Journal of Neuroimmunology 247 (2012) 63–69

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Journal of Neuroimmunology

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Neuronal antigens recognized by cerebrospinal fluid IgM inmultiple sclerosis☆,☆☆,★

Eduardo Beltrán a,c, Alberto Hernández a, Eva M. Lafuente a, Francisco Coret b, María Simó-Castelló c,Isabel Boscá c, Francisco Carlos Pérez-Miralles c, María Burgal a,1, Bonaventura Casanova c,⁎,1

a Multiple Sclerosis Laboratory, Centro de Investigación Príncipe Felipe, Valencia, Spainb Neurology Service, Hospital Clínico Universitario de Valencia, Spainc Multiple Sclerosis Unit, Hospital Universitari I Politècnic La Fe, Valencia, Spain

☆ EB, FC, BC and MB were involved in study design, daand writing of the report. EB, AH, EML, MJM and MSCprepared the figures. FC, IB, FPM and BC collected tassessed the patients.☆☆ Conflicts of Interest: This project is not involved inthors have no conflicts of interest.★ Sources of Financial Support: Ministerio de CiencSalud Carlos III–Fondo de Investigaciones Sanitarias (FPS09/00976) and by the Generalitat Valenciana–Consel⁎ Corresponding author at: Hospital Universitari i Po

València 46026, Spain.E-mail address: casanova_bon@gva.es (B. Casanova)

1 Bonaventura Casanova and María Burgal contribute

0165-5728/$ – see front matter © 2012 Elsevier B.V. Alldoi:10.1016/j.jneuroim.2012.03.013

a b s t r a c t

a r t i c l e i n f o

Article history:Received 29 January 2012Received in revised form 9 March 2012Accepted 16 March 2012

Keywords:ImmunologyMultiple sclerosisNeurodegenerationNeuronal antigensImmunoglobulin MCerebral atrophy

Axonal injury is the major cause of disability in patients with multiple sclerosis (MS), but the mechanismsleading to axonal damage are poorly understood. Oligoclonal IgM against lipids predicts an aggressive diseasecourse in MS; however, the antigen that elicits the immune response has not yet been identified. We screenedthe CSF of 12 patients with MS, 7 patients with neuromyelitis optica (NMO), and 5 controls with non-inflammatory neurological disease (NIND) for the presence of IgM-type antibodies (IgM-Ab) against neuronalsurface antigens, and analyzed the relationship between IgM-Ab level and the extent of brain atrophy. The CSFof MS patients displayed significantly higher levels of IgM-Ab compared to NIND or NMO patients. Further-more, we document for the first time that these IgM-Ab recognize neuronal surface antigens, and that thelevels of neuronal-bound IgM-Ab were independent of the IgM concentration and correlate with brain atro-phy. Our findings suggest a role for the CSF IgM-Ab in the development of MS pathophysiology.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Multiple sclerosis (MS) is a heterogeneous disease of the centralnervous system (CNS) of immunological origin. Mechanisms involvedin the disease pathogenesis of MS remain elusive, but the more ac-cepted theories implies both that an altered cellular and humoral im-mune system are in the basis of the disease.

The most consistent alteration of the immune system in MS wasdemonstrated years ago, when it was proven a restricted activationof the humoral immune response within the CNS, due to a persistentintrathecal synthesis of immunoglobulins (ITS) (Farrell et al., 1985)Since then, a laboratory hallmark of MS is the presence of immuno-globulins of intrathecal synthesis both by an altered IgG Index or by

ta analysis, data interpretation,did the laboratory studies andhe clinical data and clinically

any conflict of interest. All au-

ia e Innovación; Instituto deIS PI/052210, PS09/00551 andleria de Sanitat (AP-084/09).litècnic La Fe, Bulevar Sur s/n,

.d equally to this work.

rights reserved.

the presence of oligoclonal bands (OCBs), and it has been the morespecific and sensitive tool for MS diagnosis (Masjuan et al., 2006).

