'Connor P.W. Sandberg-Wollheim M. Thompson A.J. Weinshenker B.G. Wolinsky J.S. Diagnostic criteria for multiple sclerosis: 2005 revisions to the “McDonald Criteria”.Ann. Neurol. 2005; 58: 840-846Crossref PubMed Scopus (4361) Google Scholar). This study was approved by the Ethical Committee of our institution and the Helsinki Declaration was followed throughout the study. Although ACR nomenclature and case definitions include 12 CNS syndromes and seven peripheral nervous system syndromes (5ACR Ad Hoc Committee on Neuropsychiatric Lupus Nomenclature The American College of Rheumatology nomenclature and case definitions for neuropsychiatric lupus syndromes.Arthritis Rheum. 1999; 42: 599-608Crossref PubMed Scopus (1790) Google Scholar, 18Katsumata Y. Harigai M. Kawaguchi Y. Fukasawa C. Soejima M. Takagi K. Tanaka M. Ichida H. Tochimoto A. Kanno T. Nishimura K. Kamatani N. Hara M. Diagnostic reliability of cerebral spinal fluid tests for acute confusional state (delirium) in patients with systemic lupus erythematosus: interleukin 6 (IL-6), IL-8, interferon-alpha, IgG index, and Q-albumin.J. Rheumatol. 2007; 34: 2010-2017PubMed Google Scholar, 19Katsumata Y. Harigai M. Kawaguchi Y. Fukasawa C. Soejima M. Kanno T. Nishimura K. Yamada T. Yamanaka H. Hara M. Diagnostic reliability of magnetic resonance imaging for central nervous system syndromes in systemic lupus erythematosus: a prospective cohort study.BMC Musculoskelet Disord. 2010; 11: 13Crossref PubMed Scopus (23) Google Scholar), we used only the 12 CNS syndromes in the inclusion criteria for our study because of the substantial differences between the central and peripheral nervous systems in anatomy, function, and clinical characteristics. Slight or mild cognitive dysfunction without significant clinical impairment, as revealed by detailed neuropsychological testing, was excluded from the CNS syndromes in our study. Tension headache and episodic tension type headache were also excluded. CNS syndromes were further classified into neurologic disorders (aseptic meningitis, cerebrovascular disease, demyelinating syndrome, headache, movement disorder, myelopathy, and seizure disorders) and psychiatric disorders (acute confusional state, anxiety disorder, cognitive dysfunction, mood disorder, and psychosis) (5ACR Ad Hoc Committee on Neuropsychiatric Lupus Nomenclature The American College of Rheumatology nomenclature and case definitions for neuropsychiatric lupus syndromes.Arthritis Rheum. 1999; 42: 599-608Crossref PubMed Scopus (1790) Google Scholar). The final clinical diagnosis and classification of the various NP syndromes for inclusion in the study were made by an experienced rheumatologist (M. H.) and psychiatrist (K. N.), according to the standardized ACR nomenclature and case definitions for NP lupus syndromes (5ACR Ad Hoc Committee on Neuropsychiatric Lupus Nomenclature The American College of Rheumatology nomenclature and case definitions for neuropsychiatric lupus syndromes.Arthritis Rheum. 1999; 42: 599-608Crossref PubMed Scopus (1790) Google Scholar). These decisions were based on the medical history and neuropsychological examinations by rheumatologists, an experienced neurologist (S. U.) and a psychiatrist (K. N.) and were supported by conventional laboratory tests and appropriate complementary tests, including MRI, electroencephalography, and cerebral spinal fluid tests, as well as an assessment of the clinical course of the disease. Human neuroblastoma cell lines (IMR-32 and NB-1) and human glioblastoma cell lines (A172 and T98G) were obtained from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan). IMR-32 cells were routinely cultured in MEM (Sigma-Aldrich, St. Louis, MO) supplemented with nonessential amino acids (MP Biomedicals, Irvine, CA). NB-1 cells were cultured in a medium containing an equal amount of MEM and RPMI 1640 (Sigma-Aldrich). The human glioblastoma cell lines, A172 and T98G, were cultured in RPMI 1640. All culture media were supplemented with 10% fetal bovine serum (SAFC Biosciences, Lenexa, KS), 50 U/ml of penicillin, and 50 μg/ml of streptomycin. The total protein from human whole brain (BioChain Institute, Hayward, CA) was precipitated once using the ReadyPrep 2-D Cleanup Kit (Bio-Rad Laboratories, Hercules, CA), according to the manufacturer's protocol and used for two-dimensional polyacrylamide gel electrophoresis. The cultured cells were washed with phosphate buffered saline (PBS), scraped into a 1.5-ml tube and centrifuged to harvest cells. To prepare total cell protein, the cell pellet was sonicated on ice [output: 2; duty: 60; for 1 min (min); Sonifier 250D; Branson Ultrasonics Corporation, Danbury, CT] in 6 m urea, 2 m thiourea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), and a complete EDTA-free protease inhibitor mixture (Roche Diagnostics GmbH, Mannheim, Germany). Following centrifugation at 20,400 × g for 10–30 min at 4 °C, the supernatant was recovered as total cell protein for use in the screening of autoantigens. Similarly, as for total protein from human whole brain, the total cell protein was precipitated using the ReadyPrep 2-D Cleanup Kit. Protein concentrations were measured using the bicinchoninic acid protein assay reagent (Pierce, Rockford, IL) according to the manufacturer's protocol, using bovine serum albumin (BSA) as the standard. Human brain proteins were screened for autoantigens using two proteomic analysis techniques. First, we performed two-dimensional-PAGE/Western blots (WBs) and then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF/MS). two-dimensional-PAGE was performed as described elsewhere (24Toda T. Kimura N. Standardization of protocol for Immobiline 2-D PAGE and construction of 2-D PAGE protein database on World Wide Web home page.Jap. J. Electrophor. 1997; 41: 13-20Google Scholar). Briefly, prepared protein samples were precipitated using the ReadyPrep 2-D Cleanup Kit (Bio-Rad) according to the manufacturer's protocol. Before electrophoresis, the protein pellets were dissolved in DeStreak Rehydration Solution (GE Healthcare, Buckinghamshire, UK) containing 0.2% BioLyte 3/10 (Bio-Rad) and 50 mm dithiothreitol. Next, 50 μg of protein was applied to IPG ReadyStrip (pH 3–10, nonlinear, 7 cm long; Bio-Rad) for overnight in-gel rehydration. The proteins were then separated by one-dimensional isoelectric focusing (Multiphore II (GE Healthcare) or CoolPhoreSter IPG-IEF Type-P (Anatech, Tokyo, Japan) at 20 °C and focused with the following program: 200 V for 18 min, 500 V for 18 min, 1000 V for 18 min, 1500 V for 18 min, 2000 V for 18 min, 2500 V for 18 min, 3000 V for 18 min, and 3500 V for 90 min. Following one-dimensional isoelectric focusing, the IPG strips were equilibrated in a solution of 50 mm Tris-HCl, pH 8.8, 6 m urea, 30% glycerol, 2% SDS, and 0.002% bromphenol blue that also contained 10 mg/ml dithiotreitol for the first equilibration step and 25 mg/ml iodoacetamide in the second equilibration step; the strips were bathed in solution on an orbital shaker at room temperature for 15 min in each step. The equilibrated strips were then sealed with 1% SeaKem GTG Agarose (Lonza, Rockland, ME) in SDS running buffer at the top of two-dimensional SDS-polyacrylamide gels. SDS-PAGE was performed using precast 5–20% polyacrylamide gradient gels (SuperSep 5–20%, two-dimensional gel; Wako Pure Chemical Industries, Osaka, Japan) at a constant current of 20 mA/gel until the bromphenol blue dye front reached the lower edge of the gel. Following two-dimensional-PAGE, the separated proteins were transferred onto Immobilon-P (Millipore, Billerica, MA) with a semidry blotter (ATTO AE-6677; ATTO, Tokyo, Japan) at a constant current of 2 mA/cm2 for 1 h (h) with a solution of 100 mm Tris base, 192 mm glycine, and 5% methanol. Membranes were washed with 0.1% Tween 20 in PBS (PBS-T). Before WB, proteins on the membrane were labeled using Cy5 Mono-Reactive Dye (GE Healthcare) for 30 min in the dark at room temperature; any remaining free dye was removed by washing with PBS-T. The membranes were blocked for 1 h at room temperature with blocking buffer (1% skim milk in PBS-T). WBs were performed using the pooled sera from SLE patients with active CNS syndromes (the CNS group) or those without either current CNS syndromes or a history of CNS syndromes (the non-CNS group) diluted 1:500 in blocking buffer and incubated overnight at 4 °C. Following being washed with PBS-T, the membranes were incubated for 1 h at