Autoantibodies in lupus: Culprits or passive bystanders?

Autoimmunity Reviews 11 (2012) 596–603

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Review

Autoantibodies in lupus: Culprits or passive bystanders?☆,☆☆ Ole P. Rekvig a, b, Chaim Putterman c, d, Cinzia Casu e, Hua-Xin Gao c, Anna Ghirardello f, Elin S. Mortensen a, b, Angela Tincani e, Andrea Doria f,⁎ a

Molecular Pathology Research Group, Institute of Medical Biology, University of Tromsø, N-9037 Tromsø, Norway Trombosis and Vascular Biology Research Group, Institute of Medical Biology, University of Tromsø, N-9037 Tromsø, Norway The Department of Microbiology & Immunology, Albert Einstein College of Medicine, Bronx, NY, USA d Division of Rheumatology, Albert Einstein College of Medicine, Bronx, NY, USA e Rheumatology and Clinical Immunology, Spedali Civili and University of Brescia, Brescia, Italy f Division of Rheumatology, Department of Clinical and Experimental Medicine, University of Padova, Padova, Italy b c

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Available online 25 October 2011 Keywords: Systemic lupus erythematosus Autoantibodies Pathogenesis Lupus nephritis Neuropsychiatric SLE

a b s t r a c t Several autoantibodies are culprits in the pathogenesis of organ damage in systemic lupus erythematosus, by means of established or postulated mechanisms, whereby inducing a perturbation of cell structure and function, with consequent tissue–organ impairment. Common autoantibody-mediated mechanisms of damage include cell surface binding with or without cytolysis, immune complex-mediated damage, penetration into living cells, binding to cross-reactive extracellular molecules. Experimental data from both murine models and humans have recently clarified the key role of autoantibodies in severe organ involvements, including nephritis, neuropsychiatric (NP) dysfunction, and cerebrovascular disease (CVD). In lupus nephritis early and late phases are distinguishable and mediated by different processes in which antichromatin antibodies are both inducing and perpetuating agents, by immune-complex formation and massive deposition in mesangial matrix at first, and in glomerular basement membrane at end-stage. Also NP abnormalities occur very early, much earlier than other systemic manifestations, and exacerbate with the increase in autoantibody titers. Among the autoantibodies mainly implicated in neurolupus, anti-β2 glycoprotein I (β2GPI) antibodies are preferentially involved in focal NP events which are a consequence of noninflammatory microangiopathy; otherwise, anti-ribosomal P protein antibodies and N-methyl-D-aspartate receptor (NMDAR) antibodies cause diffuse NP events through a direct cytotoxic effect on neuronal cells at specific brain zones. © 2011 Elsevier B.V. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anti-dsDNA antibodies are really pathogenic — lesson from lupus nephritis. . . . . . . . . . . . . . 2.1. Murine lupus nephritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. The role of anti-chromatin antibodies, renal DNaseI, chromatin fragments, and (MMP) . 2.1.2. Lupus nephritis — step 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3. Lupus nephritis — step 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Human lupus nephritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Autoantibodies and neuropsychiatric systemic lupus erythematosus: lessons from murine lupus . . . . Are anti-ribosomal P protein antibodies associated with NPSLE? . . . . . . . . . . . . . . . . . . . Autoantibodies in neurolupus: pathogenic role of anti-β2GPI . . . . . . . . . . . . . . . . . . . . 5.1. Inhibition of natural anticoagulants and fibrinolysis systems . . . . . . . . . . . . . . . . .

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☆ Grant supports: Ole P Rekvig's study was supported by grants from Northern Norway Regional Health Authority Medical Research Program (Grant #s SFP-100-04, SFP-101-04), and from University of Tromsø as a Milieu support given to OPR. Chaim Putterman's study was funded in part by NIH, grants AR48692 and DK90319. ☆☆ The authors declare no conflict of interest. ⁎ Corresponding author at: Division of Rheumatology, University of Padova, Via Giustiniani, 2, 35128 PADOVA, Italy. Tel.: + 39 049 8212190; fax: + 39 049 8212191. E-mail address: [email protected] (A. Doria). 1568-9972/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.autrev.2011.10.021

