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Increased levels of CSF CD59 in neuromyelitis optica and multiple sclerosis
Clinica Chimica Acta 453 (2016) 131–133
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Increased levels of CSF CD59 in neuromyelitis optica and multiple sclerosis Akiyuki Uzawa ⁎, Masahiro Mori, Tomohiko Uchida, Hiroki Masuda, Ryohei Ohtani, Satoshi Kuwabara Department of Neurology, Graduate School of Medicine, Chiba University, Japan
a r t i c l e
i n f o
Article history: Received 27 October 2015 Received in revised form 10 December 2015 Accepted 10 December 2015 Available online 11 December 2015 Keywords: Cerebrospinal fluid CD59 Complement Interleukin-6 Multiple sclerosis Neuromyelitis optica
a b s t r a c t Background: Complement activation is important in multiple sclerosis (MS) and is essential for anti-aquaporin 4 antibodies to damage the central nervous system in neuromyelitis optica (NMO). Little is known about the role of cerebrospinal fluid (CSF) regulators of complement activation in NMO and MS. We determined whether CSF CD59, which is a complement regulator and C5b-9 formation inhibitor, is involved in the pathogenesis of NMO and MS. Methods: We analyzed CSF levels of CD59 in 30 patients with NMO, 22 patients with MS, and 24 patients with non-inflammatory neurological disorders (NINDs). Possible correlations between CSF CD59 levels and the clinical and laboratory variables in patients with NMO and MS were also reviewed. Results: CSF CD59 levels in patients with NMO and MS were higher than those in patients with NINDs (p b 0.001), and those in patients with NMO decreased after treatment. No significant correlations were found between CSF CD59 levels and clinical and laboratory parameters in NMO and MS. Conclusion: High CSF CD59 levels in NMO and MS may reflect inflammation, damage, and/or complement activation in the central nervous system. © 2015 Elsevier B.V. All rights reserved.
1. Introduction
2. Methods
Neuromyelitis optica (NMO) and multiple sclerosis (MS) are different from the viewpoints of clinical features, pathology, immunology, neuroimaging, and response to treatment but are similar from the viewpoint of autoimmune-mediated inflammatory disorders of the central nervous system (CNS) [1]. Complement is thought to play important roles in the pathogenesis of these disorders. Deposits of complement have been observed in NMO and MS lesions [2]. Increased cerebrospinal fluid (CSF) levels of soluble C5b-9 have been observed in patients with NMO and MS, indicating that complement has been activated [3]. The complement regulator CD59 is a phosphoinositol-linked glycoprotein that inhibits the terminal membrane attack complex. It is also the major complement inhibitor protein in human astrocytes and glioma cell lines [4,5]. In mice, CD59a is expressed mainly on astrocytes and weakly on oligodendrocytes and neurons within the CNS [6]. Greatly increased NMO pathology in CNS has been reported in mice with CD59 knockout or those with CD59 inhibition by a neutralizing monoclonal antibody [7]. Although the importance of complement in NMO and MS has been established, the role of complement regulation in the pathogenesis of NMO and MS is not completely understood.
2.1. Subjects NMO spectrum disorder patients (n = 30) who met the 2015 diagnostic criteria [8] and relapsing–remitting MS patients (n = 22) who met the 2005 McDonald criteria [9] were involved in this study. Twenty-four patients (13 men, 11 women; mean age, 58.9 y) with non-inflammatory neurological disorders (NINDs), including 10 with amyotrophic lateral sclerosis, 8 with spinocerebellar degeneration, 4 with Parkinson's disease, and 2 with progressive supranuclear palsy, were recruited as controls. We reviewed the gender, age, attack site, expanded disability status scale (EDSS) scores, positivity for oligoclonal bands, positivity for serum anti-aquaporin 4 (AQP4) antibody [10], and CSF variables (including cell counts, protein, interleukin (IL)-6 levels measured using a chemiluminescent enzyme immunoassay [11], CSF/serum albumin ratio (albumin quotient, QAlb), and IgG index), at the time of sampling. The ethics committee of the Chiba University School of Medicine, Chiba, Japan approved the study, and informed consent was obtained from all study subjects. 2.2. CSF CD59 measurements
⁎ Corresponding author at: Department of Neurology, Graduate School of Medicine, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail address: [email protected] (A. Uzawa).
http://dx.doi.org/10.1016/j.cca.2015.12.013 0009-8981/© 2015 Elsevier B.V. All rights reserved.