The pathogenic role of the IgG antibodies in the CSF is supportedby pathologic and molecular studies. Deposition of IgG antibodiescolocalize with activated complement fragments in active borders ofsome MS demyelinating plaques (Gay et al., 1997); and, the B lineagecells contained in the CSF have been proven to be the source of theOCBs (Obermeier et al., 2008).

But the ITS of IgG is present in nearly 95% of patients, for this rea-son it is a good tool for diagnosis but not for prognosis. In recent yearsseveral works have demonstrated that IgM-Ab directed against lipidsare present in nearly 40% of the CSF of MS patients (Villar et al., 2003),and these antibodies are related with the main known factors of poorprognosis in MS: the time to a second relapses after a clinic isolatedsyndrome of demyelianting origin (Boscá et al., 2010) the time toreach an EDSS of 4.0 (Thangarajh et al., 2008); the time to reach thesecondary progressive phase of the disease (Villar et al., 2005); theincreases in early brain atrophy and in the increases in T2 lesionload (Magraner et al., 2012).

ITS has been assessed in most MS patients, and many efforts havebeen made to identify the nature of the antigen(s) to which this B-cellresponse is directed. IgM-Ab against proteins such as alfa-B-criytallin(Langkamp et al., 2009), 2',3'-cyclic nucleotide 3' phosphodiesteras(Walsh and Murray, 1998), and lipidic components of myelin suchas gangliosides or sulfatides (Rosenbluth and Moon, 2003) havebeen found in serum or CSF of patients with MS. Also increased

64 E. Beltrán et al. / Journal of Neuroimmunology 247 (2012) 63–69

intrathecal IgM-Ab antibodies against the medium subunit of neuro-filaments have been detected in MS patients. (Bartos et al., 2007).

The presence of lipid-specific oligoclonal IgM bands (OCMB) and in-creased IgM index have been associated with poor prognosis in MS anda more rapid progression to irreversible disability (Villar et al., 2005;Thangarajh et al., 2008). These OCMB usually persist in MS patientsthroughout the course of the disease (Walsh and Tourtellotte, 1986),and their persistence indicates that they are not elements of a primaryimmune response. Persistent IgM responses are usually produced bythe long-lived, self-renewing B1 subset of B cells, responsible for the se-cretion of the so-called natural antibodies that appear in the absence ofapparent antigenic stimulation (Hamilton et al., 1994).More frequently,naturally occurring autoantibodies are IgMs rather than IgGs(Rodriguez et al., 2009). A large proportion of the natural antibodypool is polyreactive to phylogenetically conserved structures such asnucleic acids, heat shock proteins, carbohydrates, and phospholipids(Hardy and Hayakawa, 1994; Kantor and Herzenberg, 1994).

From pathologic and MRI studies, disability, the main outcome inMS patients, is related mainly to axonal loss, meanwhile the role ofmyelin damage seems to have only a protective effect over axons(Hasan et al., 2012). There are many radiologic and pathologic evi-dences that confirm this issue, but efforts on pathogenic studieshave been drive to the demonstration of antibodies against com-pounds of myelin, and only a minor of works have focused in the im-munology of axons and neurons.

In order to study the possible role of ITS of IgM isotype on axonal lossin MS, we hypothesized that the CSF fromMS patients could contain an-tibodies that recognize cell-surface antigens. For this purpose, we have

Table 1Clinical and CSF profiles of MS, NMO and NIND groups.

