Journal of Neuroimmunology 274 (2014) 185–191

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Complement activation in patients with neuromyelitis optica Petra Nytrova a,b,⁎, Eliska Potlukova c, David Kemlink a, Mark Woodhall b, Dana Horakova a, Patrick Waters b, Eva Havrdova a, Dana Zivorova d, Angela Vincent b, Marten Trendelenburg e a

Department of Neurology and Center of Clinical Neuroscience, First Faculty of Medicine, General University Hospital, Charles University in Prague, Czech Republic Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK Third Department of Medicine, General University Hospital, First Faculty of Medicine, Charles University in Prague, Czech Republic d Laboratory of Clinical Immunology, Institute of Clinical Biochemistry and Laboratory Diagnostics, General University in Prague, Czech Republic e Laboratory of Clinical Immunology, Department of Biomedicine, University Hospital Basel, Switzerland b c

a r t i c l e

i n f o

Article history: Received 19 March 2014 Received in revised form 30 June 2014 Accepted 3 July 2014 Keywords: Neuromyelitis optica Complement Aquaporin-4 IgG C1q antibodies

a b s t r a c t The role of complement has been demonstrated in experimental models of neuromyelitis optica (NMO), however, only few studies have analysed complement components longitudinally in NMO patients. We measured serum or plasma concentrations of anti-C1q antibodies and complement split products C3a and C4a and soluble C5b-9 in patients with NMO, multiple sclerosis and healthy controls. NMO patients had higher levels of C3a and anti-C1q antibodies than healthy controls. C3a levels correlated with disease activity, neurological disability and aquaporin-4 IgG in NMO patients suggesting a role of the alternative pathway of complement in the pathogenesis of NMO and supporting the strategy of therapeutic complement inhibition. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Neuromyelitis optica (NMO), also referred to as Devic's disease, is a rare, severely disabling inflammatory disorder of the central nervous system (CNS), usually with a relapsing–remitting course (Wingerchuk et al., 1999). This autoimmune disease predominantly affects the optic nerves and spinal cord. NMO is associated with serum antibodies against aquaporin-4 (also known as NMO-IgG or AQP4-IgG) in up to 80% of cases (Lennon et al., 2004; Waters et al., 2008). The pathogenesis of NMO seems to be closely linked to the activation of the complement system. In experimental models, AQP4-IgG binding to AQP4 causes cytotoxicity only in the presence of complement (Hinson et al., 2007; Saadoun et al., 2010) suggesting that this is crucial for pathogenicity in NMO. In addition, recent studies reported that exacerbations of NMO were reflected by changes in concentrations of complement activation products in sera (C4d) as well as in the cerebrospinal fluid (C5a) (Tuzun et al., 2011; Kuroda et al., 2013). Furthermore, although Veszeli et al. did not find substantial systemic complement activation in NMO patients without disease activity, the complement system was found to be abnormally affected even during

⁎ Corresponding author at: Dpt. of Neurology and Center for Clinical Neuroscience, First Medical Faculty, Charles University in Prague, Katerinska 30, 120 00 Praha 2, Czech Republic. Tel.: +420 2 2496 6422; fax: +420 2 2491 7907. E-mail address: [email protected] (P. Nytrova).

http://dx.doi.org/10.1016/j.jneuroim.2014.07.001 0165-5728/© 2014 Elsevier B.V. All rights reserved.

remission (Veszeli et al., 2014). Eculizumab, a therapeutic monoclonal IgG that neutralises the complement protein C5, has effectively reduced the relapse frequency of NMO in open label trial (Pittock et al., 2013). AQP4 is a water channel that is most abundantly expressed in the processes of astrocytes. Its monomers assemble as tetramers. These AQP4 tetramers aggregate in cell plasma membranes to form supramolecular clusters which are targeted by AQP4-IgG. These clustered AQP4IgG are crucial for complement-dependent cytotoxicity through multivalent C1q binding (Papadopoulos and Verkman, 2012). AQP4-IgG are mainly of the IgG1 subclass that usually activates the classical pathway of complement (Waters et al., 2008). Formation of AQP4:AQP4-IgG immune complexes, with subsequent complement-mediated destruction of astrocytes, is paralleled by loss of AQP4 and GFAP staining in perivascular lesions in histological sections. This loss is localised in the same areas that exhibit accumulation of eosinophils and plasma cells as well as vasculocentric deposits of immunoglobulins and products of complement activation (Lucchinetti et al., 2002; Roemer et al., 2007; Jarius et al., 2008). These characteristics also resemble the histopathological features of murine brain lesions induced by injection of human AQP4-IgG with human complement into the brain (Saadoun et al., 2010). Autoantibodies against complement C1q (anti-C1q) have been found in a number of autoimmune diseases (Wisnieski and Jones, 1992; Siegert et al., 1999; Trendelenburg, 2005; Potlukova and Kralikova, 2008). Most interestingly, anti-C1q were found in more than 97% of patients with biopsy-proven active lupus nephritis

