A subset of lupus anti-DNA antibodies cross-reacts with the NR2 glutamate receptor in systemic lupus erythematosus


In systemic lupus erythematosus, antibodies against double-stranded DNA are a major contributor to renal disease. We have previously demonstrated that the pentapeptide Asp/Glu-Trp-Asp/Glu-Tyr-Ser/Gly is a molecular mimic of double-stranded DNA. This sequence is also present in the extracellular domain of murine and human NMDA (N-methyl-d-aspartate) receptor subunits NR2a and NR2b. Here we show that the NR2 receptor is recognized by both murine and human anti-DNA antibodies. Moreover, anti-DNA antibodies with this cross-reactivity mediate apoptotic death of neurons in vivo and in vitro. Finally, we show that the cerebrospinal fluid of a patient with systemic lupus erythematosus contains these antibodies and also mediates neuronal death via an apoptotic pathway. These observations indicate that lupus antibodies cross-react with DNA and NMDA receptors, gain access to cerebrospinal fluid and may mediate non-thrombotic and non-vasculitic abnormalities of the central nervous system.


Among the protean clinical manifestations of systemic lupus erythematosus (SLE), abnormalities in the central nervous system (CNS) are often the most debilitating. In particular, it has been demonstrated that up to 80% of lupus patients experience CNS disease characterized by neuropsychiatric symptoms and cognitive decline1. Structural neuroimaging of SLE patients without overt symptoms in the CNS demonstrates non-focal atrophy; functional neuroimaging demonstrates variable cortical and subcortical abnormalities2. The mechanism of CNS injury that accounts for these cognitive and psychological impairments is unknown.

Many clinical manifestations of lupus appear to be mediated by autoantibodies, particularly those specific for native DNA. Studies show that anti-DNA antibodies correlate best with disease activity3, and they cause kidney damage by binding either directly to DNA present in tissue or to cross-reactive, non-DNA tissue antigens4. Several different antigenic cross-reactivities have been identified for anti-DNA antibodies5. These antibodies share structural similarities with antibodies against bacterial polysaccharide, and some cross-react with bacterial polysaccharide and protect mice against lethal bacterial infection6,7. Other studies have demonstrated cross-reactivity of anti-DNA antibodies with microbial protein antigens, non–nucleic-acid autoantigens, cell membranes and extracellular matrix components8,9,10,11,12.

In an effort to determine the range of antigens that anti-DNA antibodies might recognize, we screened a phage display peptide library for sequences bound by the R4A antibody13, a monoclonal murine antibody to double-stranded DNA (dsDNA) shown to deposit in glomeruli of SCID mice. The Asp/Glu-Trp-Asp/Glu-Tyr-Ser/Gly consensus sequence binds in the DNA binding site of the R4A antibody. In a multimeric configuration, this peptide elicits anti-DNA antibodies in BALB/c mice14. The consensus sequence is present in the extracellular, ligand-binding domain of mouse and human NMDA (N-methyl-d-aspartate) receptors NR2a and NR2b, residues 283–287.

NR2 receptors bind the neurotransmitter glutamate. They are expressed in a temporally regulated manner and are present on neurons broadly throughout the forebrain15,16,17,18,19. Mice with a targeted disruption of the gene encoding NR2a display a defect in hippocampal long-term potentiation20, confirming previous studies demonstrating a role for NR in learning and memory21. Glutamate receptors can display altered expression in major psychosis and over-stimulation of NR2 can cause excitotoxic neuron death through excessive entry of Ca++ into cells22,23,24.

Phenylcyclohexylpiperidine, a glutamate receptor antagonist, binds NR2's and functions as a psychotomimetic agent causing hallucinations and paranoia25,26,27. Thus, antibody reactivity with NR2a or NR2b is a plausible candidate mechanism to mediate some of the CNS disturbances in SLE28.

The R4A anti-DNA antibody binds NR2

To determine if the IgG2b R4A antibody can bind to NR2, we immunoprecipitated the NR2 receptors from PC12 cells with a polyclonal rabbit antibody and by western-blot analysis demonstrated that R4A antibody and not an isotype matched control antibody bound the receptor (Fig. 1).

Figure 1: R4A monoclonal antibody recognizes NR2.

NR2 receptors were immunoprecipitated from PC12 cells with a polyclonal rabbit anti-glutamate receptor 2/3 antibody, and probed by immunoblot with either R4A (lane 1) or isotype-matched control antibody (IgG2b) (lane 2). Lane 3 denotes PC12 whole cell extract probed with anti-GluR2/3 antibody.