CSF samples were obtained during the active disease phase (within 1 month of clinical attack and before treatment for the attack) from patients with NMO and MS. After treatment for the acute attack, CSF
132
A. Uzawa et al. / Clinica Chimica Acta 453 (2016) 131–133
Table 1 Clinical characteristics of patients with NMO and MS. NMO (n = 30)
MS (n = 22)
Men:women Age, y, mean ± SD EDSS; median (range) Positivity for anti-AQP4 antibody Positivity for oligoclonal bands
2:28 48.7 ± 14.6 7.0 (2.0–9.0) 26/30 (87%) 3/20 (15%)
Site of attack
S 21, B 3, BS 3, O 3
5:17 37.1 ± 17.2 4.5 (1.0–8.0) 0/22 (0%) 13/20 (65%) S 12, B 3, BS 2, BS and S 2, O 2, C and O 1
AQP4: aquaporin 4; B: brain (cerebrum); BS: brainstem; C: cerebellum; EDSS: Expanded Disability Status Scale; MS: multiple sclerosis; NMO: neuromyelitis optica; O: optic nerve; S: spinal cord.
samples were obtained from only four patients with NMO and none of the patients with MS. All samples were stored at − 80 °C until they were assayed. The CSF CD59 levels were measured using a CD59 ELISA kit (MyBioSource, Inc.) according to the manufacturer's instructions. The optical density was measured at 450 nm. CD59 levels were calculated with reference to a standard curve. The detection range was 31.25–2000 pg/ml.
Fig. 1. CSF CD59 levels in patients with NMO, MS, and NINDs. (a) CD59 levels in CSF of 30 NMO patients, 22 MS patients, and 24 NIND patients. Levels in patients with NMO and MS were significantly higher than those in patients with NINDs (**p b 0.001, Mann–Whitney U-test). Dashed lines indicate the mean concentration in each group. (b) The CSF CD59 levels in patients with NMO (n = 4) decreased after treatment. Pre-Tx: pre-treatment; post-Tx: post-treatment.
2.3. Correlation between the CSF CD59 levels and clinical parameters
3.2. CSF CD59 levels
We examined possible associations between the CSF CD59 levels and various clinical and laboratory variables, including EDSS, positivity for serum anti-AQP4 antibody titers, positivity for oligoclonal bands, IgG index, CSF cell counts, CSF protein levels, CSF IL-6 levels, and QAlb, in patients with NMO and MS.
CSF CD59 levels were 275.5 ± 77.4 (mean ± SD) pg/ml, 298.3 ± 67.7 pg/ml, and 181.8 ± 51.3 pg/ml in patients with NMO, MS, and NINDs, respectively. Kruskal–Wallis test revealed significant differences in NMO, MS, and NINDs group (p b 0.001). The CSF CD59 levels in patients with NMO and MS were similar and significantly higher than those in patients with NINDs (p b 0.001, Mann–Whitney U-test) (Fig. 1). We obtained CSF samples from only four patients with NMO after treatment for an acute attack (all four patients received highdose intravenous methylprednisolone treatment and one received additional plasmapheresis therapy). The CSF CD59 levels decreased after treatment (Fig. 1).