No. Age E.T. EDSSa Gender IgG Index IgM Index [IgM] CSF mg/L O

Non-inflammatory neurological diseases1 24 n.a. n.a. M 0.48 0.06 0.66 g2 59 n.a. n.a. F 0.45 0.08 0.57 g3 34 n.a. n.a. F 0.51 0.04 0.92 g4 19 n.a. n.a. F 0.53 0.05 0.30 g5 40 n.a. n.a. F 0.54 0.08 0.15 gMean 35.2 n.a. n.a. 80% 0.50 0.06 0.52 0

Neuromyelitis optica patients6 35 3 7.0 F 0.43 0.23 0.16 g7 40 2 8.0 F 0.46 0.03 0.17 g8 27 11 3.5 M 0.23 0.13 0.65 g9 36 9 3.5 F 0.58 u. u. g10 38 7 2.5 F 0.43 0.04 0.23 g11 38 10 2.0 F 0.51 0.08 0.71 g12 45 2 3.0 F 0.42 u. u. gMean 37.0 6.2 4.2 85% 0.43 0.10 0.38 0

Multiple sclerosis patients13 34 5 6.5 M 0.57 0.28 0.97 g14 48 17 3.5 F 1.37 0.04 0.29 g15 23 2 1.5 M 0.41 0.05 1.08 g16 26 10 4.5 F 0.55 0.09 0.24 g17 27 2 2.0 M 0.33 0.04 0.41 g18 34 1 1.5 F 0.72 u. u. g19 29 2 2.5 F 0.59 0.18 1.16 g20 19 5 2.0 F 0.61 0.09 0.23 g21 46 1 1.5 F 0.71 0.03 0.31 g22 30 1 3.0 F 1.33 0.09 0.67 g23 34 1 0.0 F 0.67 0.06 0.45 g24 26 1 3.0 F 0.95 0.11 0.46 gMean 31.3 4.0 2.6 75% 0.73 0.10 0.57 8P 0.451 0.072 0.082 0.883 0.0371 0.581 0.571 n

No. = patient number; Age=patient age in years; E.T. = evolution time in years; EDSSCSF_Albumin)/(Serum_IgG/Serum_Albumin); IgM Index=(CSF_IgM/CSF_Albumin)/(Serumligoclonal IgG bands in CSF; OCMB=oligoclonal IgM bands in CSF; Anti-neuronal IgM bintreatment; n.a. = not applicable; M=male; n.t. = no treatment at the time of collection ofchange 2 months before lumbar puncture; u. = unavailable; AZA=azathioprine; IvIg=bi-one year before LP; REB=rebif®; CPX=copaxone®; NTZ=natalizumab; n.a. = not applica=Expanded Disability Status Scale; 1=One-way Anova test; 2=Mann–Whitney U test;

performed an experiment in which cultures of granular cerebellar neu-rons have been exposed to the CSF from three different set of patients:MS, Devic's disease, and controls with no inflammatory neurologicaldiseases.

No previously published studies have shown the presence of IgMantibodies in the CSF of MS patients that recognize neural antigens.Herein we present our work on the presence of such IgM antibodiesin the CSF from MS patients. We further report that these antibodiesassociate with the extent of brain atrophy in these patients.

2. Materials and methods

2.1. Clinical samples

We studied a total of 24 subjects: 12 with clinically defined MS, 7with NMO, and 5 controls with non-inflammatory neurological dis-eases (Table 1). MS patients were diagnosed with relapsing-remitting MS according to the revised McDonald criteria (Polman etal., 2005). All patients were in remission and had not received steroidtreatment for at least 6 weeks. NMO patients were diagnosed accord-ing to the Wingerchuk criteria. (Wingerchuk et al., 2006).

The control group included: 1 case of posterior reversible leukoen-cephalopathy induced by cyclosporine; 1 case of central pontine mye-linolysis due to inappropriate secretion of antidiuretic hormone; 1case of traumatic myelitis; and 2 cases of migraine. There were no sig-nificant differences in age or sex between the study groups. No differ-ences between the evolution time and the Expanded Disability StatusScale (EDSS) score exist between the NMO and MS patients.

CGB OCMB Antiaquaporin Antibodies Antineuronal IgM binding (r.u) Treat.

− m− n.a. 1.010 n.t.− m− n.a. 0.999 n.t.− m− n.a. 1.060 n.t.− m− n.a. 0.938 n.t.− m− n.a. 0.984 n.t.

0 n.a. 1.020 n.t.