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supporting the hypothesis of a pathogenic role of anti-C1q in systemic lupus erythematosus (Trendelenburg et al., 2006). In other autoimmune diseases, the role of anti-C1q remains unclear. Interestingly, C1qtargeting monoclonal antibodies prevented complement-mediated damage in animal models of NMO (Phuan et al., 2013), may have a possible role in the regulation of the immune response, and providing a proof-of-concept for C1q-targeted monoclonal antibody therapies in NMO. In contrast to NMO, studies on complement activation in multiple sclerosis (MS) led to more conflicting results. As a consequence, complement activation is generally not considered to be of crucial pathogenic relevance in MS (Sellebjerg et al., 1998; Brink et al., 2005; Urich et al., 2006). The role of anti-C1q in MS remains to be determined. The aims of our study were to elucidate the role of complement activation and anti-C1q in patients with NMO by means of serological assessments during the relapse and remission of disease and to evaluate the potential use of plasma complement parameters as biomarkers of disease activity NMO. To support our observations we assessed complement binding to AQP4-IgG on frozen primate optic nerve.

2. Materials and methods 2.1. Patients The study was designed as a prospective cohort study in patients with demyelinating disorders at the Multiple Sclerosis Centre of the Department of Neurology, General University Hospital in Prague in 2010–2011. NMO and MS patients were included to the study. At inclusion, time of relapse and at 6-month follow-up, clinical data were recorded and serum as well as plasma samples were collected. The diagnosis of NMO was based on the Wingerchuk's diagnostic criteria (Wingerchuk, 2007). Plasma (n = 43) and serum samples (n = 43) were analysed from 19 AQP4-IgG positive patients. In eight patients with NMO, we obtained three or more samples at different time points (range was 2 weeks to 6 months). For the comparison of all groups, only first sample value of individual patient was included for statistical analysis. All forty-three AQP4-IgG positive samples were used for intragroup NMO analysis and correlations. The plasma samples were used for complement breakdown product assessment and the serum samples for C1q and AQP4 antibody measurement. Ten NMO patients had recently active disease (defined as ≥1 relapse during the last 6 months prior to sample collecting) and nine were recently inactive (relapse-free during the period of 6 months). In eight patients with NMO, we obtained three or more plasma samples at different time points ranging from 2 weeks to 6 months from the first. Thirty five MS patients were included in the study for the measurement of complement activation products. Furthermore, for the analysis of anti-C1q, we used additional 41 serum samples of different MS patients (in total, 76 serum samples were frozen in 2010). All MS patients fulfilled the McDonald's criteria for MS relapse remitting (n = 64), secondary (n = 9) or primary progressive form (n = 3) (Polman et al., 2005).