2.4. Statistical analyses Groups were compared using Kruskal–Wallis test and the Mann– Whitney U-test for unpaired continuous measures. For multiple comparisons, Bonferroni correction was applied. Each parameter in this study did not follow a normal distribution, hence non-parametric tests, instead of ANOVA, were used. Spearman's rank correlation coefficient was used to evaluate the statistical dependency of two variables. A p b 0.05 was considered statistically significant. 3. Results 3.1. Clinical profiles The clinical characteristics of the patients with NMO and MS are summarized in Table 1. The patients' proportion of woman, age, EDSS score, positivity for serum anti-AQP4 antibody, and negativity for oligoclonal bands at CSF sampling were higher in patients with NMO than in patients with MS. The CSF findings in patients with NMO, MS, and NINDs are summarized in Table 2. The CSF cell counts, protein levels, IL-6 levels, and QAlb in patients with NMO were significantly higher than those in patients with MS and NINDs. CSF cell counts were significantly higher in MS than in NINDs. IgG indexes were significantly increased in NMO and MS compared with NINDs.
3.3. Correlations between CSF CD59 levels and clinical variables No significant correlations were observed between CSF CD59 levels and clinical and laboratory variables (EDSS scores, IgG index, CSF cell counts, CSF protein levels, CSF IL-6 levels, Qalb, anti-AQP4 antibody titers, or oligoclonal band positivity) in patients with NMO and MS (Table 3). There was no association of CSF CD59 levels with site of lesions. 4. Discussion This study confirmed significant increases in the CSF CD59 levels in patients with NMO and MS compared with those in patients with NINDs. Anti-AQP4 antibody binds to AQP4 at the astrocytic end-feet and causes CNS inflammation mainly by complement-dependent cytotoxicity [7]. Furthermore, complement depositions in lesion areas [2] and upregulated CSF-soluble C5b-9 [3] have been reported in both NMO and MS. In this regard, these disorders are similar, and complement
Table 2 CSF findings in patients. CSF findings
Cell count (cells/mm3) Protein level (mg/dl) IL-6 level (pg/ml) QAlb × 103 IgG index
NMO (n = 30)
28.3 ± 38.0 101.1 ± 152.0 1125.9 ± 3179.0 14.3 ± 11.9 0.70 ± 0.23
MS (n = 22)
6.7 ± 9.5 31.5 ± 8.6 2.9 ± 1.1 5.1 ± 2.0 0.72 ± 0.27
NINDs (n = 24)
0.7 ± 0.7 34.4 ± 12.5 2.3 ± 1.2 4.2 ± 1.1 0.48 ± 0.08
p value NMO vs. MS
NMO vs. NINDs
MS vs. NINDs
0.008 b0.001 b0.001 b0.001 0.859
b0.001 b0.001 b0.001 b0.001 b0.001
b0.001 0.321 0.064 0.185 b0.001
MS: multiple sclerosis; NINDs: non-inflammatory neurological disorders; NMO: neuromyelitis optica; QAlb: albumin quotient = cerebrospinal/serum albumin ratio. Each value represents the mean ± SD. The Mann–Whitney U-test was used for statistical analyses.
A. Uzawa et al. / Clinica Chimica Acta 453 (2016) 131–133 Table 3 Associations between clinical and laboratory characteristics and CD59 levels in patients with NMO and MS.
EDSS score IgG index CSF cell count CSF protein level CSF IL-6 level Qalb AQP4 antibody titer Oligoclonal bands positivitya
NMO
MS
R = 0.106 (p = 0.593) R = 0.290 (p = 0.174) R = −0.307 (p = 0.098) R = −0.293 (p = 0.115) R = −0.306 (p = 0.099) R = −0.111 (p = 0.610) R = 0.026 (p = 0.890) p = 0.315
R = 0.405 (p = 0.066) R = 0.076 (p = 0.749) R = 0.209 (p = 0.340) R = −0.072 (p = 0.732) R = 0.333 (p = 0.127) R = 0.225 (p = 0.341) NA p = 0.843
AQP4: aquaporin 4; EDSS: Expanded Disability Status Scale; IL-6: interleukin 6; MS: multiple sclerosis; NA: not available; NMO: neuromyelitis optica; QAlb: albumin quotient = cerebrospinal/serum albumin ratio. Spearman's rank correlation coefficient was used to evaluate the dependency of 2 variables. a Evaluated by Mann–Whitney U-test.
