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Autoantibodies associated with neuropsychiatric systemic lupus erythematosus: the quest for symptom-specific biomarkers

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that affects multiple organs, including the central nervous system. Neuropsychiatric SLE (NPSLE) is a severe and potentially fatal condition. Several factors including autoantibodies have been implicated in the pathogenesis of NPSLE. However, definitive biomarkers of NPSLE are yet to be identified owing to the complexity of this disease. This is a major barrier to accurate and timely diagnosis of NPSLE. Studies have identified several autoantibodies associated with NPSLE;some of these autoantibodies are well investigated and regarded as symptom-specific. In this review, we discuss recent advances in our understanding of the manifestations and pathogenesis of NPSLE. In addition, we describe representative symptom-specific autoantibodies that are considered to be closely associated with the pathogenesis of NPSLE.


Introduction
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that affects multiple organs, including the central nervous system (CNS) 1) . SLE is characterized by the loss of immune tolerance to nuclear antigens due to development of autoantibodies and immune complex formation ; the resultant complement activation results in cell destruction and tissue injury 1,2) . Patients with SLE show considerable variability with respect to symptoms. In particular, neuropsychiatric (NP) SLE is a severe and potentially fatal condition that is characterized by CNS manifestations 1,2) . In 1999, the American College of Rheumatology Research Committee developed standard nomenclature and case definitions for 19 manifestations of NPSLE (Table  1) 3) . NP manifestations usually occur in the early stage of SLE and 39% -50% of patients exhibit SLE symptoms. The reported prevalence of NPSLE varies widely from 4% -91% ; this may be attributable to variability in clinical presentations, different selection criteria, and heterogeneity among study populations 4 -6) . In a recent 3 -year prospective study of 370 SLE patients with no history of CNS involvement (excluding nonspecific minor CNS complaints and peripheral nervous system symptoms), the prevalence of major CNS events was 4.3% with an estimated incidence of 7.8 events/100 personyears 7) . In spite of recent advances, the diagnosis of NPSLE is typically challenging, due in part to the absence of specific and reliable laboratory or imaging biomarkers 2,8) . The diagnosis of NPSLE requires exclusion of other causes such as infection, concurrent disease, metabolic abnormalities, or drug adverse events 1,2,5,6,8) . Therefore, investigation of dis-2 S. Sato et al. easespecific or symptomspecific autoantibodies observed in NPSLE is a key imperative to facilitate timely and accurate diagnosis. Here, we review recent NPSLE studies that focused on the pathogenesis of NPSLE and the autoantibodies associated with NPSLE manifestations, especially symptomspecific autoantibodies.

Predisposing factors
Predisposing genetic factors are believed to play an important role in SLE pathogenesis 2,6) . In NPSLE patients, mutations in TREX1, which encodes threeprime repair exonuclease 1 (DNAse III), were shown to be associated with neurological involvement. Polymorphisms in TREX1 were found to be associated with neurological involvement in European patients with SLE 10) . Lossof function mutations in TREX1 augment the production of type 1 interferons in mice and lead to earlyonset cerebral NPSLE 11,12) . In addition, HLA -DRB1*04 and rs10181656(G) alleles were shown to be associated with ischemic cardiovascular disease (CVD) in Caucasian patients with SLE 13,14) . More recently, Rullo et al. reviewed recent advances in our understanding of the genetic basis of SLE, including genetic variants in recently identified SLEassociated loci, the immunological pathways affected by these gene products, and the disease manifestations linked to these loci 15) . In addition, environmental factors and neuroendocrine factors play an important role in the development of SLE. In previous studies, silica exposure, smoking, oral contraceptives, postmenopausal hormone therapy, and endometriosis were found to be risk factors for SLE 16) . Indeed, sex steroid hormones (17β -estradiol, testosterone, prolactin, progesterone, dehydroepiandrosterone) reportedly impact the immune response and the severity of disease in SLE ; this also explains the sex disparity in SLE. Other environmental factors, such as ultraviolet radiation, vitamin D, infection (e.g., Epstein -Barr virus), vaccination, heavy metals, solvents, and pesticides are considered as risk factors for SLE 6,16) .