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5.2. Interaction with cells involved in coagulation process 5.3. Direct interaction of aPL with neuronal tissue . . . . 5.4. Pathogenic potential of anti-β2GPI antibodies . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . Take-home messages . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction The key relevance of autoantibodies in clinical assessment of systemic lupus erythematosus (SLE) is clearly established [1–3]; otherwise, more controversial is the experimental evidence of their direct pathogenetic role in the disease. Common autoantibody-mediated mechanisms of damage in SLE include immune complex-mediated damage, cell surface binding and cytotoxicity, reactivity with autoantigens expressed on apoptotic or activated cell surface, penetration into living cells, binding to crossreactive extracellular molecules (Table 1) [4]. Over the past decade major insights on the mechanisms whereby certain autoantibodies may contribute to glomerulonephritis and central nervous system (CNS) manifestations in SLE have been yielded from both murine and human lupus. In this paper, a summary of remarkable experimental evidence about the central role of autoantibody-mediated mechanisms in the pathogenesis of nephritis, CNS disease and antiphospholipid syndrome (APS) in SLE is elegantly reported by the Authors, who also gave their personal contribution to the debate. Mortensen and Rekvig described intriguing mechanistic models whereby anti-dsDNA antibodies are really pathogenic in lupus nephritis. Nephritis in SLE is caused by in situ interaction between autoantibodies to chromatin components, primarily dsDNA and nucleosomes, and glomerular cross-reactive extracellular structures. Both in murine and human lupus, early and late phases in nephritis can be distinguished and may be sustained by different processes, to which anti-chromatin antibodies participate as both inducing and perpetuating agents. Among the autoantibodies involved in the pathogenesis of neuropsychiatric SLE (NPSLE), those directed against specific neuronal molecules such as N-methyl-D-aspartate receptor (NMDAR), or targeting ubiquitary expressed intracellular antigens like ribosomal P proteins, are mainly implicated in diffuse NP events, through a direct effect on neuronal cells at specific brain zones. Otherwise, focal NP events are consequent to non-inflammatory microangiopathy, in which antiphospholipid antibodies, primarily anti-β2 glycoprotein I (β2GPI), are mainly involved.

Table 1 Major pathogenetic mechanisms of action of autoantibodies in systemic lupus erythematosus. 1. Circulating and in situ immune complexes formation, deposition in target organs, complement activation and inflammation • Anti-dsDNA antibodies • Anti-nucleosome antibodies 2. Cell surface binding and cytolysis or cytotoxicity • Anti-Ro/La antibodies • Anti-P ribosomal antibodies • Antilymphocyte antibodies • Antierythrocyte antibodies • Antiphospholipid antibodies 3. Penetration into living cells, induction of cell dysfunction and apoptosis • Anti-dsDNA antibodies • Anti-U1RNP antibodies 4. Binding to cross-reactive extracellular molecules, e.g., heparan sulfate, fragmented chromatin • Anti-chromatin antibodies • Anti-dsDNA antibodies • Antiphospholipid antibodies

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2. Anti-dsDNA antibodies are really pathogenic — lesson from lupus nephritis Lupus nephritis is caused by in situ interaction of two partners, autoantibodies to chromatin components, and exposed glomerular chromatin fragments [5]. Until recently, the basic processes accounting for lupus nephritis, were poorly understood, and conflicting models were developed [6–8]. Here, we discuss recent data obtained in our laboratory on central processes involved in lupus nephritis. 2.1. Murine lupus nephritis Substantial data have been provided during recent years related to a) how anti-dsDNA antibodies exert their clinical impact through interaction with DNA or nucleosomes, and b) description of the coherent nature of their glomerular target structures, described by quite different cutting-edge techniques (reviewed in [6]). 2.1.1. The role of anti-chromatin antibodies, renal DNaseI, chromatin fragments, and (MMP) Anti-DNA antibodies, renal DNaseI and matrix metalloprotease (MMP) enzyme activities are co-operative, interdependent and instrumental in early and late murine and human lupus nephritis [5,6,9]. However, early (step 1) and late (step 2) nephritis, although linked in a common destiny, appear from different processes. 2.1.2. Lupus nephritis — step 1 Early phases of nephritis are associated with chromatin–IgG complex deposition in the mesangial matrix. This event is basically imposed by production of anti-dsDNA (or anti-chromatin) antibodies, and by their interaction with exposed nucleosomes [10] (Fig. 1A). How these complexes reach the mesangial matrix is not clear. Mesangial cells have, however, Fcγ-receptors, meaning that they may trap chromatin–IgG complexes by these receptors. Continuous binding of immune complexes to mesangial cells may reduce their ability to engulf them, and rather transfer them to the matrix synthesized by these cells. When mesangial cells are involved in the process of binding immune complexes, they increase their matrix production. This will inevitably lead to increased binding of the immune complexes, and mild or silent mesangial nephritis is established when immune complexes activate complement and invading macrophages and granulocytes. Mesangial nephritis in the Black/White mouse always proceeds into end-stage nephritis [10,11]. This may not always be the case in human nephritis, but this is not clear in the human form since relevant studies of clinically silent kidneys are regarded unethical due to the potential procedural hazards to sample biopsies from apparently healthy (silent nephritic!) kidneys. Thus, to study disease progression, we are limited to animal studies, while we have described post-mesangial nephritis in both mice and humans [12,13]. 2.1.3. Lupus nephritis — step 2 Recent results demonstrate that DNaseI, the major renal nuclease [14], are profoundly down-regulated when mesangial nephritis is established [10,13]. We have never seen DNaseI gene shut-down prior to or during the process of mesangial nephritis. Rather, reduced DNaseI enzyme activity was strictly linked to events following immune complex deposits in glomerular basement membranes (GBM).