− m− + 1.280 RTX− m− + 1.020 PE− m+ − 0.870 n.t.− m− − 0.890 Aza− m+ + 0.860 IvIg− m+ − 0.910 n.t.− m+ − 0.890 n.t.

57% 42% 0.960 n.t.

+ m+ n.a. 1.400 ASCT+ m− n.a. 1.210 REB+ m− n.a. 1.310 n.t.− m+ n.a. 1.150 CPX+ m+ n.a. 1.390 n.t.+ m− n.a. 1.330 n.t.+ m+ n.a. 1.320 REB+ m+ n.a. 1.160 NTZ+ m+ n.a. 1.180 n.t.+ m+ n.a. 1.040 n.t.− m+ n.a. 1.140 n.t.+ m+ n.a. 1.200 n.t.3% 75% n.a. 1.280 n.t..a. n.a. n.a. 0.00011 n.a.

=Expanded Disability Status Scale; Gender=patient gender; IgG Index=(CSF_IgG/_IgM/Serum_Albumin); [IgM] CSF mg/L=IgM concentration in CSF in mg/L; OCGB=o-ding (r.u.)=IgM binding in relative units measured by confocal microscopy; Treat. =the samples; RTX=rituximab® five months before lumbar puncture; PE=plasma-ex-monthly pulsed intravenous immunoglobulin; ASCT=autologous stem cell transplantable.3=Pearson's chi-square test.

65E. Beltrán et al. / Journal of Neuroimmunology 247 (2012) 63–69

We used CSF and peripheral blood samples that had been collectedduring diagnosis. These samples were stored at −80 °C until use. Wedetermined the IgG and IgMoligoclonal bands and IgG and IgM indexes,as we described below, according to routine diagnostic criteria. Weobtained informed consent from all participants. The study was ap-proved by the ethics committees of the Hospital Universitario La Féand the Hospital Clínico Universitario, Valencia. The animal experimen-tation was approved by the committee of experimentation and animalwelfare of the Centro de Investigación Príncipe Felipe, Valencia.

2.2. Oligoclonal band detection

IgG and IgM oligoclonal bands were analyzed by isoeletric focus-ing (IEF) and immnunoblotting as previously described (Sádabaet al., 2004; Villar et al., 2001).

Briefly, sample proteins were reduced with 50 mM DTT at pH 9.5,separated by isoelectric focusing at pH 5–8 and transferred to nitrocel-lulosemembranes. IgM bandswere detected by incubation of themem-branes with anti-human-IgM antibody linked to alkaline phosphatase.

2.3. IgG and IgM indexes

Serum and CSF IgM, IgG and albumin were quantified by nephe-lometry in a Dade Behring nephelometer, except for CSF IgM, whichwas quantified by an ELISA sandwich assay involving the recognitionof IgM by a purified mouse monoclonal anti-human IgM (Tago)linked to the ELISA plate and a goat anti-human IgM labeled with per-oxidase (Jackson ImmunoResearch Laboratories Inc.). A serum samplewith a known amount of IgM was used as standard.