Patient-reported symptoms or objectively observed signs typical of an acute inflammatory demyelinating event in the CNS with a duration of at least 24 h, in the absence of fever or infection, were considered as a relapse (Polman et al., 2005). Samples were taken during both remission and relapse. Samples of the NMO or MS patients in relapse were obtained prior to the initiation of treatment by high-dose methylprednisolone, or plasma exchange. Samples of patients treated with intravenous immunoglobulins (IVIGs), cyclophosphamide, natalizumab and rituximab were drawn before regular medicament infusion. The majority of patients had long histories and had been treated with variable regimens. Six NMO patients were treated by a combination of drugs, i.e. low dose of prednisone with azathioprine. Monotherapy was used in 10 patients (low dose of prednisone, mycophenolate mofetil, cyclophosphamide, rituximab). One female patient underwent autologous stem cell transplantation three months before samples could be collected. Two patients were without treatment. MS patients were treated by a monotherapy (natalizumab, interferon beta, prednisone, IVIG, mycophenolate mofetil, cyclophosphamide) or received a combination of drugs (interferon beta and prednisone or azathioprine or methotrexate). Eleven MS patients were without treatment. Demographic data of NMO patients, MS patients, and healthy controls are summarised in Table 1. All patients were of Caucasian origin except two NMO female patients from Asia. Patients were observed by a specialist in demyelinating disorders of the CNS. Conventional brain and spinal cord MRI were used for the establishment of diagnosis and evaluation of spinal cord involvement. Neurological disability was evaluated by Kurtzke expanded disability status scale (EDSS) (Kurtzke, 1983). As controls, 40 volunteers without signs of an autoimmune disorder or infection at the time of blood sampling were included; most of them worked as health care professionals. Blood samples were collected by venipuncture in the collection tubes with EDTA. The serum and plasma samples were centrifuged at 3000 rpm for 10 min and frozen for 45 min after centrifugation and aliquoting. The samples were stored at − 80 °C and not thawed until assessment for complement split products and antibodies against AQP4 and C1q. All subjects gave an informed consent to the procedures, which were approved by the Ethical Committee of the General University Hospital and the First Faculty of Medicine, Charles University in Prague. 2.2. Serological analysis 2.2.1. Serum antibodies against aquaporin-4 and complement C1q All samples were tested in a blinded manner by a commercially available immunofluorescence cell-based assay (CBA) using recombinant human M1-AQP4 (Euroimmun, Lübeck, Germany) as antigen following the manufacturer's instructions. Sera of 19 NMO AQP4-IgG positive patients were retested by an in-house CBA using human M23-AQP4 expressed on live cells, and serial dilutions to determine the end-point titres (Waters et al., 2012). Sera of all MS and healthy controls were AQP4-IgG negative. Anti-C1q were measured in serum using a commercially available ELISA kit (Bühlmann Laboratories,

Table 1 Baseline characteristics of patients with NMO and MS. Characteristics

NMO (n = 19)

MS analysed for complement split products (n = 35)

MS analysed only for anti-C1q (n = 76)

Controls (n = 40)

p–value (ANOVA) Post hoc HSD

Females Age (years) Disease duration (years) Annual relapse rate EDSS score AQP4-IgG+

15 (79%) 44.5 (28–68) 8.9 (0.6–30) 1.58 (0–5) 4.0 (1.5–8.0) 19/19 (100)

30 (75%) 40.9 (25–65) 13.5 (3–41) 0.63 (0–3) 3.5 (1.0–7.5) 0/35 (0)

57 (75%) 39.3 (18–65) 11.9 (0.4–41) 0.65 (0–3) 3.0 (1.0–7.5) 0/76 (0)

30 (75%) 40.0 (18–74) 0 0 0 0/40 (0)

n.s. n.s. n.s. p = 0.043 n.s. p b 0.0001

Data are presented as median (range) or n (%) of patients. EDSS: Kurzke expanded disability status scale; HSD: honestly significant difference; n.s.: not significant.

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Schönenbuch, Switzerland). In this assay, undigested purified human C1q served as the antigen, and sera were diluted and incubated in a high-salt buffer (1 M NaCl). The optical densities were measured at 450 nm and converted into international units per ml (IU/ml) using the standards provided by the manufacturer. The manufacturer's cutoff of 15 IU/ml was used to determined positivity. 2.2.2. Assays for complement split products The soluble terminal complement complex (sC5b-9) as well as C3a and C4a fragments of complement proteins were measured by commercially available enzyme immunoassays (MicroVue™SC5b-9 Plus EIA Kit, MicroVue™ C3a Plus EIA Kit, MicroVue™ C4a Plus EIA Kit; Quidel, San Diego, USA) following the manufacturer's instructions. Briefly, test specimens were added to microassay wells precoated with specific monoclonal antibodies to each of the complement split products used for their capturing. Horseradish peroxidase conjugated antibodies to different epitopes of the same antigen were added to each test well. Following the addition of a chromogen, the plates were measured spectrophotometrically. The reference ranges for the complement split products for healthy individuals stated by the manufacturer were 33.8– 268.1 ng/ml (mean 129.6 ng/ml) for C3a and 383.5–8168.2 ng/ml (mean 1694.7 ng/ml) for C4a. The reference values for sC5b-9 assay were not provided by the manufacturer. Total C3 and C4 serum levels in NMO patients were measured by routine nephelometry (Dade Behring, Vienna Austria).