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death, since other renal nucleases and apoptosis-related genes were transcribed at levels similar to those observed in young pre-diseased Black/White kidneys and in age- and sex-matched BALB/c mice [11,13]. Renal DNaseI shut-down caused the transformation of mild nephritis into severe membrano-proliferative end-stage nephritis [10]. With low DNaseI enzyme activity, apoptotic chromatin will not be fragmented and will instead be transformed into secondary necrotic chromatin unmasked from apoptotic blebs [15]. Secondary to this, chromatin is exposed to the environment where it binds glomerular capillary membranes and the mesangial matrix [16] as well as skin membranes at high affinity [17]. Thus, renal DNaseI shut-down promotes massive chromatin exposure and enhances the pathogenic impact of anti-dsDNA antibodies. This is definitively determined in murine and human lupus nephritis [10,13]. Another important event in disease progression, important for development of full-blown nephritis, is executed when chromatin fragments are taken up by macrophages and dendritic cells. In these cells they bind Toll-like receptors (TLRs) [18]. Chromatin-derived peptides are subsequently presented to CD4+ T cells in the context of HLA class II molecules. The engagement of TLR serves two important functions: up-regulation of co-stimulatory molecules (CD80/CD86 or also called B7 molecules) and up-regulation of certain metalloproteinases (MMPs). One prediction was therefore that loss of renal DNaseI correlated with increased MMP activity in the kidneys. This prediction was proven true [10], and may explain two distinct features of murine lupus nephritis; generation of large chromatin fragments in the kidneys due to loss of renal DNaseI, and deposition of such fragments in glomerular basement membranes because of their disintegration due to the effect of MMPs. In this situation, anti-dsDNA antibodies exert their full pathogenic potential by binding such exposed chromatin fragments (Fig. 1B). Since these bind in GBM, this explains why anti-dsDNA antibodies gain a pathogenic potential, and how they exert it [5]. Indeed, these autoantibodies are really pathogenic, and they account for both early mesangial and end-stage lupus nephritis. Without exposed chromatin, anti-dsDNA antibodies remain nonpathogenic. In presence of chromatin exposed in glomeruli, the antibodies contribute in a direct and definitive way to initiation and progression of lupus nephritis. The interplay of the central partners involved in early and late nephritis is presented as a biplot in Fig. 2. 2.2. Human lupus nephritis

Fig. 1. Exposed, extracellular chromatin is a central factor in evolution of lupus nephritis — a model. Exposure of chromatin may have impact on the immune system. Chromatin may re-circulate as oligo-nucleosomes in this situation the antibodies are potentially pathogenic, and turn to be so if they are nucleosomes. Then they may initiate an early, mild mesangial nephritis (A). Progression of the glomerular disease depends on renal DNase1 gene shut-down, which has an immense impact on the pathogenic effect of the autoantibodies. Chromatin in cells dying from e.g. apoptosis may, due to loss of DNase1, not be degraded, and instead of clearance, they become exposed as secondary necrotic chromatin in e.g. glomerular membranes and to dendritic cells, where Toll like receptors are engaged and followed by increased expression of matrix metalloproteases. These enzymes may disintegrate membranes and matrices, and thereby open them for large chromatin fragment deposits (B). At the same time, exposed chromatin is targeted by induced anti-chromatin antibodies. Thus, chromatin fragments may exert 2 effects with fatal consequences for the kidneys: They may induce autoimmunity (nucleosomes), and they represent targets for the induced autoantibodies (chromatin fragments).