To determine if R4A antibody would bind to neurons in vivo and function as a glutamate receptor agonist to cause excitotoxic cell death22,23, we injected purified R4A antibody or irrelevant IgG2b antibody into the hippocampus and cortex of C57BL/6 mice. The R4A antibody caused neuronal loss (Fig. 2). There was minimal local activation of microglia cells and astrocytes, and no lymphocytic infiltration. Neuron degeneration in the cortex after R4A injection was characterized by shrunken, pycnotic nuclei in cells expressing glutamate receptors. Mice treated systemically with MK-801, an NR2 blocker29,30, had no neuronal injury after the injection of R4A into the cortex. MK-801 acted as a non-competitive antagonist of R4A blocking neuronal death without blocking binding of R4A to neurons (data not shown). The mechanism of cell death depended only on antibody activating cellular signaling pathways and not complement- or cell-mediated cytotoxicity, as Fab′2 fragments of R4A also caused neuronal death.

Figure 2: R4A mediates neuron death via apoptosis.

a, R4A causes death of hippocampal neurons. Photomicrograph (cresyl violet, top left, ×5) depicts focal region of neuron loss (asterisk and inset, ×40) in mouse hippocampus 2 d after intracerebral injection of R4A antibody into the CA1 region compared with IgG2b isotype control antibody injection (bottom left, cresyl violet, ×5; arrow and inset, ×40). Top right panel demonstrates Neu-N staining of the injured CA1 hippocampal region from adjacent sections (×40). The absence of Neu-N immunoreactivity in shrunken, damaged neurons in the R4A injected CA1 region is contrasted with Neu-N immunoreactivity in the CA1 region after IgG2b control antibody injection (bottom right). There was no differential immunoreactivity to isolectin and GFAP (data not shown). Data represent 4 mice. b, R4A causes death of cortical neurons through binding to NR 2. Photomicrograph (top left, cresyl violet, ×10) demonstrates mouse cortex injected with R4A. Cortex in bottom left panel is from a mouse treated with MK-801 after R4A. Cortex from the animal with MK-801 treatment is normal (bottom left, ×10) with the exception of trauma induced by pipette. Photomicrograph (top right, ×40) shows in R4A-injected cortex from alternate sections abnormally clumped DAPI-labeled nuclei (blue) in glutamate-positive neurons (red) when compared with MK-801 treated cortex (bottom right, ×40). R4A causes specific degeneration of glutamate-receptor–positive neurons in the CA1 hippocampus and the cortex. c, Fab′2 fragments are sufficient to cause neuronal death. Photomicrograph (top left, ×5) shows neuron loss in the CA1 region of the hippocampus after the injection of Fab′2 fragments (4.5 μg). Photomicrographs demonstrate the focal region of neuron loss (top right, inset, ×40; asterisk on ×5 image indicates region of interest) and adjacent normal appearing neurons (bottom right, inset at ×40; arrow on ×5 image indicates region of interest). Similar to hippocampal injection of R4A, Neu-N immunoreactivity was absent in CA1 neurons that had been exposed to Fab′2 fragment (data not shown). On adjacent sections, MAP2 positively labeled these shrunken neurons, thus the loss of Neu-N immunoreactivity occurred in degenerating neurons. Photomicrographs (right top and bottom, ×40) display MAP2 immunoreactivity in degenerating neurons in the CA1 region exposed to the Fab′2 injection (top right, region near asterisk), and in the normal CA1 neurons adjacent to the Fab′2 injection (bottom right, region near arrow).

We next asked whether SLE serum antibodies could also cause neuronal cell death. Antibodies from four lupus patients were isolated using the consensus peptide sequence in affinity chromatography. Antibodies eluted from the column bound peptide and dsDNA in an ELISA and displayed anti-nuclear antibody activity on Hep2 cells (Fig. 3). Two preparations were injected into mice and caused hippocampal damage (Fig. 4). This damage was prevented by co-administration of MK-801. Thus, lupus sera contain antibodies to the NMDA receptors NR2a and NR2b that can cause neuronal death.