2.4. Cell culture and immunostaining

To establish whether CSF contained IgM-Ab those recognize neu-ronal surface antigens we isolated cerebellar granule cells as de-scribed previously (Marcaida et al., 1995). We cultured them at1.5×106cells/ml on poly-L-lysine-coated cover glasses for 14 days.Then, the cultures were incubated for 2 h with 10% v/v CSF fromMS, NMO or MIND patients added to the culture medium. After expo-sure to the CSF, neurons on poly-L-lysine-coated cover glasses wereprefixed with 0.2 mg/ml DTSP (dithiobis-succinimidyl-propionate),a cross-linking reagent, in a humid chamber for 15 min at 37 °C, fol-lowed by rinsing twice in glycine-phosphate-buffered saline (PBS)for 5 mi (Bell et al., 1987). Then, the neurons were permeabilizedby treatment with 0.5% Triton X-100 in 1 mM EGTA, 4% polyethyleneglycol, 100 mM PIPES, pH 6.9, three times 5-min each, and post-fixedwith 4% paraformaldehyde in the same medium for 15 min. Glycine0.1 M was used to block unreacted DTSP and paraformaldehyde. Be-fore incubation with the first antibody, the cover glasses were washedthree times with 3% bovine serum albumen (BSA) in Tris-buffered sa-line (TBS), and finally with 1% BSA, 5% normal goat serum and 1% ca-sein in TBS, pH 7.4, to block nonspecific binding. Primary andsecondary antibody incubations were each done at 37 °C in a humidchamber for 1 h in PBS. Antibodies and dilutions were as follows: Pri-mary antibodies were anti-β-tubulin (mouse monoclonal, 1:250;Sigma-Aldrich) and the IgM present in the CSF of the subjects ana-lyzed. As secondary antibodies, Alexa Fluor 633-conjugated goatanti-mouse IgG (dilution 1:600; Molecular Probes) to β-tubulin anda goat anti-human-IgM FITC-conjugated (dilution 1:64; Sigma) toIgM were used. Cell nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Sigma) and the cells mounted in a drop offluorescent mounting medium (DakoCytomation).

2.5. Confocal microscopy

Images were collected with a Leica TCS SP2 AOBS (Leica Microsys-tems) inverted laser scanning confocal microscope using a 63× Plan-

Apochromat-Lambda Blue 1.4 N.A. oil objective lens. All confocalimages were obtained under identical scan settings. Images of1024×1024 pixels, 8-bits were collected for each preparation. Bestfocus was based on highest pixel intensity. Imaging conditions wereidentical for all the images, and no images were saturated. Metamorph7.0 (Molecular Devices) was used for image analysis on the images col-lected. Neuronal IgM-binding values were expressed as % of mean fluo-rescence intensity (MFI) with respect to the controls. Each value is themedian of four determinations from three independent experiments.

2.6. 4Pi microscopy

Cells for 4Pi microscopy were grown on quartz coverslips and thesamples processed in the sameway as conventional confocal microsco-py. Each coverslip was mounted on a second coverslip (with a quartermirror for the phase setting of the 4Pi set-up) of equal thickness andsealed with two components silicon (Dupliflex Silicones, Protechno,Spain). 4Pi microscopy imaging was conducted with a commercial 4PImicroscope (TCS 4Pi microscope of type A, Leica Microsystems, Germa-ny) equippedwith pair of 100×/1.35 NA glycerol immersion objectives.These yield an enhanced axial resolution of ~100 nmwith reduced sidemaxima. FITC and Texas Red fluorescences was recorded using two-photon excitation at 780 nm wavelength (Mai Tai laser, NewportSpectra-Physics, Mountain View, CA) with one photon counting ava-lanche photodiodes of the system (short pass SP700, beam splitter560, two band passes 525/50 and 607/83; Chroma Technology Corp.).The pinhole was set to 0.7 “Airy unit”. The objective correction collars,focus alignment and the interference phase of themicroscope were ad-justed for each individual sample put in the system. The samples wereembedded in Prolong Gold mounting medium (Invitrogen) with a re-fractive index of 1.46. The 3D data stacks were recorded with a voxelsize of 36×36×61 nm in the x–z–y direction. Stack dimensions wereadapted to the cell size (1.10 μm). To this end, the 4Pi raw data weresmoothed and a three-point deconvolution was applied with the LCSsoftware (Leica Microsystems).

2.7. In vivo MRI and MRI analysis

An in vivo MRI study was done with the same 1.5 T machine. Weobtained T1-weighted images with and without gadolinium, T2-weighted sequences and FLAIR sequences with slices of 3 mm. Vol-ume software is a semiautomatic method of segmentation thatallowed calculation of interpolated lesion volume from T2 MRI se-quences (Marcaida et al., 1995). Cross-sectional normalized brain vol-ume (NBV) was calculated from pre-contrast T1-weighted images byusing SIENAX-V 2.6 package (Structural Image Evaluation, using Nor-malization, of Atrophy). These are automated segmentation tech-niques, which require only minimal manual input from humanobservers (Naressi et al., 2001; Smith et al., 2001).

