Cerebrospinal BAFF and Epstein–Barr virus-specific oligoclonal bands in multiple sclerosis and other inflammatory demyelinating neurological diseases

Cerebrospinal BAFF and Epstein–Barr virus-specific oligoclonal bands in multiple sclerosis and other inflammatory demyelinating neurological diseases

Journal of Neuroimmunology 230 (2011) 160–163 Contents lists available at ScienceDirect Journal of Neuroimmunology j o u r n a l h o m e p a g e : w...

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Journal of Neuroimmunology 230 (2011) 160–163

Contents lists available at ScienceDirect

Journal of Neuroimmunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n e u r o i m

Cerebrospinal BAFF and Epstein–Barr virus-specific oligoclonal bands in multiple sclerosis and other inflammatory demyelinating neurological diseases Diego Franciotta a,⁎, Anna Luisa Di Stefano b, Sven Jarius c, Elisabetta Zardini a, Eleonora Tavazzi b, Clara Ballerini d, Enrico Marchioni b, Roberto Bergamaschi b, Mauro Ceroni b a

Laboratory of Neuroimmunology, IRCCS National Neurological Institute ‘C. Mondino’, University of Pavia, Italy Department of Neurology, IRCCS National Neurological Institute ‘C. Mondino’, University of Pavia, Italy Division of Molecular Neuroimmunology, Department of Neurology, University of Heidelberg, Germany d Department of Neurological Sciences, University of Florence, Italy b c

a r t i c l e

i n f o

Article history: Received 28 July 2010 Received in revised form 24 September 2010 Accepted 22 October 2010 Keywords: Acute disseminated encephalomyelitis BAFF Cerebrospinal fluid Epstein–Barr virus Multiple sclerosis Neuromyelitis optica

a b s t r a c t We measured circulating serum and cerebrospinal fluid (CSF) concentrations of B lymphocyte activating factor of the tumour necrosis factor superfamily (BAFF), and determined total and Epstein–Barr virus (EBV)-specific oligoclonal IgG bands (OCBs) in 43 patients with multiple sclerosis (MS), 23 patients with other inflammatory demyelinating neurological diseases, and 20 patients with non-inflammatory neurological diseases. Serum and CSF BAFF concentrations did not differ in the three studied groups. In MS, the highest BAFF concentrations were found in the CSF samples with more than 6 OCBs (233.1± 129.5 vs 79.2± 51.6 pg/mL in the samples with less than 7 OCBs, p b 0.0001). Irrespectively from BAFF levels, EBV-specific OCBs were detected in MS and in the other non-inflammatory and inflammatory demyelinating neurological diseases, with a similar frequency, and as a ‘mirror pattern’ in 30 of 33 EBV-specific OCB-positive cases (pb 0.0001). These results indicate that circulating CSF BAFF concentrations cannot help differentiate MS from other inflammatory demyelinating neurological diseases, but positively associates with the qualitative expression of elevated intrathecal IgG production in MS, and that the oligoclonal EBV-specific antibody response, when present, is mostly systemic in all the studied neurological patients, and not preferentially restricted to MS. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Multiple sclerosis (MS) is a multifactorial, demyelinating, and immunomediated neurological disease. The efficacy of a B cell targeting monoclonal antibody (Hauser et al., 2008), the appreciation of ectopic B cell follicles in the meninges of a subset of patients (Serafini et al., 2004; Magliozzi et al., 2007), and contrasting data on a dysregulated CNS infection by Epstein–Barr virus (EBV) (Serafini et al., 2007; Willis et al., 2009; Sargsyan et al., 2010), a B cell lymphotropic agent, recently sparked renewed interest in the role of humoral immunity, whether EBV-driven, or not, in MS pathogenesis (Franciotta et al., 2008). The presence of intrathecal oligoclonal IgG, detected as oligoclonal bands (OCBs) on isoelectrophoretic separation, is, on the other hand, a well established abnormality that represents an integral part of MS diagnostic iter (Freedman et al., 2005). OCBs are still considered a clue for an involvement of infectious agents in MS pathogenesis, although their specificity is largely unknown (Giovannoni et al., 2006). Data on a relatively small number of cerebrospinal fluid (CSF) samples (Cepok