activation plays a central role in their pathogenesis. Hence, we assumed that CSF complement regulators are upregulated to suppress complement activation in the CNS or in contrast, that CSF complement regulators are downregulated for an unknown reason, and this leads to complement activation in these disorders. Our results showed that CSF CD59 levels were similarly high in both NMO and MS during attack, which might be caused by issues secondary to CNS inflammation, CNS damage, and/or complement activation. Mice with a CD59 knockout or those with CD59 inhibition induced by a neutralizing monoclonal antibody showed severe deterioration of NMO pathology in the CNS [7]. However, we determined that CSF CD59 levels were not decreased but were increased during attack and decreased after treatment, suggesting that the downregulation of CD59 does not play an important role in the pathogenesis of NMO in humans, which is in contrast to the result obtained using an NMO mouse model [7]. Recently, it has been reported that CD59 is expressed in the microvascular endothelium of normal brain but is absent from NMO lesions [5]. Therefore, high CSF CD59 levels in NMO and MS may reflect damage to the vascular endothelium; however, CSF CD59 levels did not correlate with clinical variables, including Qalb (one of the markers of blood– brain barrier function). Although the increased CSF cell counts and
133
IL-6 levels in patients with NMO might be a result of their more severe CNS inflammation than patients with MS, CSF CD59 levels were not different between them. Further investigations, such as the analyses of sequential data of CSF CD59 level and simultaneous measurements of CSF CD59 and C5b-9 levels, are required. Other limitation of this study is that we have not demonstrated whether CSF CD59 levels are directly associated with CD59 expression on cell surfaces within the CNS of NMO and MS. 5. Conclusion We showed that CSF CD59 levels increased during the active phase of NMO and MS. The increase in CSF CD59 levels could be secondary to inflammation, damage, and/or complement activation within the CNS of NMO and MS. References [1] A. Uzawa, M. Mori, S. Kuwabara, Neuromyelitis optica: concept, immunology and treatment, J. Clin. Neurosci. 21 (2014) 12–21. [2] S.F. Roemer, J.E. Parisi, V.A. Lennon, et al., Pattern-specific loss of aquaporin-4 immunoreactivity distinguishes neuromyelitis optica from multiple sclerosis, Brain 130 (2007) 1194–1205. [3] H. Wang, K. Wang, C. Wang, et al., Increased soluble C5b-9 in CSF of neuromyelitis optica, Scand. J. Immunol. 79 (2014) 127–130. [4] P.F. Zipfel, C. Skerka, Complement regulators and inhibitory proteins, Nat. Rev. Immunol. 9 (2009) 729–740. [5] S. Saadoun, M.C. Papadopoulos, Role of membrane complement regulators in neuromyelitis optica, Mult. Scler. 21 (2015) 1644–1654. [6] R.J. Mead, J.W. Neal, M.R. Griffiths, et al., Deficiency of the complement regulator CD59a enhances disease severity, demyelination and axonal injury in murine acute experimental allergic encephalomyelitis, Lab. Investig. 84 (2004) 21–28. [7] H. Zhang, A.S. Verkman, Longitudinally extensive NMO spinal cord pathology produced by passive transfer of NMO-IgG in mice lacking complement inhibitor CD59, J. Autoimmun. 53 (2014) 67–77. [8] D.M. Wingerchuk, B. Banwell, J.L. Bennett, et al., International consensus diagnostic criteria for neuromyelitis optica spectrum disorders, Neurology 85 (2015) 177–189. [9] C.H. Polman, S.C. Reingold, G. Edan, et al., Diagnostic criteria for multiple sclerosis: 2005 revisions to the “McDonald Criteria, Ann. Neurol. 58 (2005) 840–846. [10] S. Hayakawa, M. Mori, A. Okuta, et al., Neuromyelitis optica and anti-aquaporin-4 antibodies measured by an enzyme-linked immunosorbent assay, J. Neuroimmunol. 196 (2008) 181–187. [11] A. Uzawa, M. Mori, S. Sawai, et al., Cerebrospinal fluid interleukin-6 and glial fibrillary acidic protein levels are increased during initial neuromyelitis optica attacks, Clin. Chim. Acta 421 (2013) 181–183.
