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Clinical features of Chinese patients with multiple sclerosis with aquaporin-4 antibodies in cerebrospinal fluid but not serum
Journal of Clinical Neuroscience xxx (2012) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Clinical Study
Clinical features of Chinese patients with multiple sclerosis with aquaporin-4 antibodies in cerebrospinal fluid but not serum Y. Long a,b, , W. Qiu a, , Z. Lu a, F. Peng a, X. Hu a,⇑ a b
Department of Neurology, The Third Affiliated Hospital of Sun Yat-Sen University, 600 Tianhe Road, Guangzhou 510630, Guangdong, China Department of Neurology, The First Affiliated Hospital of Guangdong Pharmaceutical University, Guangzhou, Guangdong, China
a r t i c l e
i n f o
Article history: Received 10 February 2011 Accepted 18 December 2011 Available online xxxx Keywords: Antibody formation Aquaporin 4 Cerebrospinal fluid Neuromyelitis optica Multiple sclerosis
a b s t r a c t The objective of this study was to determine the levels of aquaporin-4 (AQP4) antibodies in the cerebrospinal fluid (CSF) of patients meeting the diagnostic criteria for multiple sclerosis (MS) and to describe some of the clinical features of CSF-positive cases. Thirty-five patients fulfilling the diagnostic criteria for MS but not neuromyelitis optica were included in this study. AQP4 antibodies were detected using a cell-based assay. None of the serum samples were positive for AQP4 antibodies. Five CSF samples (14.3%, 5/35) were positive for AQP4 antibodies. All CSF-positive patients had atypical brain lesions in areas known to have high levels of AQP4 expression. CSF AQP4 antibody testing is an important diagnostic aid in patients meeting MS criteria but with atypical brain or spinal lesions and serum negative for AQP4 antibodies. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Diagnosis of multiple sclerosis (MS) is largely clinical and based on the finding of MS lesions in multiple areas of the central nervous system (CNS) over time.1 Neuromyelitis optica (NMO) is a severe idiopathic immune-mediated inflammatory demyelinating and necrotizing disease that is characterized by transverse myelopathy and optic neuropathy. Currently, MS and NMO are defined as two different disorders.2 The diagnostic criteria for NMO, as proposed by Wingerchuk et al., facilitate its distinction from MS.3,4 Because different treatments are used for the two diseases, it is desirable to distinguish them as early as possible. The exact etiology of MS and the mechanisms underlying it remain unknown, but clinical data from MS patients suggest that it is an immune-mediated disorder, which is the generally accepted pathophysiological model. Autoreactive T cells are thought to initiate an autoimmune response directed against components of CNS myelin.5 In NMO, serological and clinical evidence of B-cell autoimmunity has been observed in a high proportion of patients,6 suggesting a prominent role for humoral mechanisms in its pathogenesis. NMO lesions have notable immunoglobulin and complement deposits in a characteristic perivascular rosette pattern along the outer rims of thickened
⇑ Corresponding author. Tel./fax: +86 20 85252336.
E-mail address: [email protected] (X. Hu). These authors have contributed equally to this work.
vessel walls.7 Using indirect immunofluorescence, a specific immunoglobulin G (IgG) autoantibody, NMO-IgG, which selectively targets aquaporin-4 (AQP4), has been found in CNS microvessels, pia, subpia, and Virchow-Robin spaces in composite substrates of mouse tissue.8,9 Lennon and colleagues8,9 determined that NMO-IgG had a sensitivity of 73% and a specificity of 91% for differentiating patients with clinically definitive NMO from those with myelitis and optic neuritis in a North American population, but did not meet the strict criteria for the diagnosis of NMO. In a Japanese population, NMO-IgG was found to have 58% sensitivity and 100% specificity for identifying opticospinal MS. In a subsequent study, analysis of pathological brain lesions in NMO patients revealed a vasculocentric pattern of immune complex deposition and AQP4 loss identical to that seen in NMO lesions, suggesting that the NMO-IgG autoantibody is an important contributor to NMO pathology.10 Previous data strongly support the hypothesis that NMO-IgG plays a central role in NMO pathogenesis.11 However, research into the humoral immune responses involved in the immunopathogenesis of MS has led to a reconsideration of the importance of B cells and antibodies in MS.12 In a previous study, positive anti-AQP4 antibodies were detected in serum in patients with a diagnosis of classical MS.13 There is little data available on whether AQP4 antibodies can be detected in cerebrospinal fluid (CSF) in patients with serum negative for these antibodies. The aims of this study were (a) to determine the levels of AQP4 antibodies in the CSF of patients meeting the diagnostic criteria for MS and not NMO; and (b) to define any clinical features of patients with CSF positive for AQP4 antibodies.
0967-5868/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jocn.2011.12.037
Please cite this article in press as: Long Y et al. Clinical features of Chinese patients with multiple sclerosis with aquaporin-4 antibodies in cerebrospinal fluid but not serum. J Clin Neurosci (2012), http://dx.doi.org/10.1016/j.jocn.2011.12.037
2
Y. Long et al. / Journal of Clinical Neuroscience xxx (2012) xxx–xxx
Table 1 Demographic and clinical characteristics of patients with multiple sclerosis, with and without aquaporin-4 (AQP-4) antibodies in their cerebrospinal fluid
n Sex ratio (female/male) Age (years) Disease duration (months) EDSS score CSF protein (g/L) CSF cells (n/mm3) OCB (%) IgG index Brain lesions (%) MS-like Gd-enhancing Thalamus Internal capsule Corpus callosum Around ventricle Spinal lesions (%) P3 segments Gd-enhancing
Total
CSF-negative
CSF-positive
p-value
35 1.06 (18/17) 32.94 ± 11.61 44.51 ± 80.82 2.31 ± 1.67 0.22 ± 0.13 4.66 ± 10.99 25 (4/16) 0.55 ± 0.19
30 1.14 (16/14) 35.74 ± 13.14 43.70 ± 81.68 1.96 ± 1.24 0.21 ± 0.11 3.92 ± 5.91 28.6 (4/14) 0.61 ± 0.21
5 1.5 (3/2) 27.4 ± 13.60 15.8 ± 9.65 2.6 ± 1.56 0.17 ± 0.34 1.00 ± 2.24 0 (0/2) 0.52 ± 0.08
1.00 0.24 0.46 0.32 0.11 0.37 1.0 0.27
85.7 (30/35) 28.6 (10/35) 17.1 (6/35) 8.6 (3/35) 8.6 (3/35) 11.4 (4/35)
86.7 (26/30) 32.1 (9/28) 0 0 0 0
80 (4/5) 20 (1/5) 100 (5/5) 60 (3/5) 60 (3/5) 80 (4/5)
1.0 1.0 <0.001 0.002 0.002 <0.001
11.43 (4/35) 0 (0/34)
3.3 (1/30) 0 (0/30)
40 (2/5) 0
0.049 1.0
EDSS = Expanded Disability Status Scale, CSF = cerebrospinal fluid, OCB = oligoclonal bands, IgG = immunoglobulin G, MS = multiple sclerosis, Gd = gadolinium. P-values are for the comparison between the CSF-negative group and the CSF-positive group. Values are ±standard deviation; values in parentheses are n.
