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Nov 30, 2010 - Hospital, Ein–Karem, Jerusalem, Israel. Corresponding author: Adi Vaknin-Dembinsky MD, PhD, Neurology Department, Multiple. Sclerosis ...
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Preferential increase of B-cell activating factor in the cerebrospinal fluid of neuromyelitis optica in a white population Adi Vaknin-Dembinsky, Livnat Brill, Naama Orpaz, Oded Abramsky and Dimitrios Karussis Mult Scler 2010 16: 1453 originally published online 8 October 2010 DOI: 10.1177/1352458510380416 The online version of this article can be found at: http://msj.sagepub.com/content/16/12/1453

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Research Paper

Preferential increase of B-cell activating factor in the cerebrospinal fluid of neuromyelitis optica in a white population

Multiple Sclerosis 16(12) 1453–1457 ! The Author(s) 2010 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/1352458510380416 msj.sagepub.com

Adi Vaknin-Dembinsky, Livnat Brill, Naama Orpaz, Oded Abramsky and Dimitrios Karussis Abstract Background: Anti-aquaporin-4 antibodies are believed to have a central pathogenetic role in neuromyelitis optica (NMO). B-cell activating factor (BAFF) is one of the crucial factors that determines the fate and survival of B cells and may play a role in induction of antibody-mediated autoimmunity. Objectives: To evaluate the blood and cerebrospinal fluid (CSF) levels of BAFF in NMO and multiple sclerosis (MS) patients. Methods: Peripheral blood samples were collected from 21 definite NMO patients, 22 healthy controls and 45 MS patients and CSF from 8 NMO and 11 MS patients. BAFF levels were measured using an ELISA technique. Results: We found significantly higher levels of BAFF in the CSF of NMO patients compared with that in MS (215.6  41 pg/ml in NMO and 77.4  11 pg/ml in MS, p < 0.001). There were no differences in serum BAFF levels between NMO, MS and healthy donors. MS patients treated with interferon-beta (IFNb) or glatiramer acetate (GA) had significantly higher serum BAFF levels, as compared with untreated patients (1227  203 pg/ml in untreated MS, 2253  83.4 pg/ml in GA-treated, p < 0.01, and 2106  277.9 pg/ml in interferon-treated, p < 0.05) Conclusion: The presence of increased BAFF, a soluble factor associated with B-cell activation in the proximity of the disease target organ (CSF) in NMO, and its increase in association with immunomodulating treatments, may help our understanding of the immunopathogenetic mechanisms involved in this disease and contribute to more successful and targeted therapeutic intervention. Keywords anti-aquaporin-4 antibodies, autoimmunity, B-cell activating factor (BAFF), multiple sclerosis (MS), neuromyelitis optica (NMO) Date received: 12th February 2010; revised: 15th June 2010; accepted: 7th July 2010

Introduction B cells and antibodies are increasingly recognized as important players in the pathogenesis of multiple sclerosis.1 Autoantibodies are thought to have a central and more direct pathogenic role in another central nervous system (CNS) demyelinating disease, neuromyelitis optica (NMO).2 NMO, also known as Devic’s disease, is an autoimmune, severe, demyelinating disease of the CNS that preferentially affects the optic nerves and spinal cord. NMO has a worldwide distribution and poor prognosis.2 Currently, NMO can be clearly distinguished from multiple sclerosis (MS) by clinical, neuroimaging, laboratory, and pathological criteria. In particular, the

presence of a highly specific serum autoantibody targeting the water channel aquaporin-4 (AQP4) differentiates NMO from MS.2 B-cell activating factor (BAFF) is one of the crucial factors in determining the survival of late transitional B cells.3 BAFF overexpression may increase the survival of self-reactive B cells, contributing in this Department of Neurology and Laboratory of Neuroimmunology and the Agnes-Ginges Center for Neurogenetics, Hadassah-Hebrew University Hospital, Ein–Karem, Jerusalem, Israel. Corresponding author: Adi Vaknin-Dembinsky MD, PhD, Neurology Department, Multiple Sclerosis and Immunobiology Research, Hadassah University Hospital, Ein Kerem, PO Box 12000, Jerusalem 91120, Israel Email: [email protected]