Mechanisms of NPSLE
Recent reports have identified two major mechanisms for the development of NPSLE, i.e., vascular mechanisms and neuroinflammatory mechanisms ( Figure 1) 1,2,6) . Among the vascular mechanisms, vasculopathy is implicated in CNS damage in patients with NPSLE ; autopsy studies have shown pathological findings of multifocal microinfarcts, smallvessel noninflammatory vasculopathy and occlusion, embolism, cortical atrophy, and microhemorrhages 1,17,18) . Antiphospholipid antibodies (aPL) and deposition of immune complexes are likely to be associated with these conditions 1,18 -20) . Injury to large and small blood vessels mediated by aPL initiates vascular damage, finally resulting in focal, and in part, diffuse neuropsychiatric events (seizures, cognitive dysfunction, etc.). The second mechanism involves autoimmune inflammation mediated by autoantibodies, resulting in increased permeability of the BBB, intrathecal formation of immune complexes, and production of inflammatory mediators (IFNα, IL -6, IL -8, IP -10, MCP -1, etc.) 20,21) . Direct CNS tissue injury caused by excitatory amino acid toxicity, oxidative stress, plasminogen activator in-  1), and matrix metalloproteinase 9 (MMP9) activity have also been suggested 20,22,23) . These processes can cause CNS damage by activation of microglial cells and induction of neuronal cell death by apoptosis 1,6) , leading to mainly diffuse NSPLE symptoms, such as acute confusional state and psychosis 1,20) . As previously described, BBB dysfunction plays an important role in the pathogenesis of NSPLE. Normally, the brain is immunologically privileged and is sheltered from foreign substances in the circulation. The BBB limits the entry of soluble molecules and cells into brain parenchyma and regulates both uptake into and efflux out of the brain 1,20,24) . Although the precise mechanism of BBB dysfunction is still unclear, permeability of the BBB can be affected by both SLE factors (immune complex deposition, cytokine/chemokines) and non -SLE factors (smoking or hypertension) that induce endothelial dysfunction in brain vasculature 1,8,20,25) . In this regard, autoantibodies reacting with neuronal cells or those that have been reported as specific for each NPSLE symptom (from the circulation or intrathecal production) might be associated with BBB dysfunction. Here, we review the representative autoantibodies that are potentially associated with the pathogenesis of NPSLE. Table 2 shows the representative autoantibodies that have been recently described as potentially associated with NPSLE pathogenesis. More than 100 autoantibodies have been described in patients with SLE or NPSLE 26) ; however, none of these have been definitively implicated in the complex process of NPSLE pathogenesis. Therefore, extensive research is ongoing to establish distinct pathogenic roles for each autoantibody.

Antiphospholipid antibodies (aPL)
The aPL antibody family targets proteins associated with anionic phospholipids in the plasma membrane that regulate the blood clotting cascade ; subsequent activation of procoagulants promotes thrombosis and cerebral infarction 27) . Anticardiolipin (aCL), anti -β2GP1 antibodies, and lupus anticoagulant (LAC) are the most widely investigated autoantibodies targeting phospholipids. These have been recognized as major risk factors for NPSLE and are believed to contribute to the development of thrombosis and other NPSLE symptoms, such as seizures, stroke, chorea, movement disor- ders, cognitive dysfunction, and myelopathy 28 -32) .
In vitro studies have demonstrated direct binding of aPL with CNS cells ; in addition, intrathecal passive transfer of IgG isolated from aPLpositive patients was shown to induce cognitive dysfunction in mice 33) . At least, exacerbation of procoagulant state by aPL is believed to be associated with focal NPSLE causing intravascular thrombosis and cerebral ischemia 20) .

Antiribosomal P protein antibodies (antiribo P)
Antiribo P are specific autoantibodies occurring in up to 46% of patients ; target epitopes are located in the Cterminal end of three highly conserved phosphorylated proteins, P0, P1, and P2, which are present in the 60S subunit of ribosomes 6,34) . Many retrospective studies have suggested an association between elevated serum or CSF levels of antiribo P and NPSLE manifestations ; however, the results have been contested 35 -37) . Recent longitudinal studies and prospective studies have shown their association with lupus psychosis 38 -41) . In a study by Hanly et al., antiribo P was found to be a predictor of psychosis 42) . In a mouse model, antiribo P recognized neurons in the hippocampus, cingulate, and primary olfactory piriform cortex, and induced longterm depressivelike behavior when introduced into cerebral ventricles 43) . Matus et al. reported that antiribo P from psychiatric lupus induced a rapid and sustained in-crease in calcium reflux and apoptosis in rat neurons expressing cellsurface Pantigen protein P331. The death of these neurons in specific brain regions (such as hippocampus) was found to affect the memory and emotional behavior of rats 44) . Direct evidence of a pathogenic role for these antibodies in humans is still lacking ; nevertheless, experimental data and prospective studies support the role of antiribo P in the causation of diffuse NPSLE.

