Journal of Neuroimmunology 221 (2010) 81–86

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Journal of Neuroimmunology

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The antibody response to oligodendrocyte specific protein in multiple sclerosis

Muhammad Aslam, Sudhakar Reddy Kalluri, Sabine Cepok, Verena Kraus, Dorothea Buck,Rajneesh Srivastava, Bernhard Hemmer ⁎Department of Neurology, Technische Universität München, Klinikum rechts der Isar, 81675 München, Germany

⁎ Corresponding author. Department of NeurologTechnische Universität München, Ismaninger StrasseTel.: +49 8941404600; fax: +49 8941407681.

E-mail address: hemmer@lrz.tu-muenchen.de (B. He

0165-5728/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.jneuroim.2010.02.008

a b s t r a c t

a r t i c l e i n f o

Article history:Received 18 November 2009Received in revised form 7 February 2010Accepted 9 February 2010

Keywords:Oligodendrocyte specific proteinMultiple sclerosisAutoantibody response

The role of autoantibody responses in pathogenesis or progression of multiple sclerosis (MS) remainscontroversial. This is partly because the methods that can distinctly identify pathogenic antibody reactivitiestargeting native membrane proteins from the reactivities that originate as an epiphenomenon in suchdisease are just emerging. Oligodendrocyte specific protein (OSP or claudin-11) is a candidate autoantigen inMS and CSF reactivity towards OSP has been reported in MS patients. We characterized the autoantibodyresponse to OSP using sensitive cell based assays. In line with a previous report, higher antibody response toOSP 114–120 peptide and denatured protein was observed. However applying assays based on native OSPwe did not observe any specific OSP response in MS patients. Our results demonstrate that anti-OSPantibodies do not recognise native glial OSP and may therefore rather represent an epiphenomenon in MS.

y, Klinikum rechts der Isar,22, 81675 Munich, Germany.

mmer).

l rights reserved.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Multiple sclerosis (MS) is a chronic disabling central nervous system(CNS) disease characterized by inflammation, neurodegeneration andneuroregeneration (Noseworthy et al., 2000; Steinman, 2001; Sospedraand Martin, 2005). MS lesions are characterized by an inflammatoryinfiltrate composed of macrophages/microglia cells, T cells and B cells(Lassmann et al., 1998). Antibody deposition and complement productscan be demonstrated in the lesions of many MS patients (Storch et al.,1998). Moreover deletion of B cells by rituximab or removal ofantibodies from the blood by plasma exchange seems to amelioratethe course of MS (Weinshenker et al., 1999; Keegan et al., 2005; Hauseret al., 2008).

It is thought that myelin is the primary target for aberrantautoantibody responses in MS. Numerous studies have been conductedon antibody responses to various myelin antigens but no antigen hasbeen identified as a major target of the humoral response in adult MSpatients (Reindl et al., 2006). In children affected by a first demyelin-ating event high antibody titers to native myelin oligodendrocyteprotein were reported recently (McLaughlin et al., 2009; Brilot et al.,2009). In neuromyelitis optica spectrumdiseases, however, a diagnosticantibody specific for aquaporin-4 (AQP4) was identified and itsrelevance was confirmed in NMO patients worldwide (Lennon et al.,2004; Takahashi et al., 2007; Paul et al., 2007). One of the reasons for thefailure to detect relevant autoantigens in MS may be the conventionaltechniques that have been applied to study such responses. Most of the

standard assays, such as ELISA, immunoprecipitation or western blot,often only detect antibody responses to denatured, unfolded proteins orlinear epitopes exposed as a result of ongoing disease process. Onlyrecently assays have been established, that display the candidateautoantigens in its native form. Most candidate autoantibodies in MShave not been tested for their ability to bind to the native protein andtherefore it remains tobedeterminedwhether they canbind their targetin vivo.

Oligodendrocyte specific protein (OSP/claudin-11) is a transmem-brane protein expressed on oligodendrocytes, testes and the innerear (Gow et al., 1999). Due to its significance in myelin compactionand functioning, it has been considered as a strong autoantigeniccandidate protein in MS. Previously autoantibodies targeting OSPpeptides have been reported to be present in the CSF of 80% ofrelapsing–remitting MS (RRMS) patients (Bronstein et al., 1999). Theantibodies to OSP were not detected or only present at lower levels inthe CSF of controls. However, it remained unclear, whether theseautoantibodies can bind the native OSP expressed on the surface ofglial cells. In this study, we extensively characterized the antibodyresponse to OSP in MS.