teristics of each relapse and treatment required. Patients eligible for the study were required to have undergone the following studies: full blood count, clinical biochemical testing, erythrocyte sedimentation rate, antinuclear antibodies, CSF examination, urinalysis, chest X-ray and tests to exclude other diseases, especially infectious diseases, other immune diseases and CNS tumors. CSF oligoclonal bands (OCB), CSF IgG level, brainstem auditory evoked potential, visual evoked potential, somatosensory evoked potential, serum immunoglobulin, C-reactive protein and serum complement data were available for some patients. All patients were scored using the Expanded Disability Status Scale (EDSS)15 at the most recent clinical examination.
2. Patients and methods 2.1. Patients Patients were admitted to the MS center of the Third Affiliated Hospital of Sun Yat-sen University between August 2006 and March 2010. Patients were excluded from this study if they did not meet the McDonald criteria for MS14 or if at onset or during the follow-up period they met the 2006 NMO diagnostic criteria.4 There were 35 patients who met the inclusion criteria. Data were collected from patient records retrospectively. Data included age, sex, number of demyelinating events, clinical charac-
Table 2 Clinical characteristics of five patients with multiple sclerosis with aquaporin-4 (AQP4) antibodies in their cerebrospinal fluid Patient number
Age (years) Sex Disease duration (months) EDSS score Symptoms Sensory Motor Brainstem Visual Cerebellum Cognitive Sphincter Brain MRI
Spinal MRI
CSF cells (n/mm3) CSF protein (g/L) CSF OCB CSF AQP4 antibody titer
1
2
3
4
5
12 Female 24
48 Male 12
24 Male 24
19 Female 1
22 Female 18
4.5
3.5
1
1
3
Yes Yes Yes No Yes No No Periventricular white matter lesions, thalamus, periaqueductal and fourth ventricle LESCL (C1–C7) and lesions in T6–T7, T9–T10 0 0.17 Negative 1:64
Yes Yes Yes No No No No Multiple lesions in white matter of bilateral cerebral ventricles, corona radiata, internal capsule, thalamus, caudate nucleus cerebral peduncle and around fourth ventricle and corpus callosum None
No Yes Yes No No No No Lesions in white matter of bilateral ventricles, centrum semiovale, corpus callosum, thalamus
Yes Yes Yes No Yes No No Lesions in white matter of bilateral cerebral ventricles, corpus callosum, thalamus and around the aqueduct of the midbrain and cerebellum
LESCL (C3–C6)
Yes No No Yes No No No Multiple lesions in centrum semiovale, corona radiata, internal capsule, thalamus, caudate nucleus, cerebral peduncle, frontal parietal, and occipital lobe None
0 0.16 Negative 1:2
0 0.42 – 1:4
0 0.14 – 1:2
5 0.4 – 1:8
None
EDSS = Expanded Disability Status Scale, CSF = cerebrospinal fluid, LESCL = longitudinally extensive spinal cord lesions, OCB = oligoclonal bands.
Please cite this article in press as: Long Y et al. Clinical features of Chinese patients with multiple sclerosis with aquaporin-4 antibodies in cerebrospinal fluid but not serum. J Clin Neurosci (2012), http://dx.doi.org/10.1016/j.jocn.2011.12.037
Y. Long et al. / Journal of Clinical Neuroscience xxx (2012) xxx–xxx
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Fig. 1. Representative MRI of multiple sclerosis patients who tested positive for aquaporin-4 (AQP4) antibodies in their cerebrospinal fluid. (A) Sagittal T2-weighted and (B) axial fluid attenuated inversion recovery (FLAIR) images of patient 1, who had ataxia and sensory disturbance in both upper limbs (age at onset: 10 years). These images show a cervical spinal cord lesion (C1–C7) and thick lesions around the bilateral periependymal regions (arrows), which are known to have high levels of AQP4 expression. (C, D) Axial FLAIR images of patient 2, showing lesions around the fourth ventricle, internal capsule and caudate nucleus and peduncle (arrows). (E) Sagittal T2-weighted and (F) axial FLAIR images of patient 3, showing lesions in the corpus callosum, around the bilateral cerebral ventricles, thalamus and cerebellum (arrows).