room temperature with the secondary antibody goat anti-human IgG+A+M (H+L) (Zymed Laboratories Inc., San Francisco, CA), conjugated to horseradish peroxidase (HRP), and diluted 1:15,000 in blocking buffer. WBs were visualized using the ECL plus (GE Healthcare) chemifluorescence signal, and proteins on the membrane were visualized using the Cy5 fluorescence signal. These fluorescent images were scanned using a Typhoon 9400 (GE Healthcare) with excitation at 457 nm and emission filter 520BP40 (ECL plus) and with excitation at 633 nm and emission filter 670BP30 (Cy5). The resulting images were overlaid and easily matched to identical positions from the western signal and protein signal. Using this information, protein samples were prepared for mass spectrometry analysis. Using the results of the two-dimensional-PAGE/WB, the ECL plus signal spots specific for CNS lupus patients' pooled sera were detected, and their positions were identified on the Cy5-protein signals. For MALDI TOF/MS analysis, the proteins were separated using two-dimensional-PAGE, transferred onto a ProBlott membrane (Applied Biosystems, Foster City, CA) and stained with Coomassie Brilliant Blue R-250 (PhastGel Blue R, GE Healthcare). CNS lupus-specific spots were excised and washed with Milli-Q water. The isolated proteins were then digested at 37 °C for 90 min using 1 pmol/μl of lysine endopeptidase (mass spectrometry grade, Wako Pure Chemical Industries) in 50% acetonitrile. The resulting peptides were purified using NuTip NT1HIL.96 solid-phase extraction cartridges (Glygen, Columbia, MD) and mixed with α-cyano-4-hydroxycinnamic acid matrix. Peptide mass fingerprinting was performed using a MALDI-TOF mass spectrometer (Voyeger™, Applied Biosystems). Peptide mass fingerprinting data were compared with the NCBInr databases (human, 233173 sequences; date 2010/12/24) using the Mascot Search engine (Peptide Mass Fingerprint, version 2.2; Matrix Science, Boston, MA) with the following parameters: Enzyme, Lys-C; Variable modifications, Carbamidomethyl (C) and Oxidation (M); Mass values, Monoisotopic; Protein Mass, Unrestricted; Peptide Mass Tolerance: ± 0.2–0.4 Da; Peptide Charge State, 1+; and Max Missed Cleavages, 0 and exceeded the thresholds (p < 0.05). Confidence in the reliability of the identification was indicated by the number of matching and total peptides as well as the protein sequence coverage by the matching peptides. Because two-dimensional-PAGE/WB and MALDI TOF/MS analyses did not have sufficient sensitivity, we also used immunoprecipitation and protein shotgun analyses using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to screen candidate autoantigens. In the first trial, four types of cell lines (NB-1, IMR-32, A152, and T98G) were cultured, washed with PBS, and harvested. Cell pellets were suspended in a cell lysis buffer (20 mm HEPES, pH 7.5, 150 mm NaCl, 2 mm MgCl2, 10 mm NaF, 2 mm EGTA, 25 mm β-glycerophosphate, 2 mm dithiotreitol, 1% Nonidet P-40, 10% glycerol, and 0.2% benzonase nuclease [Novagen, Darmstadt, Germany]) containing a protease inhibitor mixture (Roche, Basel, Switzerland) and incubated on ice for 30 min. Following centrifugation at 20,400 × g for 10 min at 4 °C, the supernatants were recovered and protein concentrations were measured using the bicinchoninic acid protein assay. Then, 5 mg of cell lysate for each of the four cell lines was mixed. In a separate assay, 10 mg of total protein from human whole brain (BioChain Institute) was added to this mixture of lysates from the four types of cell lines (5 mg each). These antigen mixtures were precleared by incubation with 150 μl Protein G Sepharose beads (GE Healthcare) to prevent nonspecific proteins from binding to the beads and then used as antigens for immunoprecipitation. Total IgG from 67.5 μl of serum mixtures from nine SLE patients with active CNS syndromes and from nine SLE patients without active CNS syndromes was incubated with 37.5 μl of Protein G-Sepharose beads at room temperature using a gentle rocking motion for 45 min. The beads were then centrifuged and the supernatant was removed. The beads were washed twice with 0.2 m borate buffer (pH 9.0) and the IgG was crosslinked to the Protein G Sepharos