2.8. Statistics

GraphPad Prism-4.0 and SPSS-13.0 were used for statistical infer-ence analysis. The following non-parametric tests were used: theMann–Whitney U test for continuous dichotomous variables and theKruskal-Wallis test for more than two variables with Dunn post-hocanalysis. The Pearson coefficient was calculated for correlation analy-sis. The chi-square test was used to compare discrete variables. Weconsidered P values less than 0.05 to be significant.

3. Results

3.1. Demographic and clinical correlations

Demographic and clinical characteristics from MS, NMO and NINDgroups are summarized in Table 1. There were no significant

NIND

NMO

MS

Fig. 1. Immunofluorescent detection of neuroaxonal surface antigens recognized byIgMs present in the CSF of MS, NMO and NIND patients. Confocal images of rat cerebel-lar granule neurons, in primary culture, that have been exposed to 10% CSF for 2 h. IgMmolecules in green (FITC) and β-tubulin from the microtubules of the cytoskeleton inred (Texas red). This figure shows a significant increase in the amount of neuronal sur-face antigens-bound human IgM antibodies from MS-CSF.

66 E. Beltrán et al. / Journal of Neuroimmunology 247 (2012) 63–69

differences between groups in the age range, evolution time, genderratio or treatment (four patients from NMO group and five from MSgroup received treatment). As expected, there were differences be-tween MS patients and NMO/NIND patients in the IgG index (anovatest=0.037), but not in the IgM index between NMO and MS groups.None of the patients in the NIND group or NMO group presented oli-goclonal IgG bands (OCGB), in contrast to 83% in the studied MSgroup. No patients in the NIND group presented OCMB, whereasthese bands were detected in 57% and 75% of the NMO and MSgroups, respectively. It should be noted that these percentages arenot representative of the general MS or NMO populations. In 3 ofthe 7 patients (42%) from the NMO group, anti-aquaporin-4 antibodywas detected (Table 1). These results show that the IgG index is notcorrelated with clinical variables; however, the IgM index showed asignificant correlation with EDSS score (Pearson correlation 0.704,p=0.01). Neither OCGB nor OCMB showed a relationship with anyof the clinical variables. The treatments applied to the patients in-duced no observable differences in terms of IgM binding.

3.2. Presence of IgM antibodies to neuronal surface antigens in the CSF ofMS patients

IgM-Ab from CSF of MS patients showed a pattern of reactivity toneuronal surface antigens (Fig. 1). The level of binding of anti-neuronal surface IgM-Ab to neurons in culture, measured as a per-centage of average fluorescence intensity, was significantly higherfor CSF from MS patients than for CSF from either the NIND group(p=0.0004) or NMO patients (p=0.0003), in which neuronal bind-ing was detected at low levels (Fig. 2). Through 4Pi microscopy tech-nology we were able to observe with high resolution the IgM bindingto neuronal surface. The 4Pi high-resolution image (Fig. 3) showshow the IgM bind to and intercalate between a bundle of neural con-nections. These 4Pi images have allowed us to validate the studydone. No correlation exists between the IgM index and the level ofbinding of anti-neuronal surface IgM-Ab (r=0.43, p not significant)(Fig. 4).