Figure 3: Human anti-peptide antibodies bind DNA.

a, Human anti-peptide antibody binds dsDNA. Anti-peptide antibody was purified from 1 ml serum, and diluted into 2 ml PBS. Significant response against the peptide and dsDNA was demonstrated for the anti-peptide antibody. ░, normal; ░, affinity purified. Data represent 4 sera. b–e, Human anti-peptide antibody has ANA activity. Hep2 cells were stained with the prototype SLE serum (b) and the affinity-purified anti-peptide antibody (d). DNA was stained with the fluorochrome DAPI (c and e). Both sera label nuclei and chromosomes in dividing cells. Unfractionated human serum also displayed nuclear speckles and faint cytoplasmic staining.

Figure 4: Human anti-peptide antibody causes neuron death.

Photomicrograph (cresyl violet, left panel, ×5) depicts focal region of neuron loss (asterisk and inset, ×40) in mouse hippocampus 2 d after intracerebral injection of affinity-purified human antibody. Exposure to the affinity-purified human antibody caused focal neuron loss comparable to R4A or Fab′2 fragments. MK-801 co-administration (middle, ×5; inset, ×40) demonstrates protection of the CA1 hippocampal neurons. Human antibody control did not destroy CA1 hippocampal neurons (right panel, ×5; inset, ×40). Data represent 4 mice.

Neuronal death occurs by apoptosis

To understand the mechanism of neuronal cell death, we treated primary fetal neuronal cultures in vitro with R4A antibody or affinity-purified human antibodies. All four preparations of both R4A and human antibodies caused apoptotic cell death of primary neurons as evidenced by activation of caspase 3. These neurons were previously shown to be susceptible to cytokine-mediated apoptosis (Fig. 5).

Figure 5: R4A and human anti-peptide antibody causes apoptosis in primary neuron cultures.

a–d, Apoptotic cells were detected when cell cultures were treated with R4A (a and b; immunofluorescence and phase), but not with IgG2b (c and d immunofluorescence and phase). e–h, Both R4A and human affinity-purified antibodies caused apoptosis (e and g), and co-administration of R4A with MK-801 (f) or soluble R4A peptide (h) was protective. Data represent 4 sera.

Finally, we obtained cerebrospinal fluid (CSF) of a patient with SLE who had experienced progressive cognitive decline. This CSF bound both DNA and the consensus peptide in an ELISA and stained nuclei of Hep2 cells in an ANA assay (Fig. 6). Notably, CSF injected into mouse brain caused neuronal death in vivo and caused death of primary neurons in vitro (Fig. 6).

Figure 6: Lupus CSF has anti-peptide, anti-DNA and ANA activity.

a, Undiluted CSF from a lupus patient (░) but not from a non-lupus control (░) displayed anti-peptide and anti-DNA activity. b, Serum (bottom; 1:200 dilution) and undiluted CSF (top) of the lupus patient displayed nuclear staining of Hep2 cells. (Magnification, ×100) c, Lupus CSF, (left panel, cresyl violet, ×5, asterisk reflected in inset ×40) but not non-lupus CSF, (right panel, cresyl violet, ×5, asterisk reflected in inset, ×40) caused neuronal death in mouse hippocampus. d, Lupus CSF (top panels), but not normal CSF (bottom panels), caused apoptosis of primary fetal neurons, as evidenced by antibody staining of activated caspase (left, immunofluorescence; right, phase; Magnification, ×40)


The mechanism of the CNS disease in SLE is poorly understood. Thrombosis and vasculitis are responsible for catastrophic focal brain injury in approximately 20–30% of SLE patients. However, a larger number of patients display neuropsychiatric symptoms and cognitive decline. Previous studies of lupus sera have demonstrated the existence of anti-neuronal antibodies in patients with CNS disease31. Antibody specificity for neurofilaments and for a 50-kD protein present in the membrane of synaptic terminals has been identified32,33; however, there has been no demonstration of a functional consequence to this binding. Studies have correlated psychosis in SLE with the presence of antibodies to ribosomal P protein34. This correlation remains controversial and there is no mechanistic hypothesis for this possible clinical correlation. Thus, the pathogenesis of CNS disease in lupus has remained elusive.

Anti-neuronal antibodies exist in several neuropathological conditions. In the peripheral nervous system, there is substantial evidence that antibodies can mediate neuronal dysfunction35; whether they are pathogenic in the CNS remains controversial. Antibodies to another NMDA receptor, NR3, are found in patients with Rasmussen encephalitis36,37,38, but whether these antibodies contribute to the seizure disorder is still unresolved. Antibodies to neurons occur in paraneoplastic syndromes, and it is likely that the loss of these neurons underlies the CNS symptoms experienced by patients39. Finally, antibodies are present in white-matter lesions of multiple sclerosis40. Their potential role in pathogenesis needs to be further explored, although it is intriguing that one recent study demonstrated anti-DNA antibody in CSF and brain tissue of patients with multiple sclerosis41.