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Methods Subjects Peripheral blood samples were collected from 21 patients fulfilling the criteria for definite NMO7 (5 males and 16 females, with a mean age of 31  8), 22 age-matched healthy controls and 45 MS patients (23 untreated and 22 treated with immunomodulating drugs), with a median Expanded Disability Status Scale (EDSS) score of 2.95  1.8. Among the latter group, 11 patients were treated with one of the available IFNb preparations and 11 patients were treated with glatiramer acetate (GA) at the time of sampling. In patients treated with IFNb, blood was drawn 48 h after the last injection. None of the patients was treated with other immunomodulatory or immunosuppressive medications during a period of 12 weeks preceding inclusion in the study. Blood samples were obtained from MS patients during the remission/stabilization phases of the neurological disease in our Outpatients Clinic. Patients were negative for other autoantibodies except for one, in whom anti-SSA/SSB autoantibodies were detected without clinical symptoms of Sjo¨gren’s syndrome. CSF samples were obtained from 8 NMO and 11 MS patients, during the initial diagnostic workup at the time of clinical onset of the disease and before any therapeutic intervention. Fourteen out of 21 NMO patients were positive for anti-AQP4 antibodies. The study was reviewed and approved by the Ethics Committee of the Hadassah Medical Organization. Informed consent was obtained from each patient, according to the Declaration of Helsinki.

Detection of BAFF in serum and CSF For detection of BAFF levels, a standard ELISA method was used, with a BAFF pre-coated plate, according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). Serum samples

were diluted five times. CSF samples were analyzed undiluted.

Statistical analysis The results are expressed as the mean  SEM. for each group. The statistical significance was calculated using Student’s or Welch’s t-tests. Correlation analysis was made using Pearson’s correlation test.

Results CSF BAFF levels are increased in NMO patients The levels of BAFF in the CSF of NMO patients were higher than in age-matched MS patients (mean levels: 215.6  41 pg/ml in NMO and 77.4  11 pg/ml in MS) (Figure 1). This difference was highly significant statistically (p < 0.001, two tailed t-test). No increased cellularity or protein levels were detected in the CSF of NMO patients.

Serum BAFF levels in NMO and MS patients There was no significant difference between serum BAFF levels from NMO, untreated MS patients and healthy donors (the mean levels were 859.7  159.5 pg/ml in healthy controls, 1227  203 pg/ml in untreated MS patients and,

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way to the break of B-cell tolerance and development of autoimmunity. Overexpression of BAFF in mice leads to multiple autoimmune symptoms similar to human systemic lupus erythematosus (SLE) and Sjo¨gren’s syndrome.4 High serum BAFF levels have been reported in several autoimmune diseases.5 Recent studies have shown that BAFF levels in untreated MS patients are similar to those in controls, and elevated in patients treated with interferon-beta (IFNb).6 In the present study, we evaluated the levels of BAFF in the serum and the cerebrospinal fluid (CSF) of patients with NMO and compared them with those in MS patients, untreated or treated with immunomodulatory agents.

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Figure 1. Increased BAFF levels in the CSF of NMO patients. Cerebrospinal fluid (CSF) samples were obtained from 8 neuromyelitis optica (NMO) and 11 multiple sclerosis (MS) patients during the initial diagnostic work-up at the time of disease onset and before any therapeutic intervention. For detection of B-cell activating factor (BAFF) levels, a standard enzyme-linked immunosorbent assay (ELISA) method was used, with a BAFF precoated plate, as described in the Methods sxection. CSF was tested undiluted. The values (BAFF levels in pg/ml) obtained from each patient are shown as dots. The mean in each group is represented as a line.

Vaknin-Dembinsky et al.