Please cite this article in press as: Long Y et al. Clinical features of Chinese patients with multiple sclerosis with aquaporin-4 antibodies in cerebrospinal fluid but not serum. J Clin Neurosci (2012), http://dx.doi.org/10.1016/j.jocn.2011.12.037
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Y. Long et al. / Journal of Clinical Neuroscience xxx (2012) xxx–xxx
The demographic and clinical characteristics of the patients are presented in Table 1. The mean (±standard deviation) EDSS score was 2.31 ± 1.67 (range: 0–8.5) and the mean disease duration was 44.51 ± 80.82 months (range: 1–273 months). Increased total protein was detected in the CSF of five of the 35 (14%) patients and six of the 35 (17%) had an abnormal CSF white cell count. Four of 16 patients (25%) had OCB. 2.2. MRI Imaging was performed with 1.5-T MRI machines using conventional T1-, T2- and proton density-weighted sequences with gadolinium enhancement in all patients. Patients with MS were considered to have MRI activity if they had one or more enhancing lesions on T1-weighted spin-echo images after contrast injection. MS-like lesions were defined as one or more lesions located in the white matter and greater than 3 mm in diameter, of which at least one was ovoid, periventricular or infratentorial on T2weighted MRI.16 2.3. Anti-AQP4 antibody testing All CSF and serum samples were stored at 80 °C in the main laboratory of the Third Affiliated Hospital of Sun Yat-sen University. Anti-AQP4 antibody levels were determined via an assay using an AQP4-transfected cell line from a commercial BIOCHIP kit (EUROIMMUN, Lübeck, Germany) as described by Jarius et al.17,18 Serum (1:10) or CSF (1:1) was diluted using phosphate-buffered saline (PBS). The diluted sample was combined with AQP4-transfected cells on the BIOCHIP slides for 30 minutes at room temperature. The slides were then rinsed twice with PBS before incubation with fluorescein-conjugated goat anti-human IgG for 30 minutes. Finally, the slides were rinsed with PBS and fluorescence was measured under a microscope. To exclude the possibility of prozone effects,19 sera that were negative at a 1:10 dilution were also tested at 1:4, 1:32 and 1:120 dilutions. A positive reaction was deemed to have occurred when there was staining on the cell surface and in the cytoplasm of the AQP4-transfected cells (Supplementary Fig. 1), but no staining of untransfected cells in the same sample. To confirm the results, positive samples were tested again at 1:4, 1:8, 1:16: 1:32 and 1:64 dilutions. Positive samples were titrated to final dilution. 2.4. Statistical analysis All statistical analyses were performed using the Statistical Program for the Social Sciences (SPSS, version 11.0; SPSS, Chicago, IL, USA). Differences between MS patients who were CSF-positive and CSF-negative were analyzed using Fisher’s exact test and Student’s t-test. P-values of <0.05 were considered significant. 3. Results None of the serum samples tested positive for AQP4 antibodies, but five of 35 (14%) CSF samples were positive. The titers for the positive samples are shown in Table 2. Paraclinical data for patients in the CSF-positive and CSF-negative groups are shown in Table 1. All the CSF-positive patients had atypical brain lesions (Fig. 1) in regions known to have high levels of AQP4 expression (Table 2, Fig. 1).
fact, the presence of these antibodies is a current diagnostic criterion for NMO.4 More than 15 different immunoassays for the detection of AQP4 antibodies in patients with NMO have been described in previous studies, with sensitivity and specificity values varying widely (range of 33–91% for sensitivity, median of 63%; range of 85–100% for specificity, median of 99%).10 Differences in serum AQP4 levels between NMO and MS patients have been described, which suggests that NMO is distinct from MS.8,13,20–28 However, MS patients have also been found to be positive for these antibodies.8 In previous studies,8,13,20–28 1.25–9% of tested MS patients were positive for NMO-IgG or AQP4 antibodies. In the present study, no antibody-positive serum was detected among MS patients when measured using a cell-based substrate. Among the 35 patients who met the diagnostic criteria for MS, the CSF test was positive in five (14.3%) as measured using a cellbased assay. Our findings suggest a relationship between CSFAQP4 antibodies and clinical MRI features. We retrospectively compared the demographic and clinical characteristics of patients who were CSF-positive with those who were CSF-negative. Our results confirm that the brain and spinal cord lesions are distinctive in these two groups, suggesting a relationship between CNS AQP4 antibodies and lesion location (Table 1). Previous MRI studies show that such brain lesions (surrounding the ventricle and in the basal ganglia, internal capsule and thalamus) and longitudinally extensive spinal cord lesions (LESCL) (Fig. 1) are atypical for MS.21 The distribution of these characteristic lesions corresponds to sites of high AQP4 expression and they are characteristic of NMO.23,29,30 This may indicate that CSF AQP4 antibodies easily reach the lesions and react with AQP4 water channels inducing a local inflammatory response. Humoral mechanisms appear to play important immune pathogenetic roles in the initiation and propagation of lesions in our CSF-positive patients, and such lesions are likely have a similar pathogenesis to NMO. In fact, Miller et al.31 proposed the presence of AQP4 antibodies in CSF as a minor criterion for NMO diagnosis. Although our five CSF-positive patients all met the McDonald criteria for MS, one of the exclusion criteria is that there be no other more plausible explanation for the demyelinating syndrome. The findings that all five clearly had lesions that are atypical for MS (in the thalamus) and that two had LESCL call into question the diagnoses of MS. Thus, these patients may have not actually had MS and probably had NMO, particularly the two patients with LESCL. They may have been limited or inaugural syndromes of NMO. However, Miller et al.31 believe that these spatially limited syndromes should not qualify as NMO and that the subsequent development of optic neuritis may permit a later diagnosis of NMO. We hope that follow-up studies of large samples of MS patients will be performed in future, particularly those that include detailed immunohistochemical studies of autopsy and brain biopsy tissue specimens. Our results indicate that CSF AQP4 antibody testing is an important aid in diagnosing patients who meet MS criteria but have atypical brain or spinal lesions and serum negative for APQ4 antibodies.
Conflict of interest/Disclosures The authors declare that they have no financial or other conflicts of interest in relation to this research and its publication.
4. Discussion
Acknowledgment
Serum AQP4 antibodies have been shown to be highly specific and moderately to highly sensitive biomarkers for NMO;8,10,13 in
This work was supported by funds from the Technology Project of Guangzhou City (No. 2060402).