⁎ Corresponding author. Tel.: + 39 0382 380364; fax: + 39 0382 380286. E-mail address: [email protected] (D. Franciotta). 0165-5728/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2010.10.027

et al., 2005; Serafini et al., 2007) suggest that EBV proteins might be preferential targets of OCBs in MS. B lymphocyte activating factor of the tumour necrosis factor superfamily (BAFF) is a fundamental cytokine for B cell homeostasis that can play a dual role in immunity, by regulating both innate and adaptive immune responses and by sustaining autoimmunity (Mackay and Schneider, 2009). Overexpression of BAFF is involved in the pathogenesis of systemic lupus erythematosus (SLE), and high circulating serum concentrations of the cytokine have been reported in this disease, in other autoimmune connective tissue diseases, and in autoimmune diseases associated with polyclonal hypergammaglobulinaemia (Cheema et al., 2001). More in general, the presence of BAFF characterizes the inflammatory sites where lymphoid neogenesis occurs (Mackay and Schneider, 2009). These sites constitute pathological lesions that are common to chronic autoimmune diseases, such as SLE, rheumatoid arthritis, Hashimoto thyroiditis, MS (reviewed by Franciotta et al., 2008), and, more recently, myasthenia gravis (Cavalcante et al., 2010). In all these diseases, an active EBV role in the development of the lymphoid neogenesis has been hypothesized or demonstrated (reviewed by Franciotta et al., 2008; Cavalcante et al., 2010). In MS plaques, BAFF expression has been found strongly up-regulated at levels comparable to those detected in lymphatic tissues (Krumbholz et al., 2005). Similarly, the cytokine mRNA levels were increased in monocytes, and BAFF-receptor

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mRNA levels in B and T cells of MS patients (Thangarajh et al., 2004). However, BAFF protein levels in CSF and plasma were reported to be similar in MS patients vs headache controls (Thangarajh et al., 2004), and in MS patients vs other inflammatory and non-inflammatory neurological controls (Piazza et al., 2010). This study on patients with MS, patients with other inflammatory demyelinating neurological diseases, and patients with non-inflammatory neurological diseases aimed to: a) measure circulating serum and CSF BAFF concentrations; b) correlate the CSF BAFF levels with the intrathecal IgG production, calculated quantitatively and qualitatively, and with the EBV-specific OCBs, given that latent membrane protein 1, an EBV protein, induces B cells to express BAFF (He et al., 2003), and, on the other hand, latent EBV infection of B cells can promote their proliferation and periodical reactivation (reviewed by Thorley-Lawson, 2001); c) establish the frequency of EBV-specific OCBs. 2. Methods 2.1. MS patients and neurological controls Forty three patients with clinically, or laboratory supported definite MS (Poser et al., 1983), and 43 patients with other inflammatory demyelinating (10; acute disseminated encephalomyelitis (ADEM), 9; neuromyelitis optica, 4), or non-inflammatory (10; polyneuropathy, 7; benign endocranic hypertension, 2; frontotemporal dementia, 1) neurological diseases were studied. Table 1 shows their clinicodemographic features. All participants gave informed consent, on the basis of the rules by the local ethics committee. Disability of patients with demyelinating diseases was rated using Kurtzke expanded disability status score (EDSS) (Kurtzke, 1983), and Multiple Sclerosis Severity Scale (MSSS score, Roxburgh et al., 2005). Between MS patients, 33 cases were classified as relapsing–remitting (RR), 25 of whom in an active phase of disease when lumbar puncture was performed, and 10 as primary progressive (PP), in accordance with Lublin and Reingold (1996). Clinical relapse was defined using standard criteria (Poser et al., 1983). Published criteria were used to diagnose ADEM (Bradley et al., 1995; al Deeb et al., 1997), and neuromyelitis optica (Wingerchuk et al., 2006). The patients included in the study were immuno-modulating/suppressive drug-free in the three months preceding the CSF drawing. 2.2. Cerebrospinal fluid studies This was a retrospective study on banked samples (consecutively taken from those stored between 2006 and 2009). CSF and serum samples had been collected and stored in accordance with the guidelines that a posteriori matched those of an international consensus (Teunissen et al., 2009). Lumbar punctures were performed for diagnostic purposes. IgG and albumin were determined with turbidimetry (Cobas Integra, Roche, Milan, Italy). Intrathecal IgG production was calculated in accordance with Reiber and Felgenhauer (1987). OCBs were searched with isoelectric focusing in agarose gel (pH 3.0–10.0, Cambrex, Rockland, ME, USA), and capillary immunoblotting for human IgG (Dako, Milan, Italy), in accordance with the guidelines of an international consensus (Andersson et al., 1994), whose proposals for OCB pattern categorization were also