Thus, deficient DNaseI enzyme activity is a phenomenon that appeared secondary to mesangial nephritis, and is the factor that imposes endstage nephritis. Loss of renal DNaseI was not caused by massive cell

There are strong data allowing us to assume that the two-stepped process accounting for murine lupus nephritis is also relevant in human lupus nephritis [12,13,19]. Data from studies on biopsies from human cases of lupus nephritis at various stages of the disease, demonstrate that the glomerular deposits of immune complexes in the mesangial matrix and in GBM have the same composition and localization as those observed in the murine form [12,13,19]. In a study of 5 human cases with SLE and 4 normal controls, we also found that the DNaseI gene expression was severely down-regulated in patients with membranoproliferative lupus nephritis, as determined by Western blot analyses, immunohistochemistry and by quantitative PCR analyses (manuscript in preparation, [13]). In sum, these findings indicate that the mechanisms responsible for murine SLE also are pivotal in human nephritis. These data open for quite new therapeutic strategies aimed at interfering with basic disease mechanisms that will be even better understood in the near future [20]. 3. Autoantibodies and neuropsychiatric systemic lupus erythematosus: lessons from murine lupus Patients with SLE typically suffer from involvement of multiple organs, including kidney, joints, skin, and brain. Among these manifestations, NPSLE is one of most common causes of morbidity and

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Fig. 2. A principal component analysis (PCA) of parameters involved in early and late phases of lupus nephritis. This PCA biplot aims to optimally display variances and not correlations. The angles between the various biplot axes serve as good indicators of the correlations among the variables (shown as arrows). Similarly, the position of the samples of individual mice (shown as plus sign) relative to the arrows, provide good indications as to which variable(s) have had the largest effect. The result of the biplot demonstrates that groups emerging from the analysis perfectly correlated with groups of BW mice, defined as pre-nephritic BW mice (Group 1), BW mice with deposits of EDS in the mesangial matrix (Group 2) or with deposits in the GBM (Group 3). The circle identifies the mouse with the lowest renal DNase 1 mRNA level and enzyme activity, and the highest MMP2 and MMP9 mRNA levels and enzyme activities and with proteinuria. Taken from: Fenton K, Fismen S, et al. PloS One 2009; 4:e8474.

mortality, reported to affect a majority of lupus patients [21]. With recent progress in the treatment of lupus, some SLE manifestations such as nephritis are better controlled. However, the optimal therapy for NPSLE has yet to be determined. Adding to this difficulty is the fact that it can be challenging to determine whether NPSLE is a primary or secondary manifestation, since many patients with NPSLE have multiple other possible causes for NP symptoms, such as medications, hypertension, uremia, and infection [22]. Furthermore, as compared to several other aspects of disease, there has been relatively limited progress in understanding the pathogenesis of NPSLE. Studies using lupus mouse models provide great opportunity to shed light on some of these unanswered questions [23,24]. Although the pathogenesis of SLE is not fully understood, autoantibodies are believed to play an important role in target organ damage, including NPSLE as well [25–28]. Previous studies have shown that anti-ribosomal P antibodies injected intraventricularly to C3H mice induce depression and impaired smell, mediated by antibody binding to neurons in the olfactory and limbic areas [25]. In another study, direct injection into mouse brain of cross-reactive anti-double stranded (ds)DNA autoantibodies recognizing the N-methyl-D-aspartate receptor (NMDAR), causes neurotoxicity, cognition impairment and emotional behavior deficits [26]. Similar detrimental effects are induced when peripheral (intravenous) injection of anti-dsDNA antibodies follows permeabilization of the blood brain barrier [27]. Based upon the hypothesis that autoantibodies are of primary importance in the pathogenesis of NPSLE, we predicted that NP abnormalities would manifest early in lupus mice, becoming detectable once autoantibody titers begin to rise. Furthermore, we expected that gender-determined