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Increased levels of CSF CD59 in neuromyelitis optica and multiple sclerosis Akiyuki Uzawa ⁎, Masahiro Mori, Tomohiko Uchida, Hiroki Masuda, Ryohei Ohtani, Satoshi Kuwabara Department of Neurology, Graduate School of Medicine, Chiba University, Japan
a r t i c l e
i n f o
Article history: Received 27 October 2015 Received in revised form 10 December 2015 Accepted 10 December 2015 Available online 11 December 2015 Keywords: Cerebrospinal fluid CD59 Complement Interleukin-6 Multiple sclerosis Neuromyelitis optica
a b s t r a c t Background: Complement activation is important in multiple sclerosis (MS) and is essential for anti-aquaporin 4 antibodies to damage the central nervous system in neuromyelitis optica (NMO). Little is known about the role of cerebrospinal fluid (CSF) regulators of complement activation in NMO and MS. We determined whether CSF CD59, which is a complement regulator and C5b-9 formation inhibitor, is involved in the pathogenesis of NMO and MS. Methods: We analyzed CSF levels of CD59 in 30 patients with NMO, 22 patients with MS, and 24 patients with non-inflammatory neurological disorders (NINDs). Possible correlations between CSF CD59 levels and the clinical and laboratory variables in patients with NMO and MS were also reviewed. Results: CSF CD59 levels in patients with NMO and MS were higher than those in patients with NINDs (p b 0.001), and those in patients with NMO decreased after treatment. No significant correlations were found between CSF CD59 levels and clinical and laboratory parameters in NMO and MS. Conclusion: High CSF CD59 levels in NMO and MS may reflect inflammation, damage, and/or complement activation in the central nervous system. © 2015 Elsevier B.V. All rights reserved.
1. Introduction
2. Methods
Neuromyelitis optica (NMO) and multiple sclerosis (MS) are different from the viewpoints of clinical features, pathology, immunology, neuroimaging, and response to treatment but are similar from the viewpoint of autoimmune-mediated inflammatory disorders of the central nervous system (CNS) [1]. Complement is thought to play important roles in the pathogenesis of these disorders. Deposits of complement have been observed in NMO and MS lesions [2]. Increased cerebrospinal fluid (CSF) levels of soluble C5b-9 have been observed in patients with NMO and MS, indicating that complement has been activated [3]. The complement regulator CD59 is a phosphoinositol-linked glycoprotein that inhibits the terminal membrane attack complex. It is also the major complement inhibitor protein in human astrocytes and glioma cell lines [4,5]. In mice, CD59a is expressed mainly on astrocytes and weakly on oligodendrocytes and neurons within the CNS [6]. Greatly increased NMO pathology in CNS has been reported in mice with CD59 knockout or those with CD59 inhibition by a neutralizing monoclonal antibody [7]. Although the importance of complement in NMO and MS has been established, the role of complement regulation in the pathogenesis of NMO and MS is not completely understood.
2.1. Subjects NMO spectrum disorder patients (n = 30) who met the 2015 diagnostic criteria [8] and relapsing–remitting MS patients (n = 22) who met the 2005 McDonald criteria [9] were involved in this study. Twenty-four patients (13 men, 11 women; mean age, 58.9 y) with non-inflammatory neurological disorders (NINDs), including 10 with amyotrophic lateral sclerosis, 8 with spinocerebellar degeneration, 4 with Parkinson's disease, and 2 with progressive supranuclear palsy, were recruited as controls. We reviewed the gender, age, attack site, expanded disability status scale (EDSS) scores, positivity for oligoclonal bands, positivity for serum anti-aquaporin 4 (AQP4) antibody [10], and CSF variables (including cell counts, protein, interleukin (IL)-6 levels measured using a chemiluminescent enzyme immunoassay [11], CSF/serum albumin ratio (albumin quotient, QAlb), and IgG index), at the time of sampling. The ethics committee of the Chiba University School of Medicine, Chiba, Japan approved the study, and informed consent was obtained from all study subjects. 2.2. CSF CD59 measurements
⁎ Corresponding author at: Department of Neurology, Graduate School of Medicine, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail address: [email protected] (A. Uzawa).
http://dx.doi.org/10.1016/j.cca.2015.12.013 0009-8981/© 2015 Elsevier B.V. All rights reserved.