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used. Serum samples were diluted to load the gels with equal amounts of serum and CSF IgG (0.3 μg). EBV-specific OCBs were searched with the same protocol used for total IgG, except for the affinity-mediated immunoblotting protocol (Franciotta et al., 1996). The protocol foresees the use of nitrocellulose membranes that have been coated with highly pure EBV Antigen (strain P3HR1), which contains all viral antigens, and of a mouse monoclonal anti-EBV EA-D IgG as a positive control (both from Fitzgerald Industries International, Concorde, MA, USA). The serum of a patient with a monoclonal gammopathy tested on EBV-precoated papers and EBV-specific OCB-positive CSF samples tested on lanes coated with a blocking solution containing casein (WesternBreeze, Invitrogen, Carlsbad, CA, USA) only were used for assessing non-specific binding. Pre-absorption of EBV-specific OCB-positive CSF with the highly pure EBV Antigen prevented the specific binding (not shown). For each sample, 0.3 μg of IgG was used. The blots were interpreted independently by two expert laboratorists (D. F. and E. Z.), with an inter-rater agreement of 97% (disagreement resolved by consensus). BAFF was measured with a commercial ELISA, in accordance with the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA). The kit employs a mouse monoclonal antibody specific for BAFF, which is precoated onto a microplate, and horseradish peroxidase-linked antiBAFF polyclonal antibody. The detection limit was 3.0 pg/mL. Within- and between-run reproducibility values were, respectively, 5.7% and 9.0%. The calculation of intrathecal BAFF production was not applicable, as the molecular weight of the peptide is smaller than that of albumin. Accordingly, the degree of passive transfer of BAFF through the blood– CSF barrier cannot be reliably predicted on the basis of albumin quotient. 2.3. Statistical analysis Data are reported as mean± SD. Group comparisons were made by means of Mann–Whitney test. Chi square test was used for categorical variables. Correlations were analyzed with Spearman's rank correlation test. A p value lower than 0.05 was considered significant. 3. Results Mean serum and CSF BAFF concentrations were not significantly different between patients with RR-MS, PP-MS, other inflammatory demyelinating neurological diseases, or non-inflammatory neurological diseases. Between RR-MS and PP-MS patients, serum and CSF BAFF levels did not correlate with the EDSS and MSSS scores, or with disease activity in RR-MS patients. Regarding the intrathecal IgG production, CSF BAFF concentrations did not correlate with IgGLoc, but, after splitting the RR-MS patients into two groups, one with more than 6 CSF total IgG OCBs, and one with less than 7 OCBs, mean CSF BAFF concentrations in the first group (233.1±129.5 pg/mL) were higher than those in the second group (79.2±51.6 pg/mL) (Fig. 1). The six band cut-off is part of a standard categorization of oligoclonal banding in our laboratory (N6 bands denotes ‘numerous OCBs’ in a semi-quantitative report). To give consistency to such data, we tested BAFF on an independent series of 15 OCB-positive CSF samples from definite RR-MS patients, 9 with more than 6 OCBs, and 6 with less than 7 OCBs. BAFF concentrations in the first group (188.2± 88.8 pg/mL) were higher than in the second group (71.8±42.2 pg/mL;

Table 1 Clinico-demographic features and main findings of serum and cerebrospinal studies in patients with relapsing–remitting multiple sclerosis (RR-MS), primary progressive MS (PP-MS), other inflammatory demyelinating neurological diseases (OIDNDs), and non-inflammatory neurological diseases (NINDs).