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variations in autoantibody levels would translate into differential expression of NPSLE. In MRL/lpr mice, a well-characterized murine lupus model, we studied NPSLE-like manifestations in female lupus-prone mice using a battery of behavioral tests assessing cognition, emotion, as well as general locomotion. Age, gender, and background strain matched MRL/+ mice served as controls. While we found no evident abnormalities in locomotor activities, learning, and memory, MRL/lpr mice showed severe depression-like despair behavior in the forced swim test at 8 weeks of age, well before the onset of most other SLE manifestations [29]. However, while the observed depressive behavior was severe, female MRL/lpr mice did not exhibit anxiety, indicating that not all aspects of brain function are similarly affected. A significant increase in circulating ANA, anti-cardiolipin antibodies, and anti-NMDAR antibodies were detected in young MRL/lpr mice with emotional deficits. Furthermore, we found a strong correlation between autoantibody titers and depression-like behavior deficits both at this disease onset age as well in older mice when disease had progressed. Finally, as the development of NPSLE in MRL/lpr mice seemed to be independent of other organ involvement such as nephritis, NPSLE is apparently a primary SLE manifestation in this mouse model. Though MRI failed to detect any gross structural abnormalities in MRL/lpr mice, using magnetic resonance spectroscopic imaging (MRSI) we were able to detect metabolic changes in brains of mice exhibiting NPSLE. Significantly different levels of choline and Nacetyl aspartate were found in hippocampal and cortical regions, when compared to MRL/+ controls [29]. Since abnormal behavior in MRL/lpr mice was accompanied by changes in brain metabolism, if confirmed in humans our studies would suggest a possible role for non-invasive imaging such as MRSI in monitoring disease in patients with NPSLE. The strong female bias present in patients with SLE (9:1 female to male ratio) indicates a strong contribution of gender to disease pathogenesis [30]. A female bias holds true in MRL/lpr mice as well, with an earlier onset of disease and higher levels of autoantibodies found in female mice. However it was not clear whether NPSLE would follow this pattern as well. Thus, we investigated whether NPSLE is sex-dependent and develops earlier and/or more severely in females. In this study, both female and male MRL/lpr mice were behaviorally compared to the age- and sex-matched MRL/+ controls. We found that both male and female MRL/lpr mice have relatively normal locomotor activity, and visual and spatial memories. However, female mice showed severe depression-like behavior already at 5 weeks of age, a strikingly early time point for target organ involvement [31]. This behavioral deficit was not observed in the age-matched male MRL/lpr mice, which went on to develop similar symptoms by the time they were retested 13 weeks later. Interestingly, depressive female MRL/lpr mice were less anxious than the male MRL/lpr mice, or compared to MRL/+ female controls. Once again, autoantibody titers displayed a significant correlation with the severity of NPSLE deficits; depressive behavior was only observed at the time point when anti-dsDNA or anti-ribosomal P autoantibodies were significantly increased. These results indicate that NPSLE is a gender-specific manifestation, and can develop very early in the course of SLE with the rise in autoantibodies. Based on these studies, we conclude that NPSLE, especially depressive deficits, is an early SLE manifestation which is detectable once autoantibodies start to increase. ANA titers, anti-ribosomal P antibodies, anti-cardiolipin antibodies, and anti-NMDAR autoantibodies correlate with the severity of depression in lupus mice. Females are more susceptible to develop NPSLE than males, with an earlier rise in circulating autoantibodies. While these observations do support an active role for autoantibodies in NPSLE manifestations, whether increasing autoantibody titers can predict NPSLE involvement in human disease remains to be seen. Furthermore, NPSLE may present as a primary SLE

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manifestation, independent of other organ involvement in lupus. Our studies support clinical efforts for earlier diagnosis of NPSLE in lupus patients. Furthermore, it will be interesting to see whether targeted treatment of serological abnormalities may affect the severity of NPSLE in human disease. Finally, we believe that further investigation into the pathogenesis of NPSLE may provide novel and specific targets for therapeutic intervention.