CSF samples were obtained during the active disease phase (within 1 month of clinical attack and before treatment for the attack) from patients with NMO and MS. After treatment for the acute attack, CSF
132
A. Uzawa et al. / Clinica Chimica Acta 453 (2016) 131–133
Table 1 Clinical characteristics of patients with NMO and MS. NMO (n = 30)
MS (n = 22)
Men:women Age, y, mean ± SD EDSS; median (range) Positivity for anti-AQP4 antibody Positivity for oligoclonal bands
2:28 48.7 ± 14.6 7.0 (2.0–9.0) 26/30 (87%) 3/20 (15%)
Site of attack
S 21, B 3, BS 3, O 3
5:17 37.1 ± 17.2 4.5 (1.0–8.0) 0/22 (0%) 13/20 (65%) S 12, B 3, BS 2, BS and S 2, O 2, C and O 1
AQP4: aquaporin 4; B: brain (cerebrum); BS: brainstem; C: cerebellum; EDSS: Expanded Disability Status Scale; MS: multiple sclerosis; NMO: neuromyelitis optica; O: optic nerve; S: spinal cord.
samples were obtained from only four patients with NMO and none of the patients with MS. All samples were stored at − 80 °C until they were assayed. The CSF CD59 levels were measured using a CD59 ELISA kit (MyBioSource, Inc.) according to the manufacturer's instructions. The optical density was measured at 450 nm. CD59 levels were calculated with reference to a standard curve. The detection range was 31.25–2000 pg/ml.
Fig. 1. CSF CD59 levels in patients with NMO, MS, and NINDs. (a) CD59 levels in CSF of 30 NMO patients, 22 MS patients, and 24 NIND patients. Levels in patients with NMO and MS were significantly higher than those in patients with NINDs (**p b 0.001, Mann–Whitney U-test). Dashed lines indicate the mean concentration in each group. (b) The CSF CD59 levels in patients with NMO (n = 4) decreased after treatment. Pre-Tx: pre-treatment; post-Tx: post-treatment.
2.3. Correlation between the CSF CD59 levels and clinical parameters
3.2. CSF CD59 levels
We examined possible associations between the CSF CD59 levels and various clinical and laboratory variables, including EDSS, positivity for serum anti-AQP4 antibody titers, positivity for oligoclonal bands, IgG index, CSF cell counts, CSF protein levels, CSF IL-6 levels, and QAlb, in patients with NMO and MS.
CSF CD59 levels were 275.5 ± 77.4 (mean ± SD) pg/ml, 298.3 ± 67.7 pg/ml, and 181.8 ± 51.3 pg/ml in patients with NMO, MS, and NINDs, respectively. Kruskal–Wallis test revealed significant differences in NMO, MS, and NINDs group (p b 0.001). The CSF CD59 levels in patients with NMO and MS were similar and significantly higher than those in patients with NINDs (p b 0.001, Mann–Whitney U-test) (Fig. 1). We obtained CSF samples from only four patients with NMO after treatment for an acute attack (all four patients received highdose intravenous methylprednisolone treatment and one received additional plasmapheresis therapy). The CSF CD59 levels decreased after treatment (Fig. 1).
2.4. Statistical analyses Groups were compared using Kruskal–Wallis test and the Mann– Whitney U-test for unpaired continuous measures. For multiple comparisons, Bonferroni correction was applied. Each parameter in this study did not follow a normal distribution, hence non-parametric tests, instead of ANOVA, were used. Spearman's rank correlation coefficient was used to evaluate the statistical dependency of two variables. A p b 0.05 was considered statistically significant. 3. Results 3.1. Clinical profiles The clinical characteristics of the patients with NMO and MS are summarized in Table 1. The patients' proportion of woman, age, EDSS score, positivity for serum anti-AQP4 antibody, and negativity for oligoclonal bands at CSF sampling were higher in patients with NMO than in patients with MS. The CSF findings in patients with NMO, MS, and NINDs are summarized in Table 2. The CSF cell counts, protein levels, IL-6 levels, and QAlb in patients with NMO were significantly higher than those in patients with MS and NINDs. CSF cell counts were significantly higher in MS than in NINDs. IgG indexes were significantly increased in NMO and MS compared with NINDs.