RR-MS PP-MS OIDNDs NINDs

No. of pts, age (years), sex (F/M)

Serum BAFF (pg/mL)

CSF BAFF (pg/mL)

No. of pts with IgGLoc N 0 (%)

No. of pts with CSF total IgG OCBs (%)

No. of pts with CSF EBVsp IgG OCBs (%)

No. of pts with CSF = ser EBVsp IgG OCBs (%)

33, 36 ± 13, 22/11 10, 47 ± 11, 4/6 13, 40 ± 15, 4/6 10, 51 ± 10, 6/4

528.6 ± 286.6 446.0 ± 309.1 634.1 ± 330.2 456.0 ± 219.6

141.6 ± 123.4 113.4 ± 84.4 212.6 ± 137.1 91.2 ± 61.3

25 7 7 0

33 (100) 9 (90) 7 (54) 0 (0)

2 (6) 0 (0) 1 (8) 0 (0)

17 (51) 4 (40) 6 (46) 3 (30)

(75) (70) (54) (0)

Data are expressed as mean ± SD; pts, patients; BAFF, B-lymphocyte activating factor; ser, serum; CSF, cerebrospinal fluid; IgGLoc, intrathecal IgG synthesis; OCBs, oligoclonal bands; EBV, Epstein–Barr virus; EBVsp, EBV-specific; CSF = ser OCBs, OCBs that are equal in CSF and in serum (‘mirror pattern’).

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4. Discussion

Fig. 1. Cerebrospinal fluid (CSF) BAFF concentrations in patients with relapsing–remitting multiple sclerosis with unique to CSF more than 6 oligoclonal IgG bands (OCBs; OCB high) vs those with less than 7 OCBs (OCB low). Box and whiskers show medians, 25th and 75th percentiles, largest and smallest values. The between-group difference is statistically significant, p b 0.0001.

p=0.0048). There was no correlation between BAFF levels and high OCB number in the non-MS groups. CSF BAFF concentrations in patients with other inflammatory demyelinating neurological diseases tended to be higher than those in the other groups, but the difference did not reach significance (p = 0.08). Cumulatively, EBV-specific OCBs were found, whether in serum or CSF, in 33 of the 66 (50%) studied patients. However, EBV-specific OCBs, which were in the CSF only (intrathecal synthesis), were present in only 2 patients with RR-MS, and in one patient with ADEM (Table 1; difference not significant). ‘Mirror pattern’, namely the presence of EBV-specific OCBs that were equal in serum and in CSF (no intrathecal synthesis), was the most frequent OCB pattern in our series (Fig. 2). Considering the whole series of neurological patients, ‘mirror pattern’ was found in 30 cases, whereas EBV-specific OCBs that were in the CSF only (presence of intrathecal synthesis) were detected in only 3 patients (Table 1; p b 0.0001), two with RR/PP-MS, and one with ADEM. There was no correlation between the presence of EBV-specific OCBs and the EDSS and MSSS scores, disease activity in RR-MS patients, or serum and CSF BAFF levels.

Fig. 2. Epstein–Barr virus (EBV)-specific oligoclonal bands detected with agarose isoelectric focusing and affinity-mediated immunoblotting in patients with relapsing– remitting multiple sclerosis. (1) EBV-specific bands (arrows) that are equal in serum (ser) and cerebrospinal fluid (CSF) (‘mirror pattern’); controls for binding specificity: (2) monoclonal (mc) anti-EBV EA-D antibody; (3) absence of reaction of a serum of a patient with monoclonal gammopathy; (4) absence of reaction of an EBV-specific OCB-positive CSF on casein-coated nitrocellulose paper.