4. Are anti-ribosomal P protein antibodies associated with NPSLE? Neuropsychiatric (NP) involvement is a common manifestation in SLE, occurring in up to 80% of the patients at any time during the disease course. Studies on NPSLE have been improved by the 1999 ACR definitions and guidelines for the 19 major NPSLE syndromes [32]. Some NP manifestations are common such as CVD, headache and seizures. Others are relatively uncommon or rare events such as psychosis or myelopathy [33]. Although the pathophysiology of NP manifestations is poorly understood, several pathogenic mechanisms are implicated and the most relevant are vasculopathy of intracranial vessels, autoantibodymediated tissue damage, and the local production of inflammatory mediators. Among the huge panel of autoantibodies implicated in NPSLE [34], those targeting the ubiquitous ribosomal phosphorylated (P) proteins are reported in association with peculiar NP manifestations attributed to SLE. Anti-ribosomal P protein antibodies may cause diffuse NP events, i.e. psychosis, depression, cognitive impairment, through a direct effect on neuronal cells. Anti-P antibodies are directed towards the three ribosomal P proteins (P0, P1, P2) located at the stalk of the large ribosomal subunit 60S [35]. The immunodominant epitope is localized to the carboxy terminal domain (C-22), shared by the three P proteins. Moreover, epitope mapping studies have shown that the major determinant is located within the last six amino acids. Anti-P antibodies occur in 13–20% of randomly selected Caucasian SLE patients, and in more than 40% in Asians [36]. They are serological markers of SLE and lupus-like disease [37]. Although found in a relatively small proportion of patients, they are more frequent in overt and active disease, in juvenile-onset than in adult-onset disease, and associated with particular clinical features, such as renal, hepatic and primarily NP abnormalities. They are specific for the classification of SLE, similarly to anti-Sm antibody; but, they have not been included in the updated ACR classification criteria [38,39]. Since the first prospective study by Bonfa et al. in 1987 [40], reporting on a strong association between anti-P antibodies and lupus psychosis, several studies have explored the utility of anti-P in prediction of NPSLE. However, results are still inconclusive. Two large international multicenter studies led to opposite results. The first examined the association of a panel of autoantibodies and longitudinal evaluation of NP events at the time of diagnosis in an international inception cohort of SLE patients [41]. Anti-P and LAC showed evidence of an association to different NP events attributed to SLE, anti-P with psychosis, while lupus anticoagulant with CVD, suggesting different immunopathogenetic mechanisms at the basis of these NP events. Otherwise, results from a recent international meta-analysis on the accuracy of anti-P antibody testing, showed that searching for anti-P antibodies is not useful for the diagnosis of NPSLE because of the high false negative and false positive rates. An important issue was that standardization of anti-P antibody testing is essential [42]. We compared the diagnostic performance of immunoblotting and two different ELISA assays for the detection of anti-P antibodies in SLE sera. The methods resulted concordant and similar in diagnostic accuracy, although IB on P proteins from lymphoid cells was more sensitive than ELISA using synthetic peptides [43]. More recently, an international multicenter evaluation of the clinical accuracy of a new ELISA based on recombinant P polypeptides, demonstrated that a combination of all three P proteins resembling

the native heterocomplex P0(P1/P2)2 as antigen gives the best accuracy [44]. Besides the differences in test systems, many other factors may contribute to the controversy on the clinical value of anti-P antibodies in NPSLE, being the study design one of the most important. In fact, among the major studies published in the last 20 years, prospective or longitudinal ones, being more informative than cross-sectional ones for NP attribution and outcome, all have assessed the association between anti-P and NPSLE [45]. Recently, in our longitudinal study of both anti-P antibodies and NPSLE in a single-center inception cohort of 219 SLE Italian SLE patients, followed-up for over 10 years, anti-P antibodies resulted to be associated with psychosis [46]. One of the most intriguing aspects of anti-P antibodies is their pathogenic potential in NPSLE. Anti-P antibodies may exert different cellular effects, by binding to the surface of different human cells, including T cells, activated monocytes, hepatoma as well as neuroblastoma cells [47]. They are able to penetrate into living cells and cause cellular dysfunctions and tissue damage by inhibiting protein synthesis, inducing apoptosis or proinflammatory cytokine production. Anti-P binding and penetration are mediated by a cell-surface 38 kDa protein, which is assumed to correspond to the cell-surface form of P0 ribosomal protein [47]. More recently, two groups independently investigated the neuropathogenic potential of anti-P antibodies on similar murine experimental models and provided insights for their implication in NPSLE. Katzav's study [25] showed that intracerebroventricular injection of human anti-P antibodies induces depression-like behavior and olfactory dysfunction in mice. Moreover, anti-P specifically stain neurons in areas of the limbic system known to be involved in the pathogenesis of depression. In the same year, Matus at al. [48] identified a new target of human anti-P antibodies from psychiatric lupus, neuronal surface P antigen (NSPA), a protein exclusively expressed on neurons of specific rat brain zones neocortex, hippocampus, and amygdala, involved in higher brain functions such as cognition, emotion and memory, which are compromised in NPSLE. Moreover, both anti-P and anti-NSPA antibodies are capable of inducing neuronal apoptosis by a dose-dependent increase in intracellular calcium influx. Whether anti-P antibodies are detectable in the cerebrospinal fluid (CSF) has been also a matter of debate. Autoantibodies in the CSF may result from passive transfer from the circulation through an abnormally permeable blood–brain barrier or from enhanced intrathecal production. Recently, evidence is increasing on the role of anti-P and other autoantibodies in CSF as well as of CSF proinflammatory properties in neurolupus. 5. Autoantibodies in neurolupus: pathogenic role of anti-β2GPI A recent report of an EULAR task force has categorized NPSLE according to frequency. Cerebrovascular disease (CVD) and seizure were considered common events (5–15%). In particular, ischemic stroke and/or TIA comprise over 80% of CVD in patients with SLE [33]. Several mechanisms are involved in the pathogenesis of NPSLE, including autoantibody-mediated damage. Among the autoantibodies implicated in NPSLE, a peculiar role is sustained by antiphospholipid antibodies (aPL), that were found strongly associated to CVD, and in particular to ischemic events, in patients with SLE [49,50]. In NPSLE, aPL have been shown to be the most frequently relied laboratory test not only to make the diagnosis but also to decide the treatment strategies involving anticoagulant and/or antiplatelet drugs [51]. Antiphospholipid antibodies are known to react with several phospholipid binding proteins, among these β2 glycoprotein I (β2GPI) is generally considered as the main target of aPL [52,53]. In the setting of NPSLE, the pathogenetic potential of aPL is linked to their capability to cause arterial or venous thrombosis and to directly interact with neuronal tissue. Here we summarize how these pathological processes can be sustained by the specific action of anti-β2GPI antibodies.