3.3. Levels of IgM antibodies to neuronal surface antigens in the CSF ofMS patients correlate with the extent of cerebral atrophy in thesepatients

In the MS patients, no correlation was found between the pres-ence of anti-neuronal IgM-Ab and patient age, disease evolutiontime, EDSS, IgM index or CSF IgM concentration (Table 1). We mea-sured the normalized total brain volume and we found brain atrophyin MS patients. A correlation was demonstrated between the cerebralatrophy, measured by the normalized total brain volume, and theanti-neuronal IgM level (Pearson correlation −0.623, p=0.041)

0.8

1.0

1.2

1.4

1.6

Rel

ativ

e h

um

an Ig

M b

ind

ing

(% v

s. N

IND

)

NIND NMO MS

p=0.0004

p=0.0003

Fig. 2. Graphical representation of human CSF IgM binding levels to neuroaxonal sur-face antigens. Human CSF IgM binding levels are expressed as a percentage of averagefluorescence intensity relative to NIND. After incubation of CGN cultures with 10% CSFfrom MS, NMO or NIND controls. The results show a significant increase of MS CSF-IgMbinding versus both NMO and NIND CSF-IgM.

Fig. 3. Immunofluorescent detection of neuroaxonal surface antigens recognized by IgMs present in the CSF of MS patients. Central figure: Confocal image of a primary culture of ratcerebellar granule neurons that had been exposed to 10% CSF-MS for 2 h. IgM molecules in green (FITC) and β-tubulin from the microtubules of the cytoskeleton in red (Texas red).Right side figures: 4Pi confocal images showing the region framed in the central figure, with a higher resolution and different viewing angle.

67E. Beltrán et al. / Journal of Neuroimmunology 247 (2012) 63–69

(Fig. 5). Patients with greater atrophy showed higher concentrationsof anti-neuronal IgM antibodies.

4. Discussion

MS is an inflammatory disorder of the CNS of putative autoim-mune etiology (Sospedra and Martin, 2005). It is frequently associat-ed with the presence of autoantibodies in the CSF: IgG antibodiesdirected against different neuronal (Walsh and Murray, 1998;Rosenbluth and Moon, 2003; Bartos et al., 2007; Langkamp et al.,2009) and glial proteins (Gorny et al., 1990), and IgM antibodies di-rected against myelin components (Olsson et al., 1990; Annunziataet al., 1997; Mata et al., 1999; Rosenbluth and Moon, 2003;Langkamp et al., 2009), both of which are thought to be involved inthe pathogenesis of the disease.

In this study we demonstrated that IgM antibodies against neuro-nal surface can be detected in human CSF and that the presence ofthese antibodies is increased in MS patients. Moreover, these IgM an-tibodies were found to be significantly higher in MS than in NMO, an-other well-defined inflammatory demyelinating disease in whichserum IgG autoantibodies are directed against the water channel

0.0 0.1 0.2 0.30.8

1.0

1.2

1.4

1.6

IgM Index

Rel

ativ

e h

um

an Ig

M b

ind

ing

(% v

s. N

IND

)

Fig. 4. Correlation between IgM Index and antineuronal IgM binding in MS patients.r=0.43, p not significant.

aquaporin 4 in astrocytes (Polman et al., 2005), and independent ofthe concentrations of total IgM.

We decided to explore IgM antibodies for three reasons: first, theIgM response is the main effector of the innate immune system, it isthe primary response against lipids and lipoproteins of membranes,and it does not require a previous trigger to begin a humoral re-sponse; second, there are several neurological disorders in whichIgM antibodies against lipid components of the neuronal membranehave been demonstrated, including multifocal motor neuropathywith conduction block, Miller-Fisher syndrome, and Guillain-Barrésyndrome, all with an IgM-mediated immune response; and third,in MS, both IgM index and CSF OCMB have been associated with apoor prognosis (Villar et al., 2003; Villar et al., 2005; Thangarajh etal., 2008; Boscá et al., 2010; Magraner et al., 2012).

Previous studies have demonstrated that intrathecal synthesis ofoligoclonal IgM against lipids (LS-OCMB) is a highly accurate prog-nostic factor (8), and associate with higher brain atrophy in MS (9).However, it has not been proven so far the target cells of these IgMantibodies. Most of the lipids recognized by OCMS are expressed onthe surface of many cell types, particularly neurons, such as the phos-phatidylethanolamine (PE). To our knowledge, this is the first study

Pearsons -0.623, p=0.04

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1200000.0 1400000.0 1600000.0 1800000.0 2000000.0

Rel

ativ

e h

um

an Ig

M b

ind

ing

(% v

s. N

IND

)

Total brain volume (mm3)

Fig. 5. Correlation between total brain volume and antineuronal IgM binding in MS pa-tients. r=−0,623, p=0.04.