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For each serum (diluted 1:10), indirect immunofluorescence was carried out on individual chips: A) diluted sample/FITC-conjugated anti-human IgG (Euroimmun), B) diluted sample/serum of blood donors as complement source (Euroimmun)/FITC-conjugated anticomplement antibodies [polyclonal rabbit anti-human C1q (Dako, Glostrup, Denmark), polyclonal rabbit anti-human C3c (Euroimmun), polyclonal rabbit anti-human C4c (Dako)]. Since C3b and C4b were progressively degraded, we used anti-C3c and anti-C4c antibodies for immunohistochemistry analysis. The lyophilized complement source (Euroimmun) from 10 healthy donors was resuspended in sterile water prior using. The patterns were visually interpreted by fluorescence microscopy (Zeiss LSM 700 Confocal, Carl Zeiss Microscopy GmbH, Jena, Germany). 2.4. Statistical analysis Statistical intergroup analyses were conducted by Kruskal Wallis non-parametric ANOVA and its post hoc multiple comparison z-test. For correlation statistics we used the Spearman's test. For NMO group comparisons we used Mann–Whitney non-parametric tests. Tests were performed with the help of StatSoft, Inc. (2011) STATISTICA (Statsoft, Tulsa, OK, USA), version 10 (www.statsoft.com) and GraphPad PRISM 5. For the construction of receiver operating characteristics (ROC) and its statistics we used the programme JROCFIT1.0.2 (www. jrocfit.org). Differences were considered as being statistically significant in case of p b 0.05.

2.3. Immunohistochemistry-indirect immunofluorescence 3. Results Fourteen serum samples from NMO patients and 20 healthy blood donors were tested on multiplex biochip based slides (Euroimmun). Each field, that is screened with an individual serum, consisted of four individual chips with fixed frozen tissue (rat hippocampus, primate optic nerve, primate and rat cerebellum) and four individual chips with HEK cells transfected with human M1-AQP4, LGI1, CASRP2 and GlyR-alpha1, giving a total of 8 biochips per field. Phosphate-buffered saline (PBS) containing 5% Tween-20 was used for samples dilution.

3.1. Complement activation products analysis Plasma levels of complement activation products differed substantially between groups (Fig. 1A–C). Both NMO and MS patients had significantly higher levels of C3a and sC5b-9 and lower levels of C4a compared with controls. However, patients with NMO had lower levels of C4a than the MS patients (p b 0.05). Serum levels of anti-C1q were

Fig. 1. Comparison of plasma levels of complement activation products and serum levels of anti-C1q antibodies in patients with demyelinating disorders versus normal controls. A: C3a; B: C4a; C: sC5b-9; D: anti-C1q. For this analysis, only the first samples of each patient (before escalation of treatment) were used. Horizontal lines depict the medians of the measurements, the dotted horizontal lines represent the mean of normal controls (C3a and C4a, not available for sC5b-9) or the upper limit of normal values (anti-C1q) as stated by the manufacturer. (n.s.: not significant).

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Table 2 Spearman rank order coefficients of correlations between complement split products, AQP4-IgG, anti-C1q and EDSS in NMO.

EDSS score AQP4-IgG titres Anti-C1q

EDSS score

Anti-C1q

C3a

C4a

sC5b-9

AQP4-IgG titres

C3a/C3

C4a/C4

– 0.590 p b 0.05 n.s.

n.s. n.s.

0.683 p b 0.05 0.447 p b 0.05

n.s. n.s.

n.s. n.s.

0.590 p b 0.05 –

0.705 p b 0.05 0.482 p b 0.05

n.s. n.s.



n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

For this analysis, all available samples were used, including those from follow-up (n = 43). EDSS: Kurzke expanded disability status scale; n.s. not significant. R values were capture in bold if p was b 0.05.

higher in the NMO group compared to both MS and controls (Fig. 1D). Surprisingly, the MS patients had lower anti-C1q levels than controls.

correlation between complement split products or C1q antibodies and the clinical activity or severity of disease including EDSS score in MS patients.