Please cite this article in press as: Long Y et al. Clinical features of Chinese patients with multiple sclerosis with aquaporin-4 antibodies in cerebrospinal fluid but not serum. J Clin Neurosci (2012), http://dx.doi.org/10.1016/j.jocn.2011.12.037
Y. Long et al. / Journal of Clinical Neuroscience xxx (2012) xxx–xxx
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jocn.2011.12.037. References 1. Poser CM, Brinar VV. Diagnostic criteria for multiple sclerosis. Clin Neurol Neurosurg 2001;103:1–11. 2. de Seze J, Lebrun C, Stojkovic T, et al. Is Devic’s neuromyelitis optica a separate disease? A comparative study with multiple sclerosis. Mult Scler 2003;9:521–5. 3. Wingerchuk DM, Hogancamp WF, O’Brien PC, et al. The clinical course of neuromyelitis optica (Devic’s syndrome). Neurology 1999;53:1107–14. 4. Wingerchuk DM, Lennon VA, Pittock SJ, et al. Revised diagnostic criteria for neuromyelitis optica. Neurology 2006;66:1485–9. 5. O’Brien K, Gran B, Rostami A. T-cell based immunotherapy in experimental autoimmune encephalomyelitis and multiple sclerosis. Immunotherapy 2010;2:99–115. 6. Hamnik SE, Hacein-Bey L, Biller J, et al. Neuromyelitis optica (NMO) antibody positivity in patients with transverse myelitis and no visual manifestations. Semin Ophthalmol 2008;23:191–200. 7. Lucchinetti CF, Mandler RN, McGavern D, et al. A role for humoral mechanisms in the pathogenesis of Devic’s neuromyelitis optica. Brain 2002;125:1450–61. 8. Lennon VA, Wingerchuk DM, Kryzer TJ, et al. A serum autoantibody marker of neuromyelitis optica: distinction from multiple sclerosis. Lancet 2004;364:2106–12. 9. Lennon VA, Kryzer TJ, Pittock SJ, et al. IgG marker of optic-spinal multiple sclerosis binds to the aquaporin-4 water channel. J Exp Med 2005;202:473–7. 10. Jarius S, Wildemann B. AQP4 antibodies in neuromyelitis optica: diagnostic and pathogenetic relevance. Nat Rev Neurol 2010;6:383–92. 11. Jarius S, Paul F, Franciotta D, et al. Mechanisms of disease: aquaporin-4 antibodies in neuromyelitis optica. Nat Clin Pract Neurol 2008;4:202–14. 12. Reindl M, Kuenz B, Berger T. B cells and antibodies in MS. Results Probl Cell Differ 2010;51:99–113. 13. Matsuoka T, Matsushita T, Kawano Y, et al. Heterogeneity of aquaporin-4 autoimmunity and spinal cord lesions in multiple sclerosis in Japanese. Brain 2007;130:1206–23. 14. Polman CH, Reingold SC, Edan G, et al. Diagnostic criteria for multiple sclerosis: 2005 revisions to the ‘‘McDonald Criteria’’. Ann Neurol 2005;58:840–6. 15. Kurtzke JF. Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS). Neurology 1983;33:1444–52.
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16. Barkhof F, Polman CH, Radue EW, et al. Magnetic resonance imaging effects of interferon beta-1b in the BENEFIT study: integrated 2-year results. Arch Neurol 2007;64:1292–8. 17. Jarius S, Probst C, Borowski K, et al. Standardized method for the detection of antibodies to aquaporin-4 based on a highly sensitive immunofluorescence assay employing recombinant target antigen. J Neurol Sci 2010;291:52–6. 18. Jarius S, Franciotta D, Paul F, et al. Cerebrospinal fluid antibodies to aquaporin-4 in neuromyelitis optica and related disorders: frequency, origin, and diagnostic relevance. J Neuroinflammation 2010;7:52. 19. Dubois-Galopin F, Beauvillain C, Dubois D, et al. New markers and an old phenomenon: prozone effect disturbing detection of filaggrin (keratin) autoantibodies. Ann Rheum Dis 2007;66:1121–2. 20. Fazio R, Malosio ML, Lampasona V, et al. Antiacquaporin 4 antibodies detection by different techniques in neuromyelitis optica patients. Mult Scler 2009;15:1153–63. 21. Nakashima I, Fujihara K, Miyazawa I, et al. Clinical and MRI features of Japanese patients with multiple sclerosis positive for NMO-IgG. J Neurol Neurosurg Psychiatry 2006;77:1073–5. 22. Takahashi T, Fujihara K, Nakashima I, et al. Establishment of a new sensitive assay for anti-human aquaporin-4 antibody in neuromyelitis optica. Tohoku J Exp Med 2006;210:307–13. 23. Paul F, Jarius S, Aktas O, et al. Antibody to aquaporin 4 in the diagnosis of neuromyelitis optica. PLoS Med 2007;4:e133. 24. Hayakawa S, Mori M, Okuta A, et al. Neuromyelitis optica and anti-aquaporin-4 antibodies measured by an enzyme-linked immunosorbent assay. J Neuroimmunol 2008;196:181–7. 25. Tanaka K, Tani T, Tanaka M, et al. Anti-aquaporin 4 antibody in selected Japanese multiple sclerosis patients with long spinal cord lesions. Mult Scler 2007;13:850–5. 26. Jarius S, Franciotta D, Bergamaschi R, et al. NMO-IgG in the diagnosis of neuromyelitis optica. Neurology 2007;68:1076–7. 27. Marignier R, De Seze J, Vukusic S, et al. NMO-IgG and Devic’s neuromyelitis optica: a French experience. Mult Scler 2008;14:440–5. 28. Marnetto F, Hellias B, Granieri L, et al. Western blot analysis for the detection of serum antibodies recognizing linear Aquaporin-4 epitopes in patients with Neuromyelitis Optica. J Neuroimmunol 2009;217:74–9. 29. Pittock SJ, Weinshenker BG, Lucchinetti CF, et al. Neuromyelitis optica brain lesions localized at sites of high aquaporin 4 expression. Arch Neurol 2006;63:964–8. 30. Pittock SJ, Lennon VA, Krecke K, et al. Brain abnormalities in neuromyelitis optica. Arch Neurol 2006;63:390–6. 31. Miller DH, Weinshenker BG, Filippi M. Differential diagnosis of suspected multiple sclerosis: a consensus approach. Mult Scler 2008;14:1157–74.
