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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

Neuromyelitis optica (NMO) and multiple sclerosis (MS) are two of the autoimmune inflammatory demyelinating diseases in the central nervous system. Complement is thought to have an important role in pathogenesis of these diseases, especially in NMO. However, the change of terminal complement complex (TCC, C5b-9) in patients with NMO is still unclear. Cerebrospinal fluid (CSF) C3a, C5a, sC5b-9 were measured by enzyme-linked immunosorbent assay in patients with NMO (n = 26), MS (n = 25) and other neurological disease (OND, n = 19). CSF levels of C5a in patients with NMO were higher than patients with OND (= 0.006). Increased CSF sC5b-9 were found in the patients with NMO compared with patients with MS (= 0.029) and OND (= 0.0001). CSF sC5b-9 in patients with MS were also higher than patients with OND (= 0.030). Patients with NMO revealed a trend to an increased disease disability with increased CSF sC5b-9 during relapse but not in MS (NMO:= 0.006, MS: = 0.097). CSF levels of sC5b-9 are increased in patients with NMO and reflect the activation of complement in NMO.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

Neuromyelitis optica (NMO) and multiple sclerosis (MS) are acquired immune-mediated inflammatory disorders of the peripheral nervous system. Compared with MS, NMO often affects the optic nerve and spinal cord [1-3]. The pathogenesis of NMO and MS is not clear. An autoantibody against aquaporin 4 (AQP4-Ab) in NMO, which is absent in MS, has been reported [4]. Several studies have suggested that NMO may be an inflammatory demyelinating disease mediated by the humoral immune system [5-7].

AQP4 is a water channel expressed on astrocytic endfeet. Active NMO lesions are characterized by severe astrocytic damage with extensive loss of AQP4 accompanied by perivascular deposition of activated complement and immunoglobulin (Ig) [8, 9]. The complement activation is part of the humoral immune response, which appears to play an important part in the development of inflammatory diseases of the central nervous system. In complement activation, a terminal product of the complement response, membrane-attack complex (MAC; C5b-9), damages the cell membrane directly. Increased levels of soluble C5b-9 (sC5b-9) have been detected in the cerebrospinal fluid (CSF) of patients with MS during relapse [10, 11] and correlate with neurological disability as measured by the Expanded Disability Status Scale (EDSS) [12]. These findings suggest that complement activation and sC5b-9 assembly take place also in the CSF of patients with MS and breakdown the blood–brain barrier [13]. In NMO, reports of changes in serum levels of CH50 and complement are controversial [14-16]. Kuroda et al. found a significant increase in C5a levels in CSF during exacerbations in patients with NMO [17]. C5a and C5b (which are the cleavage products of C5) lead to the formation of C5b-9. However, the change of sC5b-9 levels in the CSF of patients with NMO is not clear.

In this study, we analysed the sC5b-9 levels in the CSF of patients with NMO or MS. We wished to ascertain if sC5b-9 levels in CSF could be used as sensitive biomarkers for disease activity.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

The study protocol was approved by the Ethics Committee of the Third Affiliated Hospital of Sun-yatsen University (Guangzhou, China).

Patients and controls

Twenty-six relapsing patients with NMO fulfilling the diagnostic criteria set by Wingerchuk et al. [18] and 25 relapsing–remitting patients with MS based on the diagnostic criteria by McDonald et al. [19] were enrolled. Patients enrolled from the database were diagnosed by two very experienced neurologists. All samples were taken during relapse before treatment. A ‘clinical relapse’ was defined as a sudden appearance of new symptoms lasting for ≥24 h with an increase in EDSS >1.0 before sampling. Patients were not receiving interferon-β therapy before lumbar puncture. None of the patients were seropositive for human T cell leukaemia virus type I. Nineteen subjects with other neurological diseases (OND; 6 subjects with amyotrophic lateral sclerosis; multiple system atrophy, 3; sciatica, 4; cervical spondylosis, 6) were recruited as controls. The demographic and clinical features of patients are shown in Table 1. There was no significant difference in age and sex between the groups. The EDSS scores of all patients were reviewed. In the relapsing stage, the EDSS scores of patients with NMO and MS were scored when they peaked.

Table 1. Demographic and clinical features of the patients and controls
 NMO (n = 26)MS (n = 25)OND (n = 19)
  1. AQP4-Ab, autoantibody of aquaporin 4; CSF, cerebrospinal fluid; TP, total protein in CSF; WBC, white blood cell in CSF; MS, multiple Sclerosis; NMO, neuromyelitis optica; OND, other neurological disease.

  2. Age (years) refers to age at sampling time point; Disease duration (years) refers to years from disease onset to sampling.

Gender, female/male19/717/813/6
Age (years)36.54 ± 15.0534.56 ± 12.5436.21 ± 11.12
Disease duration (years)4.46 ± 3.403.92 ± 1.572.15 ± 0.89
CSF TP0.30 ± 0.200.29 ± 0.200.25 ± 0.19
CSF WBC5.85 ± 9.075.80 ± 7.322.26 ± 2.18
AQP4-Ab (positive)2100
Enzyme-linked immunosorbent assay (ELISA)

Cerebrospinal fluid samples were obtained during diagnostic lumbar punctures. Samples were processed within 30 min of withdrawal and stored at −80 °C until assay. ELISA was used to quantify the levels of C3a (Bender MedSystems, Vienna, Austria), C5a (BD Biosciences, San Diego, CA, USA) and sC5b-9 (BD Biosciences) in CSF according to manufacturer instructions.