2. Materials and methods

2.1. Patients and controls

Patients and controls were recruited at the Department ofNeurology in Munich and Duesseldorf. Paired serum and CSF samplesfrom MS patients (n=40) diagnosed with RRMS (n=10), PPMS(n=10), SPMS (n=10), clinically isolated syndrome (CIS, n=10)and serum from children diagnosed with inflammatory CNS demy-elinating disease (n=10), were obtained. Serum and CSF of age

82 M. Aslam et al. / Journal of Neuroimmunology 221 (2010) 81–86

matched patients with inflammatory (n=10) and non-inflammatory(n=8) neurological disease and serum from healthy individuals(n=10) served as controls (n=28). Clinical and CSF data of ourpatients and controls are summarized in Table 1. All MS patients gavetheir written informed consent and the ethics committee of theTechnische Universität in Munich approved the study.

2.2. Cloning and expression of OSP/claudin-11

Cloning and expressionwasperformed aspreviously reported (Zhouet al., 2006). A human brain cDNA library prepared from total brain RNA(BD Biosciences) was used to get OSP cDNA. SpeI and SacII restrictionsites were added to the OSP cDNA, using 5′-actagtcatggtggccacgtgcctg-3′ and 5′-ccgcgggccctcttatacgtgggcactc-3′ as forward and reverseprimers, respectively. The PCR product was cloned into the plasmidpLenti6/V5 (Invitrogen). The resultant pLenti6/V5-OSP construct andthe viral packaging mix were used to transfect a 293FT cell line withLipofectamine 2000 (Invitrogen). Virus-containing supernatant wascollected andwas used to transduce human LN18 glioblastoma cell line.As a control, we transduced the LN18 cells with an empty vectorpLenti6/V5 containing viral supernatant. Both LN18OSP and LN18CTR celllines were maintained under the same culture conditions in RPMI 1640medium(PAA) supplementedwith 10% FCS, 1%penicillin–streptomycinand 1% L-glutamate (Invitrogen).

2.3. Immunofluorescence staining, flow cytometry and western blotting

For Immunofluorescence staining cells were grown overnightin chamber slides (Nalge Nunc). Staining was performed by using0.3 μg/ml anti-OSP monoclonal antibody (LifeSpan BioSciences) andan Alexa 488-conjugated goat anti-mouse IgG (Invitrogen) assecondary antibody. DAPI (Invitrogen) was used for the nuclearstaining. Images were captured and analyzed by an Olympus IX71microscope system.

For flow cytometry staining and titration experiment, 20 µl ofdiluted primary monoclonal antibody (10 dilutions from 0.001 to1 mg/ml), was added in duplicates to 96-well plates containing 30,000LN18OSP or LN18CTR cells in 20 µl of RPMI 1640 growth medium. After20 min incubation on ice cells werewashed twice with PBS containing1% FCS. Alexa Fluor 488-labelled goat anti-mouse IgG (Invitrogen) wasapplied as secondary antibody. After final washing steps cell surfacestaining was analyzed on a FACS cell analyzer (CyAn ADP, Beckman

Table 1Clinical and CSF data of patients and controls.

MS (n=50)

RRMS(n=10)

SPMS(n=10)

PPMS(n=10)

CIS(n=1

Age(Mean/range)

40 years(30–48)

44 years(27–66)

46 years(21–68)

34 yea(27–5

Gender(F/M)

6/4 6/4 5/5 4/6

EDSS(Mean/range)

1.8(1–3)

6(3.5–8.5)

5(3.5–6.5)

1.8(1–2)

CSF cells/µl(Mean/range)

17(3–81)

2(1–5)

8(1–25)

8.0(4–21

QIgG(Mean/range)

6.0(3.3–9.5)

8(3–20)

9(2–20)

6.0(3.6–9

QAlb(Mean/range)

5.0(3–13)

9(5–17)

8(4–14)

7.0(2–29

IgG index(Mean/range)

0.97(0.4–1.8)

1(0.6–1.6)

1.13(0.5–3)

1.05(0.56–

Demographic and clinical data is shown for patients (n=50) and controls (n=28). CSF frelapsing–remitting multiple sclerosis, PPMS: primary-progressive multiple sclerosis, SPMSpaediatric/children MS, OIND: other inflammatory neurological diseases, NIND: non-inflamm

a OIND include meningo-encephalomyelitis (4), cerebral vasculitis (1), herpes-encephalib NIND includes pseudotumor cerebri (4), headache (3) and ideopathic facial palsy (1).