3.2. Correlation of complement activation products with disease activity 3.3. Immunohistochemical studies In NMO patients (Table 2) there was a significant correlation between EDSS score and C3a or the ratio C3a:C3 (Fig. 2A). This correlation was as strong as the correlation between EDSS score and titres of AQP4IgG (Fig. 2B). In addition, plasma levels of C3a in NMO patients with a clinically recently active disease were significantly higher than in NMO patients with an inactive disease (Fig. 3A), and C3a levels were higher during relapses than during remission periods (Fig. 3B). Using ROC curves, the best cut-off for the distinction between patients with a recently active disease compared to those who were inactive was 500 ng/ml with a sensitivity of 94.7% and a specificity of 90.3% (Fig. 4). However, we did not find any correlation between C3a levels and the time to the next relapse after plasma sampling. In contrast to C3a, the other complement split products and anti-C1q did not correlate with disease activity. We also did not find any

Using diluted patients' sera and FITC-conjugated anti-human IgG, all patients with NMO were found to be AQP4-IgG positive, showing weak reactivity with optic nerve and cerebellum, but strong membrane reactions to the AQP4-expressing recombinant cells which have high AQP4 expression (Fig. 5 rows I–III: B). All control sera remained negative (Fig. 5 rows I–III: A). We did not find any reactivity of patients' or healthy control sera with LGI-1, CASPR2 and GlyR-alpha1 antigen when assessed by CBA (data not shown). To assess complement activation by the antibodies, patients' sera together with a complement source and FITC-conjugated anti-human complement (anti-human C1q, anti-human C3c, anti-human C4c, in separate assays) were applied to the cells and the tissue sections. First, as a positive control, the recombinant AQP4 expressing cells were tested

Fig. 2. Linear regression between neurological disability as expressed by EDSS score and plasma levels of C3a (A) and AQP4-IgG (B). The analysis was performed using only the first samples available.

Fig. 3. Plasma C3a levels in NMO patients with 1≥ relapse within the last 6 months prior to sample collecting (Group I) versus patients N 6 months relapse-free (Group II) (A); and during the relapse as compared to remission periods (B). Analysis in Fig. 3A was performed using only the first samples available whereas in Fig. 3B all available samples from 19 patients were used (i.e. including those at follow-up).

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4. Discussion

Fig. 4. ROC curve of complement C3a levels for the distinction between patients with ≥1 relapse during the last 6 months prior to sampling and patients that remained free of relapse during the last 6 months. AUC: area under curve, TPF: true positive fraction and FPF: false positive fraction.

in this three-step staining procedure (Fig. 5, row I: C–E). All NMO sera but none of the controls produced fluorescent signals on the AQP4expressing cells indicating complement activation by the bound human antibodies. The primate optic nerve transverse sections fluoresced in amesh-like staining pattern (Fig. 5, row II: C-E) corresponding to the glial supporting meshwork that separates the optic nerve fibres. The monkey and the rat cerebellums (Fig. 5, row III: C–E) exhibited mainly membrane staining, particularly in the granule cell layers of cerebellum. The rat hippocampal sections showed intensive staining in the subgranular zone of dentate gyrus particularly for complement split product C3c (data not shown).

Previous studies have implicated complement-activating IgG1 AQP4 antibodies in the pathogenesis of NMO, but these have concentrated on the classical pathway. Here we provide clinical, serological and immunohistochemical data suggesting that activation of C3 is implicated in the pathogenesis of NMO, and might provide a biomarker of disease activity. Moreover, by immunofluorescence staining we show that patients' AQP4-IgG form immune complexes, including C1q, C3c and C4c, not only on AQP4 transfected cells but also on native neuronal tissues when normal human serum is added as a source of complement. The staining pattern was similar to that described by Waters et al. on mouse brain tissue sections (Waters et al., 2008). Based on the similar staining patterns observed for AQP4 and AQP4:AQP4-IgG complexes and complement deposits, particularly C3c, we conclude that AQP4IgG binds to brain tissue AQP4 with subsequent complement activation of the alternative pathway as well as the better classical pathway. We did not find any correlation between C3a and sC5b-9 levels, which might suggest a more complex mechanism of astrocyte damage including the possibility of C3a-mediated recruitment of eosinophils and neutrophils into NMO lesions. C3a is proinflammatory mediator and anaphylotoxin with immune and non-immune biological functions. For instance, C3a is involved in excitotoxicity-mediated neuronal death through astrocytes stimulation (van Beek et al., 2001). Receptor for C3aR is expresses by monocytes/macrophages, microglia, astrocytes, neurons and endothelial cell too (Klos et al., 1992; Ischenko et al., 1998; Davoust et al., 1999). Interestingly, C3a has been shown to induce an increase in interleukin 6 (IL-6) mRNA expression by astrocyte cell lines (Sayah et al., 1999; Jauneau et al., 2003), which is in line with the observation that cerebrospinal fluid concentrations of IL-6 are increased during the initial attack of NMO (Uzawa et al., 2013). Low C4a levels as observed in our NMO patients might be the result of complement dysregulation and/or the more complex interplay between complement-activating AQP4-IgG and anti-C1q. The biological function of C4a in NMO remains more speculative but might be linked