3.3. Correlations between CSF CD59 levels and clinical variables No significant correlations were observed between CSF CD59 levels and clinical and laboratory variables (EDSS scores, IgG index, CSF cell counts, CSF protein levels, CSF IL-6 levels, Qalb, anti-AQP4 antibody titers, or oligoclonal band positivity) in patients with NMO and MS (Table 3). There was no association of CSF CD59 levels with site of lesions. 4. Discussion This study confirmed significant increases in the CSF CD59 levels in patients with NMO and MS compared with those in patients with NINDs. Anti-AQP4 antibody binds to AQP4 at the astrocytic end-feet and causes CNS inflammation mainly by complement-dependent cytotoxicity [7]. Furthermore, complement depositions in lesion areas [2] and upregulated CSF-soluble C5b-9 [3] have been reported in both NMO and MS. In this regard, these disorders are similar, and complement
Table 2 CSF findings in patients. CSF findings
Cell count (cells/mm3) Protein level (mg/dl) IL-6 level (pg/ml) QAlb × 103 IgG index
NMO (n = 30)
28.3 ± 38.0 101.1 ± 152.0 1125.9 ± 3179.0 14.3 ± 11.9 0.70 ± 0.23
MS (n = 22)
6.7 ± 9.5 31.5 ± 8.6 2.9 ± 1.1 5.1 ± 2.0 0.72 ± 0.27
NINDs (n = 24)
0.7 ± 0.7 34.4 ± 12.5 2.3 ± 1.2 4.2 ± 1.1 0.48 ± 0.08
p value NMO vs. MS
NMO vs. NINDs
MS vs. NINDs
0.008 b0.001 b0.001 b0.001 0.859
b0.001 b0.001 b0.001 b0.001 b0.001
b0.001 0.321 0.064 0.185 b0.001
MS: multiple sclerosis; NINDs: non-inflammatory neurological disorders; NMO: neuromyelitis optica; QAlb: albumin quotient = cerebrospinal/serum albumin ratio. Each value represents the mean ± SD. The Mann–Whitney U-test was used for statistical analyses.
A. Uzawa et al. / Clinica Chimica Acta 453 (2016) 131–133 Table 3 Associations between clinical and laboratory characteristics and CD59 levels in patients with NMO and MS.
EDSS score IgG index CSF cell count CSF protein level CSF IL-6 level Qalb AQP4 antibody titer Oligoclonal bands positivitya
NMO
MS
R = 0.106 (p = 0.593) R = 0.290 (p = 0.174) R = −0.307 (p = 0.098) R = −0.293 (p = 0.115) R = −0.306 (p = 0.099) R = −0.111 (p = 0.610) R = 0.026 (p = 0.890) p = 0.315
R = 0.405 (p = 0.066) R = 0.076 (p = 0.749) R = 0.209 (p = 0.340) R = −0.072 (p = 0.732) R = 0.333 (p = 0.127) R = 0.225 (p = 0.341) NA p = 0.843
AQP4: aquaporin 4; EDSS: Expanded Disability Status Scale; IL-6: interleukin 6; MS: multiple sclerosis; NA: not available; NMO: neuromyelitis optica; QAlb: albumin quotient = cerebrospinal/serum albumin ratio. Spearman's rank correlation coefficient was used to evaluate the dependency of 2 variables. a Evaluated by Mann–Whitney U-test.