Our results indicate that mean levels of circulating CSF BAFF are not elevated in the inflammatory demyelinating neurological diseases that are characterized by activation of humoral immunity, differently from what was reported in serum samples of patients with B cell involving autoimmune connective tissue diseases (Cheema et al., 2001). Our findings are in line with those by Piazza et al. (2010), who reported high CSF BAFF concentrations in acute viral meningoencephalitis, but not in MS, and with those by Krumbholz et al. (2008), who demonstrated that serum BAFF concentrations and BAFF transcript were not increased in untreated MS patients, as compared to healthy controls (2008). In our series, there was no difference in circulating BAFF levels even when we stratified the RR-MS patients for disease activity, namely for a variable that has been never addressed previously (Thangarajh et al., 2004; Krumbholz et al., 2008; Piazza et al., 2010). In SLE and Sjögren syndrome, increased serum levels of BAFF correlated with disease activity (Mackay and Schneider, 2009). We first show data on circulating BAFF in a relatively large series of patients with non-MS, inflammatory demyelinating neurological diseases, namely ADEM and neuromyelitis optica. Mean levels of CSF BAFF in these patients were higher than those in RR-/PP-MS patients, but the difference did not reach statistical significance. In Japanese patients with neuromyelitis optica, BAFF levels were found to be elevated in both serum and CSF samples, in comparison with RR-MS patients, whose serum and CSF BAFF levels, in turn, were higher than those in non-inflammatory neurological controls (Okada et al., 2010). Small case series, ethnicity, and lack of test standardization can account for the discrepancies between such data (Okada et al., 2010) and the here-reported findings. However, we did find a correlation between an index of the degree of intrathecal IgG synthesis, namely a high number of OCBs, and CSF BAFF concentrations in RR-MS patients. Ectopic B cell follicles, which contain long-lived plasma cells and plasmablasts, are preferentially localized to the subarachnoid space in the cerebral sulci of MS brains, and it is likely that they sustain a compartmentalized humoral immune response which can drive intrathecal OCB production and neuropathological cortical damage (Magliozzi et al., 2007). Findings on a relatively small series of autoptic CSF samples of MS patients showed that the number of CSF OCBs was significantly higher in the cases with higher brain inflammation which, in turn, colocalized with ectopic B cell follicles (Serafini et al., 2007). This view is supported by the lack of correlation between BAFF levels and high OCB number in the here studied non-MS neurological diseases, which could lack meningeal B cell follicles. Recent data also indicate that BAFF is expressed in such follicles in the MS brain (Serafini et al., 2010). Therefore, the higher CSF BAFF concentrations, which we found in our RR-MS patients with higher number of OCBs, could testify a crucial role for BAFF in supporting humoral autoimmunity at ectopic follicle level. From a clinical point of view, such abnormality, however, demonstrated no prognostic value, as it was not associated with worse clinical courses. The demonstration of EBV infection of B cells and plasma cells in the brain of MS patients is controversial (Pender, 2009). Here, we first studied EBV-specific OCBs in a relatively large number of paired CSF and serum samples of RR-/PP-MS patients and neurological controls. An OCB pattern associated with intrathecal EBV-specific OCB production was found in only 2 RR-MS patients, and in one patient with ADEM, whereas the most frequently detected pattern, namely EBV-specific OCBs that were equal in CSF and serum, indicated no intrathecal OCB production, and was present at a similar frequency in all the studied groups. These findings do not support the hypothesis that an intrathecal and oligoclonal EBV-specific humoral immune response is preferentially present in MS. Previous studies gave only partial contributions to this topic, as they did not include the analysis of serum samples (Cepok et al., 2005; Serafini et al., 2007), or, when serum samples were included, the