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5.1. Inhibition of natural anticoagulants and fibrinolysis systems Anti-β2GPI were described by different authors as able to bind several members of serine protease (SP) family, that participate in hemostasis and fibrinolysis. This is the case of activated protein C (APC), thrombin, plasmin, tissue-type plasminogen, and fibrin. It has been suggested that aPL recognize conformational epitopes shared by β2GPI and the catalytic domain of SP [54,55]. Anti-β2GPI antibodies binding impairs the function of the above quoted proteins resulting in a procoagulant state. In fact, as an example, anti-β2GPI antibodies bound to β2GPI may either compete with components of the APC complex for limited phospholipid binding sites or disrupt the interaction within the APC complex [56]. Therefore, in this case, APC cannot exert its physiological role of binding and inactivating the pro-coagulant factors Va and VIIIa. The alterations in fibrinolytic pathway can well contribute to thrombosis occurrence. Fibrinolysis is the process by which fibrin thrombi are remodeled and degraded; it involves the conversion of plasminogen to plasmin by tissue plasminogen activator (tPA). The activity of tPA is regulated by plasminogen activator inhibitor (PAI1). Takeuchi showed how monoclonal anti-β2GPI antibodies significantly suppress the intrinsic fibrinolytic system [57]. Furthermore it was suggested that β2GPI protects tPA from inhibitory effect of PAI1. In the presence of anti-β2GPI antibodies, β2GPI is not more available to protect tPA resulting in the enhancement of PAI-1 inhibition [58]. In addition, a recent study has shown that anti-β2GPI block the ability of β2GPI to stimulate tPA-mediated plasminogen activation [59]. 5.2. Interaction with cells involved in coagulation process The thrombogenic activity of anti-β2GPI is also based on their ability to recognize β2GPI expressed on the surface membranes of cell types involved in the coagulation cascade [60]. In vitro experiments have shown that affinity purified anti-β2GPI antibodies can induce a proadhesive phenotype by up-regulation of adhesion molecules (E-selectin, ICAM-1, VCAM-1) and can enhance the synthesis and secretion of proinflammatory cytokines (IL-6, IL1β) [61]. This effect is mediated by the presence of β2GPI on endothelial surface where it strongly binds annexin A2 [62] and/or Toll like receptor-4 [60]. Anti-β2GPI/β2GPI complexes, in vitro, have been shown to be able to stimulate monocytes with the consequent enhanced expression of tissue factor (TF). This protein is committed to start coagulation process after vessel injury. The enhanced production of TF by monocytes and endothelial cells has been suggested to be one of the possible causes of thrombosis in APS [63]. It is thought that anti-β2GPI lead to basic platelet activation. Indeed, the involvement of platelets in anti-β2GPI mediated thrombosis, has been studied in different settings. It was shown by in vitro studies that anti-β2GPI is able to potentiate thrombin platelet activation, leading to increased production of thromboxane A2, a major proaggregant eicosanoid [64]. Furthermore, Lutters suggested that anti-β2GPI/ β2GPI complexes linking apolipoprotein E receptor 2 on platelets may contribute to an enhanced activation [65]. Finally platelet particularly rich thrombi were described in hamsters carotid arteries primed with photochemical injury, after infusion of monoclonal antiβ2GPI antibodies [66]. 5.3. Direct interaction of aPL with neuronal tissue Shoenfeld et al. investigated the pathogenic potential of aPL by intracerebro-ventricular administration of immunoglobulins (IgG) from a patient with APS in mice [67]. Animals injected with this IgG performed worse in the water maze than the controls with significant effects attributed to aPL IgG regarding the overall performance of the

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mice. IgG from one APS patient was found to bind best to neuronal structures in the hippocampus and the cerebral cortex. These results support the hypothesis that aPL that gain access to the central nervous system may play a direct role in the pathogenesis of neurological manifestation of APS. Even if the described models imply the use of the whole IgG fraction, containing several antibodies besides antiβ2GPI, some experiments suggest a specific role for these antibodies. In fact a direct binding between purified anti-β2GPI and astrocytes and neurons was observed by indirect immunofluorescence and/or streptavidin-biotin-peroxidase techniques [68].