Please cite this article in press as: Long Y et al. Clinical features of Chinese patients with multiple sclerosis with aquaporin-4 antibodies in cerebrospinal fluid but not serum. J Clin Neurosci (2012), http://dx.doi.org/10.1016/j.jocn.2011.12.037
Contents lists available at SciVerse ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Clinical Study
Clinical features of Chinese patients with multiple sclerosis with aquaporin-4 antibodies in cerebrospinal fluid but not serum Y. Long a,b, , W. Qiu a, , Z. Lu a, F. Peng a, X. Hu a,⇑ a b
Department of Neurology, The Third Affiliated Hospital of Sun Yat-Sen University, 600 Tianhe Road, Guangzhou 510630, Guangdong, China Department of Neurology, The First Affiliated Hospital of Guangdong Pharmaceutical University, Guangzhou, Guangdong, China
a r t i c l e
i n f o
Article history: Received 10 February 2011 Accepted 18 December 2011 Available online xxxx Keywords: Antibody formation Aquaporin 4 Cerebrospinal fluid Neuromyelitis optica Multiple sclerosis
a b s t r a c t The objective of this study was to determine the levels of aquaporin-4 (AQP4) antibodies in the cerebrospinal fluid (CSF) of patients meeting the diagnostic criteria for multiple sclerosis (MS) and to describe some of the clinical features of CSF-positive cases. Thirty-five patients fulfilling the diagnostic criteria for MS but not neuromyelitis optica were included in this study. AQP4 antibodies were detected using a cell-based assay. None of the serum samples were positive for AQP4 antibodies. Five CSF samples (14.3%, 5/35) were positive for AQP4 antibodies. All CSF-positive patients had atypical brain lesions in areas known to have high levels of AQP4 expression. CSF AQP4 antibody testing is an important diagnostic aid in patients meeting MS criteria but with atypical brain or spinal lesions and serum negative for AQP4 antibodies. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Diagnosis of multiple sclerosis (MS) is largely clinical and based on the finding of MS lesions in multiple areas of the central nervous system (CNS) over time.1 Neuromyelitis optica (NMO) is a severe idiopathic immune-mediated inflammatory demyelinating and necrotizing disease that is characterized by transverse myelopathy and optic neuropathy. Currently, MS and NMO are defined as two different disorders.2 The diagnostic criteria for NMO, as proposed by Wingerchuk et al., facilitate its distinction from MS.3,4 Because different treatments are used for the two diseases, it is desirable to distinguish them as early as possible. The exact etiology of MS and the mechanisms underlying it remain unknown, but clinical data from MS patients suggest that it is an immune-mediated disorder, which is the generally accepted pathophysiological model. Autoreactive T cells are thought to initiate an autoimmune response directed against components of CNS myelin.5 In NMO, serological and clinical evidence of B-cell autoimmunity has been observed in a high proportion of patients,6 suggesting a prominent role for humoral mechanisms in its pathogenesis. NMO lesions have notable immunoglobulin and complement deposits in a characteristic perivascular rosette pattern along the outer rims of thickened
⇑ Corresponding author. Tel./fax: +86 20 85252336.
E-mail address: [email protected] (X. Hu). These authors have contributed equally to this work.
vessel walls.7 Using indirect immunofluorescence, a specific immunoglobulin G (IgG) autoantibody, NMO-IgG, which selectively targets aquaporin-4 (AQP4), has been found in CNS microvessels, pia, subpia, and Virchow-Robin spaces in composite substrates of mouse tissue.8,9 Lennon and colleagues8,9 determined that NMO-IgG had a sensitivity of 73% and a specificity of 91% for differentiating patients with clinically definitive NMO from those with myelitis and optic neuritis in a North American population, but did not meet the strict criteria for the diagnosis of NMO. In a Japanese population, NMO-IgG was found to have 58% sensitivity and 100% specificity for identifying opticospinal MS. In a subsequent study, analysis of pathological brain lesions in NMO patients revealed a vasculocentric pattern of immune complex deposition and AQP4 loss identical to that seen in NMO lesions, suggesting that the NMO-IgG autoantibody is an important contributor to NMO pathology.10 Previous data strongly support the hypothesis that NMO-IgG plays a central role in NMO pathogenesis.11 However, research into the humoral immune responses involved in the immunopathogenesis of MS has led to a reconsideration of the importance of B cells and antibodies in MS.12 In a previous study, positive anti-AQP4 antibodies were detected in serum in patients with a diagnosis of classical MS.13 There is little data available on whether AQP4 antibodies can be detected in cerebrospinal fluid (CSF) in patients with serum negative for these antibodies. The aims of this study were (a) to determine the levels of AQP4 antibodies in the CSF of patients meeting the diagnostic criteria for MS and not NMO; and (b) to define any clinical features of patients with CSF positive for AQP4 antibodies.
0967-5868/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jocn.2011.12.037
Please cite this article in press as: Long Y et al. Clinical features of Chinese patients with multiple sclerosis with aquaporin-4 antibodies in cerebrospinal fluid but not serum. J Clin Neurosci (2012), http://dx.doi.org/10.1016/j.jocn.2011.12.037
2
Y. Long et al. / Journal of Clinical Neuroscience xxx (2012) xxx–xxx
Table 1 Demographic and clinical characteristics of patients with multiple sclerosis, with and without aquaporin-4 (AQP-4) antibodies in their cerebrospinal fluid
n Sex ratio (female/male) Age (years) Disease duration (months) EDSS score CSF protein (g/L) CSF cells (n/mm3) OCB (%) IgG index Brain lesions (%) MS-like Gd-enhancing Thalamus Internal capsule Corpus callosum Around ventricle Spinal lesions (%) P3 segments Gd-enhancing
Total
CSF-negative
CSF-positive
p-value
35 1.06 (18/17) 32.94 ± 11.61 44.51 ± 80.82 2.31 ± 1.67 0.22 ± 0.13 4.66 ± 10.99 25 (4/16) 0.55 ± 0.19
30 1.14 (16/14) 35.74 ± 13.14 43.70 ± 81.68 1.96 ± 1.24 0.21 ± 0.11 3.92 ± 5.91 28.6 (4/14) 0.61 ± 0.21
5 1.5 (3/2) 27.4 ± 13.60 15.8 ± 9.65 2.6 ± 1.56 0.17 ± 0.34 1.00 ± 2.24 0 (0/2) 0.52 ± 0.08
1.00 0.24 0.46 0.32 0.11 0.37 1.0 0.27
85.7 (30/35) 28.6 (10/35) 17.1 (6/35) 8.6 (3/35) 8.6 (3/35) 11.4 (4/35)
86.7 (26/30) 32.1 (9/28) 0 0 0 0
80 (4/5) 20 (1/5) 100 (5/5) 60 (3/5) 60 (3/5) 80 (4/5)
1.0 1.0 <0.001 0.002 0.002 <0.001
11.43 (4/35) 0 (0/34)
3.3 (1/30) 0 (0/30)
40 (2/5) 0
0.049 1.0
EDSS = Expanded Disability Status Scale, CSF = cerebrospinal fluid, OCB = oligoclonal bands, IgG = immunoglobulin G, MS = multiple sclerosis, Gd = gadolinium. P-values are for the comparison between the CSF-negative group and the CSF-positive group. Values are ±standard deviation; values in parentheses are n.