Our data demonstrate that a subset of anti-DNA antibodies cross-react with NMDA receptors and can signal neuronal death through an excitotoxic mechanism. Moreover, the data show that such antibodies can be present in the CSF of patients with lupus. Thus, some antibodies to NR2 function as agonists; some may also function as receptor antagonists like phenylcyclohexylpiperidine and meditate lupus psychosis25,26,27. It is not yet known whether the antibodies are produced in situ in the brain or cross the blood–brain barrier42. There is evidence for both possibilities. In either case, these data provide the rationale for longitudinal studies of anti-peptide reactivity and non-focal CNS disease in SLE, and suggest a strategy for neuroprotection.

We suggest that reactivity with the glutamate receptor may also underlie other clinical manifestations of SLE. Recent studies show that NMDA receptors, including NR2, are expressed on multiple cell types and on platelets43,44. Thus, the glutamate receptor might provide a general target for antibody-mediated damage in SLE. Furthermore, receptors on such non-neuronal tissues may act as initial triggers for these autoantibodies.


Immunoprecipitation of the glutamate receptor.

PC12 cells were lysed in NP-40 lysis buffer, in the presence of protease inhibitors: (50 μg/ml aprotinin, 10 μg/ml leupeptin, 50 μg/ml pepstatin A, and 1 mM PMSF). Glutamate receptors from PC12 cells were precipitated with 8 μl of rabbit anti-Glu 2/3 (Chemic-Con, Temecula, California) bound to 40 μl of protein A-Sepharose (50% suspension; Pharmacia, Uppsala, Sweden). After an overnight incubation, immunoprecipitates were washed. Bound proteins were eluted with SDS sample buffer, electrophoresed on a 10% SDS gel and transferred to nitrocellulose. The nitrocellulose blot was stained with R4A or IgG2b control antibody (Sigma) and peroxidase labeled anti-mouse IgG (Southern Biotechnology, Birmingham, Alabama) or rabbit anti-Glu R 2/3 and peroxidase labeled anti-rabbit IgG (Southern Biotechnology) and developed by chemiluminescence (ECL Plus Amersham, Buckinghamshire, UK).

ELISAs and ANA assay.

SLE serum was affinity-purified on a DWEYSVWLSN peptide-conjugated sepharose column (Pharmacia). Serum was incubated with gel overnight at 4°C. The bound fraction was eluted with 5% acetic acid, 0.5% NaCl, neutralized to pH 7 1M Tris (pH 8.5) and dialyzed overnight against PBS.

The anti-peptide antibody ELISA was performed as described14. Affinity-purified antibody was used at 1 μg/ml. Normal serum was used at a 1:1000 dilution. SLE and normal CSF were used undiluted.

The ELISA assay for dsDNA binding was performed as described14. Hep2 slides (Bion, Park Ridge, Illinois) were incubated with affinity-purified antibody or lupus serum (1:200) followed by FITC-labeled goat anti-human IgG (Jackson ImmunoResearch, West Grove, Pennsylvania). These assays were also performed with undiluted CSF of a patient meeting ACR criteria for SLE (ref. 45) who exhibited decline in intellectual function over a 10-year period and control CSF from a non-lupus patient with migraine headaches.


Adult male C57BL/6 mice (30–34 g) were anesthetized with 2.5% Avertin (i.p.) and placed in a stereotaxic frame46,47. These procedures were in compliance with the Animal Welfare Act, the Public Health Service Policy on Humane Care and Use of Laboratory Animals, the US Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training, the NIH Guide for the Use and Care of Laboratory Animals and the RARC Users Guide. Experimental mice received a unilateral cortical injection of mouse R4A antibody (IgG2b; 1 μg in 3 μl sterile 0.1 M PBS and were killed at 1 d post-lesion (d.p.l.) (n = 4), or 2 d.p.l. (n = 7). Five control mice were injected with comparable amounts of normal mouse IgG2b antibody (Sigma), and killed in tandem with the experimental groups. Stereotaxic coordinates for cortex were A/P, –0.7 mm; Lat, 0.5 mm; D/V, –0.6 mm. The same protocol was used to inject the hippocampus with R4A (5 μg, n = 4), R4A Fab′2 fragments (4.5 μg, n = 3), affinity-purified human SLE antibody (5 μg; n = 4) and undiluted CSF of a lupus patient (n = 3). Control mice were injected with either normal mouse IgG2b (n = 3) normal human IgG (n = 7) (Sigma) or non-lupus CSF (n = 3). Stereotaxic coordinates for hippocampi were A/P, –2.0mm; Lateral, –1.3 mm; D/V, –1.1 mm and –1.4. These mice were killed 2 d after injection. In all control mice, damage was limited to injection trauma. MK-801 treatment in 3 mice followed past protocols47. The MK-801 was begun 2 h after R4A injection (5 mg/kg, i.p.) and continued bid until mice were killed.