Fig. 5. Row I: Cell-based assay for AQP4-IgG. AQP4 transfected HEK cells incubated with serum of a healthy control and a patient's serum and FITC-conjugated anti-hIgG (A,B); AQP4 transfected HEK cells incubated with patient's serum, complement source and FITC-conjugated anti-hC1q (C), anti-hC3c (D) and anti-hC4c (E). Row II: Primate optic nerve tissue staining. Primate optic nerve transversal section was incubated with serum of a healthy control and a patient's serum and FITC-conjugated anti-hIgG (A,B); with patient's serum, complement source and FITC-conjugated anti-hC1q (C), anti-hC3c FITC (D) and anti-hC4c (E). Row III: Rat cerebellum tissue staining. Rat cerebellum section was incubated with serum of a healthy control and a patient's serum and FITC-conjugated anti-hIgG (A,B); with patient's serum, complement source and FITC-conjugated anti-hC1q (C), anti-hC3c (D) and anti-hC4c (E).

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to regulatory properties of the fragment. Recombinant C4a has been demonstrated to inhibit C3a and C5a-stimulated degranulation of mast cells (Xie et al., 2012). In addition, an anti-inflammatory role of C4a in glomerulonephritis has been described (Welch et al., 2001). Considering that the lack of early components of the classical pathway of complement (C1q and C4) are associated with systemic autoimmunity (Pickering et al., 2000), these components might also have a protective effects in NMO. To date, only a few analyses of complement split products in plasma/ serum or cerebrospinal fluid have been reported and led to conflicting results. Tuzun et al. described an increased activity of the classical pathway during relapses of NMO by measuring the levels of breakdown products for the classical (C4d), alternative (fragment Bb) and terminal complement (sC5b-9) pathways (Tuzun et al., 2011). Moreover, they described a negative correlation between EDSS and levels of C4d, fragment Bb and sC5b-9. The differing results of our study could partially be explained by the use of different assays, by different sampling time points, differences in the accompanying treatment of the patients and the assessment in sera of AQP4-IgG negative NMO patients. More in line with our findings is the report demonstrating significantly elevated C5a levels in the cerebrospinal fluid of NMO patients, in particular in patients with multiple enhanced lesions on MRI (Kuroda et al., 2013). Independent of the complement activation products, we found that anti-C1q Abs levels were significantly higher in NMO patients as compared to the MS patients and healthy controls, respectively. This might be of particular interest with regard to the recent study by Phuan et al. who demonstrated that monoclonal neutralising antibodies targeting C1q significantly improved the course of NMO in an experimental model of NMO (Phuan et al., 2013). However, the effects of anti-C1q on C1q could be different from neutralizing monoclonal antibodies as mentioned before. Again, data derived from studies in systemic autoimmunity point towards a disease exacerbating effect of autoantibodies against C1q (Trendelenburg et al., 2006), e.g. by the inhibition of protective effects of the C1q molecule. MS patients had lower anti-C1q levels. However, the main differences were observed in the range of very low anti-C1q levels making it difficult to draw any definite conclusions. Potentially, the very low anti-C1q levels are the consequence of immunomodulatory (interferon beta) or immunosuppressive therapy as administered in most MS patients but not in the healthy controls. Our study has several limitations: the descriptive character of our data does not allow final statements on the role of complement in NMO. In addition, the relatively small and locally recruited cohort of NMO patients, due to the fact that NMO is a rare disease in Caucasians, does not allow definite conclusions on the correlation between plasma/serum parameters and disease activity. The limited relapse samples and the time frame of the study may preclude the finding of associations between these products and disease activity. Finally, different treatment profiles in MS and NMO patients might have strongly affected our comparative analyses. Thus, further studies on the role of complement in NMO, in particular with a focus on outcome measures such as disability, will be necessary. In conclusion, our data strongly support the hypothesis that complement activation is involved in the pathogenesis of NMO in vivo. Independently, complement C3a as a biomarker might play an important role not only for the diagnosis of NMO but also for the evaluation of disease activity at follow-up. Moreover, our data support strategies of therapeutic complement inhibition in NMO patients in particular during relapses of disease. Abbreviations AQP4 aquaporin-4 AQP4-IgG antibodies against aquaporin-4 Anti-C1q antibodies against C1q AUC area under curve CASPR2 contactin-associated protein 2