teristics of each relapse and treatment required. Patients eligible for the study were required to have undergone the following studies: full blood count, clinical biochemical testing, erythrocyte sedimentation rate, antinuclear antibodies, CSF examination, urinalysis, chest X-ray and tests to exclude other diseases, especially infectious diseases, other immune diseases and CNS tumors. CSF oligoclonal bands (OCB), CSF IgG level, brainstem auditory evoked potential, visual evoked potential, somatosensory evoked potential, serum immunoglobulin, C-reactive protein and serum complement data were available for some patients. All patients were scored using the Expanded Disability Status Scale (EDSS)15 at the most recent clinical examination.
2. Patients and methods 2.1. Patients Patients were admitted to the MS center of the Third Affiliated Hospital of Sun Yat-sen University between August 2006 and March 2010. Patients were excluded from this study if they did not meet the McDonald criteria for MS14 or if at onset or during the follow-up period they met the 2006 NMO diagnostic criteria.4 There were 35 patients who met the inclusion criteria. Data were collected from patient records retrospectively. Data included age, sex, number of demyelinating events, clinical charac-
Table 2 Clinical characteristics of five patients with multiple sclerosis with aquaporin-4 (AQP4) antibodies in their cerebrospinal fluid Patient number
Age (years) Sex Disease duration (months) EDSS score Symptoms Sensory Motor Brainstem Visual Cerebellum Cognitive Sphincter Brain MRI
Spinal MRI
CSF cells (n/mm3) CSF protein (g/L) CSF OCB CSF AQP4 antibody titer
1
2
3
4
5
12 Female 24
48 Male 12
24 Male 24
19 Female 1
22 Female 18
4.5
3.5
1
1
3
Yes Yes Yes No Yes No No Periventricular white matter lesions, thalamus, periaqueductal and fourth ventricle LESCL (C1–C7) and lesions in T6–T7, T9–T10 0 0.17 Negative 1:64
Yes Yes Yes No No No No Multiple lesions in white matter of bilateral cerebral ventricles, corona radiata, internal capsule, thalamus, caudate nucleus cerebral peduncle and around fourth ventricle and corpus callosum None
No Yes Yes No No No No Lesions in white matter of bilateral ventricles, centrum semiovale, corpus callosum, thalamus
Yes Yes Yes No Yes No No Lesions in white matter of bilateral cerebral ventricles, corpus callosum, thalamus and around the aqueduct of the midbrain and cerebellum
LESCL (C3–C6)
Yes No No Yes No No No Multiple lesions in centrum semiovale, corona radiata, internal capsule, thalamus, caudate nucleus, cerebral peduncle, frontal parietal, and occipital lobe None
0 0.16 Negative 1:2
0 0.42 – 1:4
0 0.14 – 1:2
5 0.4 – 1:8
None
EDSS = Expanded Disability Status Scale, CSF = cerebrospinal fluid, LESCL = longitudinally extensive spinal cord lesions, OCB = oligoclonal bands.
Please cite this article in press as: Long Y et al. Clinical features of Chinese patients with multiple sclerosis with aquaporin-4 antibodies in cerebrospinal fluid but not serum. J Clin Neurosci (2012), http://dx.doi.org/10.1016/j.jocn.2011.12.037
Y. Long et al. / Journal of Clinical Neuroscience xxx (2012) xxx–xxx
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Fig. 1. Representative MRI of multiple sclerosis patients who tested positive for aquaporin-4 (AQP4) antibodies in their cerebrospinal fluid. (A) Sagittal T2-weighted and (B) axial fluid attenuated inversion recovery (FLAIR) images of patient 1, who had ataxia and sensory disturbance in both upper limbs (age at onset: 10 years). These images show a cervical spinal cord lesion (C1–C7) and thick lesions around the bilateral periependymal regions (arrows), which are known to have high levels of AQP4 expression. (C, D) Axial FLAIR images of patient 2, showing lesions around the fourth ventricle, internal capsule and caudate nucleus and peduncle (arrows). (E) Sagittal T2-weighted and (F) axial FLAIR images of patient 3, showing lesions in the corpus callosum, around the bilateral cerebral ventricles, thalamus and cerebellum (arrows).