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1372  188 pg/ml in NMO patients, p ¼ 0.27; p ¼ 0.08 MS and NMO vs. healthy donors, respectively) (Figure 2A). BAFF serum levels of both MS and NMO patients did not correlate with disease activity and severity (EDSS) or with disease duration. Furthermore, no correlation was found between serum and CSF BAFF levels. Serum BAFF levels from anti-AQP4 positive NMO patients were not significantly different from the sero-negative ones. Treatment of MS patients with IFNb and GA was associated with higher levels of BAFF in the serum. As shown in Figure 2B, MS patients treated with IFNb or GA had significantly elevated BAFF levels, compared with the levels in untreated patients; the mean levels in untreated MS patients were 1227  203 pg/ml; in GA-treated MS patients, 2253  83.4 pg/ml (p < 0.01, two-tailed t-test) and in

interferon-treated MS patients, 2106  277.9 pg/ml (p < 0.05, two-tailed t-test). Since this is not a longitudinal follow-up study, there is no definite proof for a causal association between the increased BAFF levels and the immunomodulatory treatment. However, we checked BAFF levels before and 6 months following interferon treatment in two patients and indeed detected an increase, from 482 pg/ml before treatment to 3585 pg/ml following IFNb, in the first patient, and from 265 pg/ml to 4335 pg/ml in the second one.

Discussion In the current study we report a preferential increase of BAFF levels in the CSF of NMO patients, compared with those in MS. Similarly to previous studies, we could not detect any elevation in serum BAFF levels

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Figure 2. Serum BAFF levels in NMO and MS patients. (A) Serum B-cell activating factor (BAFF) levels are not elevated in neuromyelitis optica (NMO) patients. Serum samples were obtained from 21 NMO, 22 age-matched healthy controls and 23 untreated multiple sclerosis (MS) patients. The values (BAFF levels in pg/ml) obtained from each patient are shown as dots. The mean in each group is represented as a line. (B) Serum BAFF levels are increased in MS patients treated with interferon beta or glatiramer acetate. Serum samples were obtained from 23 untreated MS patients, 11 treated with interferon beta and 11 treated with glatiramer acetate. For detection of BAFF levels, a standard ELISA method was used, with a BAFF pre-coated plate, as described in the Methods section. The serum was diluted five times. The values (BAFF levels in pg/ml) obtained from each patient are shown as dots. The mean in each group is represented as a line.

1456 in untreated MS patients, whereas, significantly higher levels were found in the patients treated with interferon beta or Glatiramer acetate. Serum BAFF levels in NMO patients were slightly higher than those in the controls, similarly to what previously reported (8), but in our study this difference did not reach statistical significance. BAFF is a B-cell activating factor that plays a major role in peripheral B-cell survival, maturation, and differentiation. In mice, dysregulation of BAFF expression leads to inappropriate survival of self-reactive B cells and the development of a severe autoimmune syndrome.4 Moreover, several human systemic autoimmune diseases, including SLE and Sjo¨gren’s, are associated with higher than normal serum BAFF levels.5 On the other hand, in organ-specific autoimmune diseases such as primary biliary cirrhosis and diabetes the serum levels of BAFF were not elevated.5 An increased coincidence of antibody-mediated autoimmune diseases autoimmune diseases such as Sjo¨gren’s syndrome and SLE has been reported in NMO patients.7,9 In others, autoantibodies such as anti-nuclear and anti-SSA/SSB antibodies, without overt clinical signs of SLE or Sjogren’’s disease, can be detected.10,11 It is possible that changes in BAFF are more relevant and occur inside the target organs in organ-specific antibody-mediated diseases. Our finding, therefore, of elevated BAFF only in the CSF and not in the serum of NMO patients is not surprising and is in line with our previous assumption. Several lines of evidence suggest that astrocytes play a central role in the pathogenesis of NMO. NMO has been considered a primary astrocyte disease.12 Astrocytes express AQP4, the target for autoantibodies in NMO patients. Astrocytes are able to initiate an immune response after minor activation. They can secrete proinflammatory cytokines, act as antigen-presenting cells and secrete B-cell activation factors such as BAFF.13 We have detected BAFF secretion from astrocytes cultured in the presence of serum from two NMO patients (preliminary observation, data not shown). It is therefore possible that increased CSF BAFF in NMO patients is the result of astrocyte activation and/or damage. Subsequently, locally excessive BAFF production may maintain or support the survival of self-reactive B cells, which, in addition to other dysregulated immune mediators, may accelerate or upregulate the anti-AQP4 antibody production. On the other hand, we found only slightly increased BAFF serum concentrations in MS patients compared with that in healthy controls. BAFF serum levels are mainly increased in systemic autoimmune diseases such as SLE, but not necessarily in diseases targeting directly and solely the CNS, such as multiple sclerosis or NMO. The finding of increased BAFF levels in the CSF in