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case series were small, and no EBV-specific band was reported in any serum sample (Rand et al., 2000). This latter finding suggests that Rand and colleagues focused their attention to the CSF samples only. Indeed, serum EBV-specific OCBs seem to be a common feature, as we found them in 30–51% of serum samples in our series of neurological patients. The EBV latent membrane protein 1 is a potent inducer for BAFF production by B cells (He et al., 2003), but we found no association between CSF BAFF levels and EBV-specific OCBs, in line with the fact that EBV-specific OCBs were mostly detected in both serum and CSF samples, without intrathecal production. Our data suggest that, differently from what reported in some connective tissue autoimmune diseases (Mackay and Schneider, 2009), circulating BAFF is not elevated at the CSF level in inflammatory demyelinating neurological diseases characterized by the involvement of the humoral immune response and sustained immunoglobulin production. However, when considering BAFF as a pathogenetic factor, its emerging and multifaceted functions, such as its role in silencing autoreactive B cells (Meffre and Wardemann, 2008), should be considered. Our findings on EBV-specific OCBs add information to the puzzling case of EBV involvement in MS. Acknowledgments The study was supported by grants from FISM – Fondazione Italiana Sclerosi Multipla – Cod. 2007/R/17/C3, and from the Italian ‘Ministero della Salute’, Progetto Strategico 2007, n. 9ACF/P3. References al Deeb, S.M., Yaqub, B.A., Bruyn, G.W., Biary, N.M., 1997. Acute transverse myelitis. A localized form of postinfectious encephalomyelitis. Brain 120, 1115–1122. Andersson, M., Alvarez-Cermeño, J., Bernardi, G., Cogato, I., Fredman, P., Frederiksen, J., Fredrikson, S., Gallo, P., Grimaldi, L.M., Grønning, M., Keir, G., Lamers, K., Link, H., Magalhães, A., Massaro, A.R., Ohman, S., Reiber, H., Rönnbäck, L., Schluep, M., Schuller, E., Sindic, C.J.M., Thompson, E.J., Trojano, M., Wurster, U., 1994. Cerebrospinal fluid in the diagnosis of multiple sclerosis: a consensus report. J. Neurol. Neurosurg. Psychiatry 57, 897–902. Bradley, W.G., Daroff, R.B., Fenichel, G.M., Marsden, C., 1995. Neurology in Clinical Practice: Principles of Diagnosis and Management, 2nd ed. Butterworth-Heinemann, Boston. Cavalcante, P., Serafini, B., Rosicarelli, B., Maggi, L., Barberis, M., Antozzi, C., Berrih-Aknin, S., Bernasconi, P., Aloisi, F., Mantegazza, R., 2010. Epstein–Barr virus persistence and reactivation in myasthenia gravis thymus. Ann. Neurol. 67, 726–738. Cepok, S., Zhou, D., Srivastava, R., Nessler, S., Stei, S., Büssow, K., Sommer, N., Hemmer, B., 2005. Identification of Epstein–Barr virus proteins as putative targets of the immune response in multiple sclerosis. J. Clin Invest. 115, 1352–1360. Cheema, G.S., Roschke, V., Hilbert, D.M., Stohl, W., 2001. Elevated serum B lymphocyte stimulator levels in patients with systemic immune-based rheumatic diseases. Arthritis Rheum. 44, 1313–1319. Franciotta, D., Salvetti, M., Lolli, F., Serafini, B., Aloisi, F., 2008. B cells and multiple sclerosis. Lancet Neurol. 7, 852–858. Franciotta, D., Zardini, E., Bono, G., Brustia, R., Minoli, L., Cosi, V., 1996. Antigen-specific oligoclonal IgG in AIDS-related cytomegalovirus and toxoplasma encephalitis. Acta Neurol. Scand. 94, 215–218. Freedman, M.S., Thompson, E.J., Deisenhammer, F., Giovannoni, G., Grimsley, G., Keir, G., Ohman, S., Racke, M.K., Sharief, M., Sindic, C.J., Sellebjerg, F., Tourtellotte, W.W., 2005. Recommended standard of cerebrospinal fluid analysis in the diagnosis of multiple sclerosis: a consensus statement. Arch. Neurol. 62, 865–870. Giovannoni, G., Cutter, G.R., Lunemann, J., Martin, R., Münz, C., Sriram, S., Steiner, I., Hammerschlag, M.R., Gaydos, C.A., 2006. Infectious causes of multiple sclerosis. Lancet Neurol. 5, 887–894. Hauser, S., Waubant, E., Arnold, D., Vollmer, T., Antel, J., Fox, R., Bar-Or, A., Panzara, M., Sarkar, N., Agarwal, S., Langer-Gould, A., Smith, C., 2008. B-cell depletion with rituximab in relapsing–remitting multiple sclerosis. N. Engl. J. Med. 358, 676–688. He, B., Raab-Traub, N., Casali, P., Cerutti, A., 2003. EBV-encoded latent membrane protein 1 cooperates with BAFF/BLyS and APRIL to induce T cell-independent Ig heavy chain class switching. J. Immunol. 171, 5215–5224.

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