Please cite this article in press as: Long Y et al. Clinical features of Chinese patients with multiple sclerosis with aquaporin-4 antibodies in cerebrospinal fluid but not serum. J Clin Neurosci (2012), http://dx.doi.org/10.1016/j.jocn.2011.12.037
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Y. Long et al. / Journal of Clinical Neuroscience xxx (2012) xxx–xxx
The demographic and clinical characteristics of the patients are presented in Table 1. The mean (±standard deviation) EDSS score was 2.31 ± 1.67 (range: 0–8.5) and the mean disease duration was 44.51 ± 80.82 months (range: 1–273 months). Increased total protein was detected in the CSF of five of the 35 (14%) patients and six of the 35 (17%) had an abnormal CSF white cell count. Four of 16 patients (25%) had OCB. 2.2. MRI Imaging was performed with 1.5-T MRI machines using conventional T1-, T2- and proton density-weighted sequences with gadolinium enhancement in all patients. Patients with MS were considered to have MRI activity if they had one or more enhancing lesions on T1-weighted spin-echo images after contrast injection. MS-like lesions were defined as one or more lesions located in the white matter and greater than 3 mm in diameter, of which at least one was ovoid, periventricular or infratentorial on T2weighted MRI.16 2.3. Anti-AQP4 antibody testing All CSF and serum samples were stored at 80 °C in the main laboratory of the Third Affiliated Hospital of Sun Yat-sen University. Anti-AQP4 antibody levels were determined via an assay using an AQP4-transfected cell line from a commercial BIOCHIP kit (EUROIMMUN, Lübeck, Germany) as described by Jarius et al.17,18 Serum (1:10) or CSF (1:1) was diluted using phosphate-buffered saline (PBS). The diluted sample was combined with AQP4-transfected cells on the BIOCHIP slides for 30 minutes at room temperature. The slides were then rinsed twice with PBS before incubation with fluorescein-conjugated goat anti-human IgG for 30 minutes. Finally, the slides were rinsed with PBS and fluorescence was measured under a microscope. To exclude the possibility of prozone effects,19 sera that were negative at a 1:10 dilution were also tested at 1:4, 1:32 and 1:120 dilutions. A positive reaction was deemed to have occurred when there was staining on the cell surface and in the cytoplasm of the AQP4-transfected cells (Supplementary Fig. 1), but no staining of untransfected cells in the same sample. To confirm the results, positive samples were tested again at 1:4, 1:8, 1:16: 1:32 and 1:64 dilutions. Positive samples were titrated to final dilution. 2.4. Statistical analysis All statistical analyses were performed using the Statistical Program for the Social Sciences (SPSS, version 11.0; SPSS, Chicago, IL, USA). Differences between MS patients who were CSF-positive and CSF-negative were analyzed using Fisher’s exact test and Student’s t-test. P-values of <0.05 were considered significant. 3. Results None of the serum samples tested positive for AQP4 antibodies, but five of 35 (14%) CSF samples were positive. The titers for the positive samples are shown in Table 2. Paraclinical data for patients in the CSF-positive and CSF-negative groups are shown in Table 1. All the CSF-positive patients had atypical brain lesions (Fig. 1) in regions known to have high levels of AQP4 expression (Table 2, Fig. 1).
fact, the presence of these antibodies is a current diagnostic criterion for NMO.4 More than 15 different immunoassays for the detection of AQP4 antibodies in patients with NMO have been described in previous studies, with sensitivity and specificity values varying widely (range of 33–91% for sensitivity, median of 63%; range of 85–100% for specificity, median of 99%).10 Differences in serum AQP4 levels between NMO and MS patients have been described, which suggests that NMO is distinct from MS.8,13,20–28 However, MS patients have also been found to be positive for these antibodies.8 In previous studies,8,13,20–28 1.25–9% of tested MS patients were positive for NMO-IgG or AQP4 antibodies. In the present study, no antibody-positive serum was detected among MS patients when measured using a cell-based substrate. Among the 35 patients who met the diagnostic criteria for MS, the CSF test was positive in five (14.3%) as measured using a cellbased assay. Our findings suggest a relationship between CSFAQP4 antibodies and clinical MRI features. We retrospectively compared the demographic and clinical characteristics of patients who were CSF-positive with those who were CSF-negative. Our results confirm that the brain and spinal cord lesions are distinctive in these two groups, suggesting a relationship between CNS AQP4 antibodies and lesion location (Table 1). Previous MRI studies show that such brain lesions (surrounding the ventricle and in the basal ganglia, internal capsule and thalamus) and longitudinally extensive spinal cord lesions (LESCL) (Fig. 1) are atypical for MS.21 The distribution of these characteristic lesions corresponds to sites of high AQP4 expression and they are characteristic of NMO.23,29,30 This may indicate that CSF AQP4 antibodies easily reach the lesions and react with AQP4 water channels inducing a local inflammatory response. Humoral mechanisms appear to play important immune pathogenetic roles in the initiation and propagation of lesions in our CSF-positive patients, and such lesions are likely have a similar pathogenesis to NMO. In fact, Miller et al.31 proposed the presence of AQP4 antibodies in CSF as a minor criterion for NMO diagnosis. Although our five CSF-positive patients all met the McDonald criteria for MS, one of the exclusion criteria is that there be no other more plausible explanation for the demyelinating syndrome. The findings that all five clearly had lesions that are atypical for MS (in the thalamus) and that two had LESCL call into question the diagnoses of MS. Thus, these patients may have not actually had MS and probably had NMO, particularly the two patients with LESCL. They may have been limited or inaugural syndromes of NMO. However, Miller et al.31 believe that these spatially limited syndromes should not qualify as NMO and that the subsequent development of optic neuritis may permit a later diagnosis of NMO. We hope that follow-up studies of large samples of MS patients will be performed in future, particularly those that include detailed immunohistochemical studies of autopsy and brain biopsy tissue specimens. Our results indicate that CSF AQP4 antibody testing is an important aid in diagnosing patients who meet MS criteria but have atypical brain or spinal lesions and serum negative for APQ4 antibodies.
Conflict of interest/Disclosures The authors declare that they have no financial or other conflicts of interest in relation to this research and its publication.
4. Discussion
Acknowledgment
Serum AQP4 antibodies have been shown to be highly specific and moderately to highly sensitive biomarkers for NMO;8,10,13 in
This work was supported by funds from the Technology Project of Guangzhou City (No. 2060402).