Multiple Sclerosis 16(12) NMO raises the possibility that in such antibodymediated CNS-specific autoimmune diseases, B-cell abnormalities can be detected only locally in the target organ. In MS, which is also a CNS-specific, autoimmune disease, the lack of a similar increase in the CSF may indicate a difference in the pathogenetic mechanisms between these two demyelinating diseases and point to a more significant role of localized B-cell activation in NMO than in MS and to less pronounced astrocytic involvement in MS. A recent article,8 submitted in parallel to ours, showed that BAFF levels were high not only in the CSF but also in the serum of NMO patients. In the same research the investigators reported mildly elevated CSF levels of BAFF in MS patients as well. This seeming discrepancy can be explained by the small numbers of patients in both studies and the variability in MS pathogenesis in distinct populations. It is indeed accepted that whereas in the majority of MS patients T cells play a major role, in some of the cases B cells are also important in its pathogenesis.14 The unique MS population in Japan (where the neuromyelitic type of MS, with a putative antibody-mediated mechanism and a great overlap with NMO, is dominant), compared with our cases with European classical type of MS, may also explain these discrepancies. In any case, even in this study, the increase of BAFF in the CSF was much more pronounced,8 similarly to our data. Our study also shows an increase in BAFF levels in MS patients treated with IFNb or GA. Although an increase following IFNb treatment has been described previously, the higher BAFF levels in patients treated with Copaxone (GA), compared with that in untreated patients, is a novel finding, not presented elsewhere, and could be related to the induction of anti-GA antibodies in patients treated with Copaxone, as we have shown previously.15 The reportedly low efficacy of IFNb or GA treatment in NMO (and even clinical deterioration under these treatments) may be related to the activation of B cells through upregulation of BAFF production, following treatment with these immunomodulators. In support of this possibility is the finding of an increased incidence of other antibody-mediated autoimmune diseases, including thyroiditis, or the increased autoantibody production following treatment with interferon.16 Therefore, testing BAFF levels in patients with MS treated with IFNb or GA could predict the response to these treatments and be an additional indication of the biological activity of IFNb and GA. BAFF could serve as a surrogate biomarker not only for the response to treatment but, most importantly, for the possible induction of secondary B-cell-associated autoimmunity in MS patients treated with GA or IFNb. Finally, measurement of BAFF levels could represent an additional biomarker for distinguishing MS from NMO. This is of practical

Vaknin-Dembinsky et al. clinical importance since treatment of NMO patients misdiagnosed as MS with IFNb or GA may cause a deterioration in their disease. In conclusion, the increased BAFF levels in the CSF (in the proximity of the target organ) in NMO may represent one of the first steps in the pathogenetic immunological cascade in CNS antibody-mediated diseases such as NMO. Our findings could offer a plausible link between BAFF, astrocyte dysfunction and B-cell-related autoimmunity, opening new perspectives in our understanding of the pathogenetic mechanisms involved, in NMO and similar diseases.

Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Conflict of interest statement Dr Karussis has received honoraria from pharmaceutical companies for invited lectures. All other authors have nothing to declare.

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