Anti -Nmethyl -Daspartate receptor antibodies (anti -NMDA)
Anti -NMDA antibodies occur in 30% -40% of patients with SLE ; these have been demonstrated as a subset of doublestranded DNA (dsDNA) antibodies that crossreact with NMDA receptors, specifically with the NMDA receptor subunit 2 (NR2) 45) . NMDA receptors are widely distributed in the brain and localized within glutamatergic synapses ; a particularly high density is observed in the amygdala and hippocampus, which modulate cognitive function, emotional processes, and memory 46,47) . Activation of NMDA receptors is critical in learning and memory ; however, prolonged stimulation can cause apoptotic death of neuronal cells. The potential pathogenic role of anti -NMDA detected in CSF has been demonstrated in both in vitro and in vivo studies 48,49) . Interestingly, no neuronal damage was observed if the BBB remained intact ; however, several pathological changes were detected when the  46 -48) . Correlation between CSF anti -NMDA and diffuse NPSLE manifestations has also been reported 50) . Furthermore, it has been suggested that anti -NR2/dsDNA antibodies may also help distinguish SLE patients with central diffuse NPSLE manifestations from patients with peripheral manifestations ; the pathogenic factors and mechanisms underlying these manifestations are probably different 51) . Then, how do these autoantibodies gain access to the brain in SLE? These results indicate that activation of BBB endothelial cells by anti -NMDA may cause inflammation, disrupt the BBB, and promote entry of autoantibodies into the CSF in patients with SLE.

Antimicrotubuleassociated protein 2 antibodies (anti -MAP -2)
MAP -2 is one of the abundant groups of cytoskeletal components predominantly expressed in neurons 54) . MAP -2 regulates the nucleation and stabilization of microtubules, organelle transport protein kinases that are involved in signal transduction 55) . In a study by Yamada et al., 33.3% of NPSLE patients tested positive for anti -MAP -2 in the CSF 54) . Williams also reported that 17% of SLE patients had anti -MAP -2 in contrast to 4% of neurologic injury/disease control patients 56) . Patients who tested positive for anti -MAP -2 exhibited neuropsychiatric symptoms (psychosis, seizures, neuropathy, and cerebritis). Moreover, both antiribo P titers and IL -6 levels in CSF were significantly higher in NPSLE patients having anti -MAP -2, indicating some association between them 54) .

Anti -U1 ribonucleoprotein antibodies (anti -U1RNP)
Anti -U1RNP reacts with proteins that are associated with U1 RNA and form U1 small nuclear ribonucleoprotein (snRNP) ; these are detectable in 25% -47% of SLE patients 57) . The snRNP are RNAprotein complexes that are abundant in the nucleus ; these are involved in the nuclear processing of pre -mRNA along with other proteins compris-ing the spliceosome 58) . In a study by Sato et al., anti -U1RNP in CSF, but not that in serum, was associated with NPSLE and mixed connective tissue disease 59) . Recent studies have shown that anti -RNP as well as anti -Sm antibodies were less likely to be produced within the CNS ; this was assessed by calculating anti -RNP and anti -Sm indices in the CSF (indices of each reflect the intrathecal production of these antibodies) 60) . Nevertheless, the detailed role of anti -U1RNP should be further investigated.