CNS EDSS FPF GlyR HSD IVIG LGI1 MS NMO ROC sC5b-9 TPF

central nervous system expanded disability status scale false positive fraction glycine receptor honestly significant difference intravenous immunoglobulins leucine-rich, glioma inactivated 1 multiple sclerosis neuromyelitis optica receiver operating characteristic soluble terminal complement complex true positive fraction

Competing interests Nytrova Petra, her research stay at Dpt. of Clinical Neurosciences at University of Oxford was supported by Euroimmun and a John Newsom-Davis Fellowship from the Guarantors of Brain. Potlukova Eliska has nothing to disclose. Kemlink David has nothing to disclose. Woodhall Mark has nothing to disclose. Waters Patrick is a named inventor on patents for antibody assays and has received royalties, and he has received a speaker honorarium from Biogen-Idec Japan. Horakova Dana received speaker honoraria and consultant fees from Biogen Idec, Novartis, Merck Serono, Teva and Bayer Healthcare and financial support for research activities from Biogen Idec. Havrdova Eva received speaker and consulting honoraria from Biogen Idec, Novartis, Sanofi Genzyme, Roche, Merck Serono, Teva and Bayer Healthcare. Zivorova Dana has nothing to disclose. Vincent Angela has received funding from Euroimmun AG and is a consultant for Athena Diagnostics. The University of Oxford holds patents and receives royalties and payments for antibody tests. Trendelenburg Marten receives financial support for research activities from Roche Pharma and is a member of the Swiss advisory board of GSK. Acknowledgments Supported by Grant Agency of the Charles University (grant GAUK 132010), the Czech Ministries of Education and Health (PRVOUK-P26/ LF1/4, NT13237-4/2012) and by the National Health Service National Specialised Commissioning Group for Neuromyelitis Optica and the National Institute for Health Research Oxford Biomedical Research Centre for funding. The research stay of main author PN at Dpt. of Clinical Neuroscience at University of Oxford was supported by a John NewsomDavis Fellowship from the Guarantors of Brain, Fond Mobility of the Charles University and Euroimmun. MT is a recipient of a grant from the Swiss National Foundation (310030_134900/1). References Brink, B.P., Veerhuis, R., Breij, E.C., van der Valk, P., Dijkstra, C.D., Bo, L., 2005. The pathology of multiple sclerosis is location-dependent: no significant complement activation is detected in purely cortical lesions. J. Neuropathol. Exp. Neurol. 64, 147–155. Davoust, N., Jones, J., Stahel, P.F., Ames, R.S., Barnum, S.R., 1999. Receptor for the C3a anaphylatoxin is expressed by neurons and glial cells. Glia 26, 201–211. Hinson, S.R., Pittock, S.J., Lucchinetti, C.F., Roemer, S.F., Fryer, J.P., Kryzer, T.J., Lennon, V.A., 2007. Pathogenic potential of IgG binding to water channel extracellular domain in neuromyelitis optica. Neurology 69, 2221–2231. Ischenko, A., Sayah, S., Patte, C., Andreev, S., Gasque, P., Schouft, M.T., Vaudry, H., Fontaine, M., 1998. Expression of a functional anaphylatoxin C3a receptor by astrocytes. J. Neurochem. 71, 2487–2496. Jarius, S., Paul, F., Franciotta, D., Waters, P., Zipp, F., Hohlfeld, R., Vincent, A., Wildemann, B., 2008. Mechanisms of disease: aquaporin-4 antibodies in neuromyelitis optica. Nat. Clin. Pract. Neurol. 4, 202–214.

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Complement activation in patients with neuromyelitis optica.

The role of complement has been demonstrated in experimental models of neuromyelitis optica (NMO), however, only few studies have analysed complement ...
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