68 E. Beltrán et al. / Journal of Neuroimmunology 247 (2012) 63–69

to show the presence in CSF of IgM antibodies directed against neuro-nal surface antigens in MS patients (Figs. 1–3). Although our resultsdo not provide either the target antigen(s) or an indication as towhether these anti-neuronal surface IgM antibodies play a role inneuroaxonal MS pathology, the relationship between NBV, a reliablemeasure of brain atrophy, and the presence of anti-neuronal IgM an-tibodies (Fig. 5) suggests that anti-neuronal surface IgM antibodiesagainst neurons are not simply a result of nonspecific inflammatoryactivity or due to the presence of natural antibodies.

As expected, we observed differences in both IgG index and OCGBbetween MS and NMO/NIND patients. In fact, no patients with NMOor NIND showed OCGB, and their IgG index was normal. IgM indexhas been associated with high disability (Villar et al., 2002; Perini etal., 2006). Our results also indicate a correlation between IgM indexand disability, which supports other studies published previously(Perini et al., 2006). Despite the relatively low number of patientsstudied, it seems that the different treatments listed in the Table 1did not significantly change the concentration of IgM bound to neu-rons in culture. Other possible confounding factors such as sex, age,and disability (in cases of NMO and MS) did not influence the results,and point to the robustness of these findings (Table 1).

The presence of CSF-IgM against the neuronal surface appeared tobe significantly greater in MS compared to NMO and NIND. It shouldbe noted that, although the production of specific autoantibodies isthe hallmark of autoimmune diseases, the presence of such anti-bodies is also part of the body's normal immunological defense mech-anisms (Heymanb, 2000; Mongini et al., 2005). According to thisreasoning, the increased level of IgM found in the CSF of MS patientsthat recognizes neuronal surface antigens clearly differentiates theMS population from both the NMO and NIND populations, but itdoes not provide information about whether these IgM are pathogen-ic, neuroprotective, or result from a bystander phenomenon.

Considering that the manifestation of acute axonal injury iscomplement-dependent and that IgM antibodies are the most effec-tive in activating the complement system (Heymanb, 2000), our re-sults suggest that the IgM-type antibodies against surface neuronalantigens identified in our work could be involved in axonal destruc-tion through complement activation. On the other hand, previouswork demonstrated the presence in human serum of natural IgM au-toantibodies that bind to neuronal surface antigens and that protectneurons and extend neuronal processes in CNS axonal disorders(Annunziata et al., 1997). In the cited work, patients with monoclonalgammopathies without neurologic disease were used. Our results,based on the use of antibodies from patients with multiple sclerosisdo not allow us to conclude whether the IgM antibodies present inthe CSF of MS patients play a pathological or neuroprotective role;either remains a possibility.

In summary, the demonstration of an increased concentration ofIgM antibodies present in the CSF of MS patients that recognize neu-ronal membrane antigens provides new insights into the identifica-tion of potential antigens involved in some of the pathogenic eventsin MS. Whether these IgM antibodies play a pathogenic role, a regula-tory role or a neuroprotective role, we believe that their identificationis extremely important. The main limitation of our study is the lowernumber of patients studied. For this reason, an expanded study, nowunderway, that includes a larger number of patients should allow usto identify the neuronal surface antigens in MS.

Acknowledgments

We thank María José Agulló for her valuable technical assistance.This work was supported by the Ministerio de Ciencia e Innovación;Instituto de Salud Carlos III–Fondo de Investigaciones Sanitarias (FISPI/052210, PS09/00976 and PS09/00551) and by the GeneralitatValenciana–Conselleria de Sanitat (AP-084/09).

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