Please cite this article in press as: Long Y et al. Clinical features of Chinese patients with multiple sclerosis with aquaporin-4 antibodies in cerebrospinal fluid but not serum. J Clin Neurosci (2012), http://dx.doi.org/10.1016/j.jocn.2011.12.037
Y. Long et al. / Journal of Clinical Neuroscience xxx (2012) xxx–xxx
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jocn.2011.12.037. References 1. Poser CM, Brinar VV. Diagnostic criteria for multiple sclerosis. Clin Neurol Neurosurg 2001;103:1–11. 2. de Seze J, Lebrun C, Stojkovic T, et al. Is Devic’s neuromyelitis optica a separate disease? A comparative study with multiple sclerosis. Mult Scler 2003;9:521–5. 3. Wingerchuk DM, Hogancamp WF, O’Brien PC, et al. The clinical course of neuromyelitis optica (Devic’s syndrome). Neurology 1999;53:1107–14. 4. Wingerchuk DM, Lennon VA, Pittock SJ, et al. Revised diagnostic criteria for neuromyelitis optica. Neurology 2006;66:1485–9. 5. O’Brien K, Gran B, Rostami A. T-cell based immunotherapy in experimental autoimmune encephalomyelitis and multiple sclerosis. Immunotherapy 2010;2:99–115. 6. Hamnik SE, Hacein-Bey L, Biller J, et al. Neuromyelitis optica (NMO) antibody positivity in patients with transverse myelitis and no visual manifestations. Semin Ophthalmol 2008;23:191–200. 7. Lucchinetti CF, Mandler RN, McGavern D, et al. A role for humoral mechanisms in the pathogenesis of Devic’s neuromyelitis optica. Brain 2002;125:1450–61. 8. Lennon VA, Wingerchuk DM, Kryzer TJ, et al. A serum autoantibody marker of neuromyelitis optica: distinction from multiple sclerosis. Lancet 2004;364:2106–12. 9. Lennon VA, Kryzer TJ, Pittock SJ, et al. IgG marker of optic-spinal multiple sclerosis binds to the aquaporin-4 water channel. J Exp Med 2005;202:473–7. 10. Jarius S, Wildemann B. AQP4 antibodies in neuromyelitis optica: diagnostic and pathogenetic relevance. Nat Rev Neurol 2010;6:383–92. 11. Jarius S, Paul F, Franciotta D, et al. Mechanisms of disease: aquaporin-4 antibodies in neuromyelitis optica. Nat Clin Pract Neurol 2008;4:202–14. 12. Reindl M, Kuenz B, Berger T. B cells and antibodies in MS. Results Probl Cell Differ 2010;51:99–113. 13. Matsuoka T, Matsushita T, Kawano Y, et al. Heterogeneity of aquaporin-4 autoimmunity and spinal cord lesions in multiple sclerosis in Japanese. Brain 2007;130:1206–23. 14. Polman CH, Reingold SC, Edan G, et al. Diagnostic criteria for multiple sclerosis: 2005 revisions to the ‘‘McDonald Criteria’’. Ann Neurol 2005;58:840–6. 15. Kurtzke JF. Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS). Neurology 1983;33:1444–52.
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16. Barkhof F, Polman CH, Radue EW, et al. Magnetic resonance imaging effects of interferon beta-1b in the BENEFIT study: integrated 2-year results. Arch Neurol 2007;64:1292–8. 17. Jarius S, Probst C, Borowski K, et al. Standardized method for the detection of antibodies to aquaporin-4 based on a highly sensitive immunofluorescence assay employing recombinant target antigen. J Neurol Sci 2010;291:52–6. 18. Jarius S, Franciotta D, Paul F, et al. Cerebrospinal fluid antibodies to aquaporin-4 in neuromyelitis optica and related disorders: frequency, origin, and diagnostic relevance. J Neuroinflammation 2010;7:52. 19. Dubois-Galopin F, Beauvillain C, Dubois D, et al. New markers and an old phenomenon: prozone effect disturbing detection of filaggrin (keratin) autoantibodies. Ann Rheum Dis 2007;66:1121–2. 20. Fazio R, Malosio ML, Lampasona V, et al. Antiacquaporin 4 antibodies detection by different techniques in neuromyelitis optica patients. Mult Scler 2009;15:1153–63. 21. Nakashima I, Fujihara K, Miyazawa I, et al. Clinical and MRI features of Japanese patients with multiple sclerosis positive for NMO-IgG. J Neurol Neurosurg Psychiatry 2006;77:1073–5. 22. Takahashi T, Fujihara K, Nakashima I, et al. Establishment of a new sensitive assay for anti-human aquaporin-4 antibody in neuromyelitis optica. Tohoku J Exp Med 2006;210:307–13. 23. Paul F, Jarius S, Aktas O, et al. Antibody to aquaporin 4 in the diagnosis of neuromyelitis optica. PLoS Med 2007;4:e133. 24. Hayakawa S, Mori M, Okuta A, et al. Neuromyelitis optica and anti-aquaporin-4 antibodies measured by an enzyme-linked immunosorbent assay. J Neuroimmunol 2008;196:181–7. 25. Tanaka K, Tani T, Tanaka M, et al. Anti-aquaporin 4 antibody in selected Japanese multiple sclerosis patients with long spinal cord lesions. Mult Scler 2007;13:850–5. 26. Jarius S, Franciotta D, Bergamaschi R, et al. NMO-IgG in the diagnosis of neuromyelitis optica. Neurology 2007;68:1076–7. 27. Marignier R, De Seze J, Vukusic S, et al. NMO-IgG and Devic’s neuromyelitis optica: a French experience. Mult Scler 2008;14:440–5. 28. Marnetto F, Hellias B, Granieri L, et al. Western blot analysis for the detection of serum antibodies recognizing linear Aquaporin-4 epitopes in patients with Neuromyelitis Optica. J Neuroimmunol 2009;217:74–9. 29. Pittock SJ, Weinshenker BG, Lucchinetti CF, et al. Neuromyelitis optica brain lesions localized at sites of high aquaporin 4 expression. Arch Neurol 2006;63:964–8. 30. Pittock SJ, Lennon VA, Krecke K, et al. Brain abnormalities in neuromyelitis optica. Arch Neurol 2006;63:390–6. 31. Miller DH, Weinshenker BG, Filippi M. Differential diagnosis of suspected multiple sclerosis: a consensus approach. Mult Scler 2008;14:1157–74.
Please cite this article in press as: Long Y et al. Clinical features of Chinese patients with multiple sclerosis with aquaporin-4 antibodies in cerebrospinal fluid but not serum. J Clin Neurosci (2012), http://dx.doi.org/10.1016/j.jocn.2011.12.037