Statistical analyses

Data are the mean ±1 SD. Differences in the levels of C3a, C5a and sC5b-9 in CSF between subgroups were analysed using one-way anova. Correlations between the sC5b-9 in CSF and EDSS score were analysed using Spearman's rank test. < 0.05 was considered significant. Statistical analyses were conducted using spss, version 16.0 (SPSS, Chicago, IL, USA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

Levels of C3a, C5a and sC5b-9 in the CSF of patients and controls

Mean levels of C3a in CSF (ng/ml) were 9.85 ± 6.46 for patients with NMO, 8.24 ± 2.85 for patients with MS and 9.77 ± 3.61 for patients with OND (Fig. 1). There was no significant different between in the three groups.

image

Figure 1. Cerebrospinal fluid (CSF) levels of C5a in patients with neuromyelitis optica (NMO) were higher than patients with other neurological disease (OND) (= 0.006). CSF sC5b-9 levels were increased in the patients with NMO compared with patients with multiple sclerosis (MS) (= 0.029) and OND (= 0.0001). CSF sC5b-9 in patients with MS were also higher than patients with OND (= 0.030).

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Mean levels of C5a in CSF (ng/ml) were 6.54 ± 3.27 for patients with NMO, 4.94 ± 2.85 for patients with MS and 4.08 ± 1.77 for patients with OND. The levels of C5a in the CSF of patients with NMO were higher than those for patients with OND (= 0.006).

Mean levels of sC5b-9 in CSF (ng/ml) were 20.23 ± 6.03 for patients with NMO, 16.55 ± 4.65 for patients with MS and 13.37 ± 3.60 for patients with OND. The levels of sC5b-9 in CSF were higher in patients with NMO compared with patients with MS (= 0.029) and OND (= 0.0001). When comparing sC5b-9 levels in CSF between patients with MS and OND, there was also a significant difference (= 0.030).

sC5b-9 level in CSF and EDSS scores

Cerebrospinal fluid sC5b in patients with NMO had significant correlation with EDSS scores, which was not seen in patients with MS (NMO: = 0.006, = 0.521; MS: = 0.097, = 0.340; Fig. 2).

image

Figure 2. Cerebrospinal fluid (CSF) sC5b in patients with neuromyelitis optica (NMO) had significant correlation with Expanded Disability Status Scale (EDSS) scores, which was not seen in patients with multiple sclerosis (MS) (NMO:= 0.006, = 0.521; MS:= 0.097, = 0.340).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

In the present study, sC5b-9 levels in CSF increased significantly in patients with NMO during relapse and had significant correlation with EDSS scores. In acute NMO lesions, loss of AQP4 and glial fibrillary acidic protein, but relatively preserved myelin basic protein (MBP), has been observed [20, 21]. Immunohistochemical findings have revealed activated complement C9neo to be stained diffusely, but particularly around numerous dilated vessels [20]. Activation of the complement system may play an important part in the immunological mechanisms of NMO.

Activation of complement by classical or alternative pathways results in the cleavage of C3 to C3b and C3a. C3b is a critical factor in furthering complement activation as a material constituent of C5 convertase. C5 convertase cleaves C5 to C5a and C5b. C5a, a potent anaphylatoxin, is bound rapidly by cellular receptors. Kuroda et al. [17] reported C5a levels in the CSF of patients with NMO to be increased. One consequence of C5b generation, after sequential interaction with four other proteins, is the formation of MAC in cell membranes. However, much of the C5b generated does not result in the formation of MAC, but instead is diverted by control proteins (S proteins) to form a soluble, lytically inert complex called sC5b-9 [22]. Thus, sC5b-9 levels can be used as an indication of the status of total complement activity [23, 24]. Increased levels of sC5b-9 have been detected in autoimmune diseases such as systemic lupus erythematosus [25], rheumatoid arthritis [26] and Behçet's disease [15]. Serum levels of sC5b-9 in patients with NMO are also higher than those in patients with MS [16]. However, the CSF levels of sC5b-9 in patients with NMO have rarely been studied.

Saadoun et al. [27] found that co-injection of IgG from patients with NMO with human complement into mice could reproduce the key histological features of NMO. Hinson et al. [28] demonstrated a statistically significant association between the severity of NMO attacks and measures of complement-mediated injury to AQP4-expressing cells. Sabater et al. [29] showed that AQP4-Ab induces the complement-dependent cytotoxicity of astrocytes in vitro. In the present study, we measured the levels of complement breakdown products in CSF. According to our results, we repute the notion that sC5b-9 levels in CSF reflect the activation of complement in NMO and are related to the level of disability. In acute relapse of NMO, the complement system was activated. Higher expression of sC5b-9 could mean a severe inflammatory response.

We did not find an obvious association between sC5b-9 levels in CSF and EDSS scores in patients with MS. Intrathecal complement activation occurs in MS [30]. However, complement may have a protective role by promoting oligodendrocyte survival, axon preservation and providing protection from gliosis in MS [31].

Taken together, our results suggest that sC5b-9 levels in CSF are increased in patients with NMO and may be a sensitive biomarker for disease activity. Further investigation on the precise role of complement in NMO may provide insight into the drug targets for treatment.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

This study was supported by the Natural Science Foundation of China (grant no.81200920) and Research Fund of young scholars for the Doctoral Program of Higher Education of China (no. 20123420120009).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References