Antiendothelial cell antibodies (AECA)
AECAs target a heterogenous group of antigens including structural endothelial proteins (ranging from 10 to 200 kDa) as well as adhesion molecules to endothelial cells ; these are found in a variety of diseases that are characterized by vessel wall damage 61) . The reported prevalence of AECA in SLE patients ranges from 17% to 75%. Conti et al. found an association of serum AECA with psychosis and depression in NPSLE 62) . AECA can activate endothelial cells by inducing the expression of adhesion molecules (ICAM -1 and VCAM -1) and stimulating the production of cytokines (IL -1, IL -8) and chemokines [such as monocytechemotactic protein 1 (MCP -1)]. AECA can also enhance the production of tissue factor and von Willebrand factor, promoting thrombosis. However, low specificity due to the lack of a standardized detection method and possible presence of natural AECAs (seen in a small percentage of healthy individuals and showing low affinity for their target antigens) limit the use of AE-CAs as diagnostic and prognostic markers 61,62) .

Antitriosephosphate isomerase antibodies (anti -TPI)
Triosephosphate isomerase (TPI) is an important glycolytic enzyme that catalyzes the interconversion of dihydroxyacetone phosphate and Dglyceraldehyde -3 -phosphate 63) . TPI is mediated solely by glycolysis in red blood cells and in brain cells, and its deficiency is associated with hemolytic anemia and neurological disorders. TPI is involved in the stability of neuronal microtubules 63 -66) . Watanabe et al. have suggested some association with the pathogenesis of NPSLE 64 -66) . Furthermore, in our previous study, anti -TPI -TPI immune complex was detected in the CSF of anti -TPIpositive NPSLE patients 65) . In a lupus model mouse, anti -TPI was detected in MRL/lpr mice ; in addition, anti -TPI was shown to bind to brain tissue in the meninges, choroid plexus, hippocampus, and periventricular le-sions using anti -TPIproducing hybridoma inoculation into the brain hemisphere 66) . Recently, we reported a higher frequency of aseptic meningitis in anti -TPIpositive NPSLE patients ; in addition, serum anti -TPI index showed a positive correlation with serum IgG levels 8) . These results indicate that anti -TPI may be associated with NPSLE, mainly focal NPSLE (aseptic meningitis) ; the underlying mechanism may involve disruption of the BBB via formation of immune complexes in the CNS. Indeed, the underlying mechanism of aseptic meningitis in SLE patients is still unclear. A possible mechanism can be considered along with meningeal inflammation similar to that proposed for multiple sclerosis 67) : immune cells first pass through the meninges via the bloodstream to the choroid plexus, as a part of immune surveillance. The interaction of autoimmune myelinspecific T cells with myelinloaded antigenpresenting cells can induce T cell reactivation and production of inflammatory cytokines/ chemokines in the meninges. The additional inflammatory cells compromise local BBB integrity, which finally triggers immune cell infiltration into the CNS 8,67) . In any case, further investigation is needed to clarify the pathogenic role of anti -TPI in NPSLE.

Antiglyceraldehyde -3 -phosphate dehydrogenase antibodies (anti -GAPDH)
GAPDH is a glycolytic enzyme ; however, more recent evidence indicates that mammalian GAPDH performs a number of other functions 68) . Its activity contributes to membrane fusion, microtubule bundling, phosphotransferase activity, nuclear RNA export, DNA replication and repair, and further, to neuronal cell death 68,69) . Takasaki et al. first reported that anti -GAPDH is one of the elements of proliferating cell nuclear antigens, specifically those reactive with serum from SLE patients 69) . Furthermore, Delunardo et al. demonstrated the reaction of anti -GAPDH with neuronal cells and its association with cognitive dysfunction in patients with SLE 70) . Recently, Sun et al. also reported a positive correlation of serum anti -GAPDH with SLEDAI -2K, ESR, IgG, and IgM ; in addition, anti -GAPDH showed an association with increased intracranial pressure, indicating its potential role in the induction of brain tissue damage 71) . Although the direct effect of anti -GAPDH is yet to be elucidated, it may serve as a useful biomarker for cerebrovascular damage in NPSLE patients.

Conclusion
Neuropsychiatric sequelae are among the main causes of morbidity and mortality in patients with SLE, but they are the least wellunderstood aspect of the disease. Appropriate evaluation and accurate classification of NP manifestations is an important focus of NPSLE treatment and research. However, its complex pathogenesis and polymorphic phenotype hampers the identification of pertinent, robust biomarkers. Further investigation of biomarkers (such as diseasespecific or symptomspecific autoantibodies and cytokine/chemokine expression profiles) is required to increase our knowledge and improve the management of NPSLE.