5.4. Pathogenic potential of anti-β2GPI antibodies Despite the previously described mechanisms about the pathogenic role of anti-β2GPI antibody, it is a common observation that thrombotic events take place only occasionally although antibodies are persistently present in the circulation [69]. This suggests that aPL are necessary but not sufficient to trigger clotting. A second hit is required in order to show the thrombogenic activity of the antibodies. Indeed, it has been reported that the arterial infusion of anti-β2GPI IgG fraction in naïve rats does not induce any vascular effects in mesenteric microcirculation unless a small amount of lipopolysaccharide (LPS) is injected intra-peritoneally. LPS alone is not able to mediate any vascular significant effect. In this model aPL represent the first hit and LPS the second hit [70]. In humans, the second hit could be represented by infections, hypertension, smoke, pregnancy, oral contraceptives, hormone therapy, etc. Anti-β2GPI has been reported in non-autoimmune conditions and detected also in healthy individuals. This has raised the hypothesis that different specificities of anti-β2GPI may carry a different pathogenetic potential. A recent study has investigated the fine specificity of anti-β2GPI. It showed that children without thrombotic events often carry antiβ2GPI antibodies but these antibodies are mainly directed to the domain (D) 4/5 of anti-β2GPI molecule, while in subjects with definite APS there was a predominance of anti-D1 [71,72]. Therefore, in the not too distant future, the anti-β2GPI antibodies potentially responsible for thrombotic events could be different from those not associated with thrombosis (“innocent”). This classification might greatly help to identify patients at high risk for developing future thrombosis, including CVD, from those with low risk.

6. Conclusions The role of autoantibodies in the pathogenesis of SLE is really of relevance in order to understand the etiology of organ damage and propose effective therapeutic strategies. The main mechanisms of autoantibody-mediated tissue damage in nephritis and CNS disease are on the way to being elucidated in both murine and human lupus. In lupus nephritis, anti-chromatin antibodies are crucial either as determinants in the early phase of the disease, or as effectors in the end stage. Several hypotheses have been proposed concerning the pathogenesis of NPSLE, including the generation of specific autoantibodies. Both systemic and brain-specific antibodies have been identified, exerting direct neurotoxic effects by cross-reacting with neuronal-specific surface targets (i.e. NSPA, NMDAR), causing neuronal injury and loss at specific brain zones where an insult at the blood–brain barrier integrity has occurred. Antiphospholipid antibodies are involved in CVD and are also implicated in the pathogenesis of focal damage in NPSLE. In particular, anti-β2GPI antibodies are the most thrombogenic and may exert a pathogenetic potential in NP involvement either as a strong procoagulant factor in the systemic circulation, or as a perturbating/interacting factor in situ, at the neuronal tissue milieu.

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The therapeutic benefit of vitamin D for systemic lupus erythematosus could depend on its ability to counteract monocyte maturation induced by lupus sera immunostimolatory milieu Vitamin D exerts immunomodulatory effects on many different cell types, both in vitro and in vivo, including the inhibition of antigen-presenting cell (APC) activation and differentiation. Type 1 interferons are potent stimulators of monocyte-derived APCs, and by this as well as other immunological mechanisms, they have a key role in systemic lupus erythematosus (SLE) pathogenesis. As SLE patients are at high risk of vitamin D deficiency, Lerman et al. (Lupus 2011;20:749-53) have postulated that vitamin D could be of therapeutic benefit for SLE by inhibiting INFα-induced overactivation of peripheral APCs. They analyzed activation/differentiation cell surface markers of peripheral human healthy monocytes using flow cytometric analysis, after stimulation in vitro by INFα or GM-CSF/IL-4 and in the presence or absence of 1,25 dihydroxyvitaminD3 (1,25(OH)2D3). Moreover, the effects of 1,25(OH)2D3 on monocyte maturation were analyzed after the addition of sera from pediatric SLE patients or healthy subjects to culture medium. The authors demonstrated that treatment with 1,25(OH)2D3 of human monocytes activated by lupus sera is able of limiting monocyte maturation, as assessed by the reduction of cell surface activation/ maturation markers, i.e. MHC Class II, CD40 and CD86 molecules. This could represent a physiopathological rationale of the beneficial effects of vitamin D supplementation in SLE. Anna Ghirardello