activation plays a central role in their pathogenesis. Hence, we assumed that CSF complement regulators are upregulated to suppress complement activation in the CNS or in contrast, that CSF complement regulators are downregulated for an unknown reason, and this leads to complement activation in these disorders. Our results showed that CSF CD59 levels were similarly high in both NMO and MS during attack, which might be caused by issues secondary to CNS inflammation, CNS damage, and/or complement activation. Mice with a CD59 knockout or those with CD59 inhibition induced by a neutralizing monoclonal antibody showed severe deterioration of NMO pathology in the CNS [7]. However, we determined that CSF CD59 levels were not decreased but were increased during attack and decreased after treatment, suggesting that the downregulation of CD59 does not play an important role in the pathogenesis of NMO in humans, which is in contrast to the result obtained using an NMO mouse model [7]. Recently, it has been reported that CD59 is expressed in the microvascular endothelium of normal brain but is absent from NMO lesions [5]. Therefore, high CSF CD59 levels in NMO and MS may reflect damage to the vascular endothelium; however, CSF CD59 levels did not correlate with clinical variables, including Qalb (one of the markers of blood– brain barrier function). Although the increased CSF cell counts and
133
IL-6 levels in patients with NMO might be a result of their more severe CNS inflammation than patients with MS, CSF CD59 levels were not different between them. Further investigations, such as the analyses of sequential data of CSF CD59 level and simultaneous measurements of CSF CD59 and C5b-9 levels, are required. Other limitation of this study is that we have not demonstrated whether CSF CD59 levels are directly associated with CD59 expression on cell surfaces within the CNS of NMO and MS. 5. Conclusion We showed that CSF CD59 levels increased during the active phase of NMO and MS. The increase in CSF CD59 levels could be secondary to inflammation, damage, and/or complement activation within the CNS of NMO and MS. References [1] A. Uzawa, M. Mori, S. Kuwabara, Neuromyelitis optica: concept, immunology and treatment, J. Clin. Neurosci. 21 (2014) 12–21. [2] S.F. Roemer, J.E. Parisi, V.A. Lennon, et al., Pattern-specific loss of aquaporin-4 immunoreactivity distinguishes neuromyelitis optica from multiple sclerosis, Brain 130 (2007) 1194–1205. [3] H. Wang, K. Wang, C. Wang, et al., Increased soluble C5b-9 in CSF of neuromyelitis optica, Scand. J. Immunol. 79 (2014) 127–130. [4] P.F. Zipfel, C. Skerka, Complement regulators and inhibitory proteins, Nat. Rev. Immunol. 9 (2009) 729–740. [5] S. Saadoun, M.C. Papadopoulos, Role of membrane complement regulators in neuromyelitis optica, Mult. Scler. 21 (2015) 1644–1654. [6] R.J. Mead, J.W. Neal, M.R. Griffiths, et al., Deficiency of the complement regulator CD59a enhances disease severity, demyelination and axonal injury in murine acute experimental allergic encephalomyelitis, Lab. Investig. 84 (2004) 21–28. [7] H. Zhang, A.S. Verkman, Longitudinally extensive NMO spinal cord pathology produced by passive transfer of NMO-IgG in mice lacking complement inhibitor CD59, J. Autoimmun. 53 (2014) 67–77. [8] D.M. Wingerchuk, B. Banwell, J.L. Bennett, et al., International consensus diagnostic criteria for neuromyelitis optica spectrum disorders, Neurology 85 (2015) 177–189. [9] C.H. Polman, S.C. Reingold, G. Edan, et al., Diagnostic criteria for multiple sclerosis: 2005 revisions to the “McDonald Criteria, Ann. Neurol. 58 (2005) 840–846. [10] S. Hayakawa, M. Mori, A. Okuta, et al., Neuromyelitis optica and anti-aquaporin-4 antibodies measured by an enzyme-linked immunosorbent assay, J. Neuroimmunol. 196 (2008) 181–187. [11] A. Uzawa, M. Mori, S. Sawai, et al., Cerebrospinal fluid interleukin-6 and glial fibrillary acidic protein levels are increased during initial neuromyelitis optica attacks, Clin. Chim. Acta 421 (2013) 181–183.