Brains were fixed with 4% paraformaldehyde, following replacement of brain circulation with heparanized normal saline. Brains were removed, post-fixed for 1 h, and infiltrated with 30% sucrose overnight at 4 °C. A 3-mm cortical block containing the injection site was isolated in a brain mold and sectioned at 40 μm on a freezing stage microtome. Sections were collected in 0.1 M PB (pH 7.4). Every fourth section was mounted in 0.05 M PB on gelatin-coated slides, air-dried and stained with cresyl violet. Alternate sections were immunostained.


For identification of chromatin clumps in cortical neurons, sections were washed in 0.1 M PBS and permeabilized for 10 min with 0.1% Triton X-100 in PBS containing 1% BSA. Following PBS rinses, non-specific binding sites were blocked with 1% BSA in PBS for 45 min, and then incubated overnight at 4 °C with rabbit anti–l-glutamate antibody (1:250; Sigma). After 3 rinses as above, sections were incubated for 1 h with Texas-Red–conjugated donkey anti-rabbit IgG diluted in 0.1 M PB (1:200; Jackson Labs, West Grove, Pennsylvania). Preparations were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI, 1 μg/ml; Sigma) for 30 min, extensively rinsed, mounted and coverslipped with Cytoseal (Stephens Scientific, Kalamazoo, Michigan). For cell-type identification, the ABC detection method (Vector Labs, Burlingame, California) was used as reported48. Neuronal markers were mouse anti-Neu-N (1:1000; Chemicon, Temecula, California) and mouse anti-MAP2 (1:500; Sigma). Other antibodies included astrocyte rabbit anti-glial fibrillary acidic protein (GFAP, 1:5000; DAKO, Carpinteria, California) and biotinylated anti-B220 (T cell; 1:100), anti-CD3 (B cell; 1:100) and anti-isolectin (microglia; 1:100; Sigma).

Apoptosis of primary fetal neurons.

Human mixed neuronal-astroglia cultures were propagated as described49 and replated in 96-well flat-bottom tissue culture plates (Falcon, Franklin Lanes, New Jersey) for immunohistochemistry and on glass cover slips (Corning) for immunofluorescence.

Cells were pre-incubated for 12–16 h at 37 °C with R4A (10 μg/ml) or IgG2b (10 μg/ml) or undiluted CSF. Peptide in an octameric form (5 μg/ml) and MK-801 (200 μg/ml) were used in inhibition experiments.

For immunofluorescence, cover slips were rinsed briefly with ice-cold PBS and fixed at 12 °C with methanol (5 min) and acetone (2 min). Cover slips were incubated with polyclonal rabbit antibody to activated caspase at 25 °C for 1 h, and then with Cy3-conjugated goat anti-rabbit antibodies (1:5000, Jackson ImmunoResearch), prior to mounting on slides with Slow-Fade Light Antifade kit (Molecular Probes, Eugene, Oregon).

For immunohistochemistry, cells were fixed with ice-cold methanol for 30 min, rinsed with PBS and blocked with 5% NGS, 1% BSA, 0.05% Triton X-100 for 1 h. Anti-caspase antibody was applied for 1 h (1:5000, Idun Pharmaceuticals, La Jolla, California) in blocking buffer. Secondary antibodies were peroxidase-conjugated goat anti-rabbit IgG (1:5000, Southern Biotechnology). Diaminobenzidine was used as chromogen substrate (DAB, Vector Laboratories).


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We thank I. Joshi and W. Shen for technical help. This work was supported by grants from the National Institutes of Health, the SLE Foundation and the American Heart Association.

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Correspondence to Betty Diamond.

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