Coulter) using summit software (Beckman Coulter). Difference of themedian fluorescence intensities of LN18OSP and LN18CTR cell lines(ΔMFI) served as a measure of the level of OSP surface expression. Toscreen sera and CSF samples for anti-OSP reactivity, 20,000 LN18OSP

and LN18CTR cells in 20 μl of RPMI medium were added to each wellcontaining 20 μl serum or CSF dilution (5 mg/l total IgG). Alexa Fluor-488-labelled goat anti-human IgG (Invitrogen) was used as secondaryantibody. Difference of the median fluorescence intensities of LN18OSP

and LN18CTR cell lines (ΔMFI) for a particular serum or CSF stainingserved as a measure of its anti-OSP reactivity.

For western blotting, LN18OSP and LN18CTR cells were lysed withmammalian protein extraction solution (Thermo Fisher Scientific Inc.USA). Cell lysates were heat denatured at 75 °C in the presence ofa reducing agent containing 500 mM dithiothreitol (DTT) and LDSSample Buffer (both from Invitrogen) and loaded onto 4–12% SDS-PAGE(Invitrogen). Western blotting was carried out using standard protocol.Briefly, after transfer to PVDF membrane and blocking with 4%milk, rabbit anti-OSP polyclonal antibody (Santa Cruz Biotechnology)was used in combination with HRP labelled anti-rabbit IgG antibody(Invitrogen).

2.4. Enzyme linked immunosorbent assays

For peptide ELISA, 96-well plates (Nalge Nunc) were coated with100 µl (10 µg/ml) OSP 114–120 peptide in bicarbonate buffer (pH9.6). To block unreacted sites, the wells were treated with 2% BSA for2 h at room temperature. Washing was performed with PBS-T (0.05%Tween 20 in PBS). After washing four times, serum (100 µl/well,20 mg/l IgG) or CSF (100 µl/well, 20 mg/l IgG) was added andincubated for 1h at room temperature. The wells were washed andhorseradish peroxidise labelled rabbit anti-human IgG antibody(DAKO) was added. After the final incubation and four washes,100 µl of HRP substrate (KPL Inc.) was added. After 20 mindevelopment of colour reaction was stopped with of 2 N sulfuricacid (50 µl/well) and the plates were read using an automated ELISAplate reader (Tecan).

For native ELISA LN18CTR and LN18OSP were grown to 90confluence. Medium was removed and cells were washed twicewith PBS. Following washing cells were incubated in 12 ml Sulfo-NHS-SS-Biotin solution in PBS (Thermo Fisher Scientific) at 4 °C for30 min. After quenching the biotinylation reaction cells werescrapped from the flask and washed twice with TBS buffer (Thermo

Controls (n=28)

0)Child-MS(n=10)

OINDa

(n=10)NINDb

(n=8)HC(n=10)

rs7)

12 years(7–19)

40 years(27–76)

40 years(20–56)

33 years(22–45)

5/5 6/2 6/4 9/1

1.1(0–5)

– – –

)NA 92

(29–206)2.0(0–6)

–

.7)NA 35

(8.4–45)5(3–9.6)

–

.1)NA 17

(2.4–55)2(1.3–5.1)

–

3.59)NA 0.82

(0.4–1.6)0.47(0.41–0.53)

–

rom childhood MS patients and healthy control individuals was not available. RRMS:: secondary-progressive multiple sclerosis, CIS: clinically isolated syndrome, Child-MS:atory neurological disease, HC: healthy controls. NA: not available, “–”: not applicable.

tis (2), limbic encephalitis (2) and HIV (1).

83M. Aslam et al. / Journal of Neuroimmunology 221 (2010) 81–86

Fisher Scientific) and 50,000 biotinylated cells per well were added tostreptavidin coated microwell plates (Thermo Fisher Scientific). Aftercoating wells were washed twice and then blocked with 2% BSA.Staining was then carried out as described for peptide ELISA. Specificnative OSP antibody reactivity (ΔOD) was defined by OD obtained bya particular serum or CSF sample with LN18OSP minus the OD obtainedby the same sample with LN18CTR cell line.

For denatured antigen ELISA, LN18OSP and LN18CTR cells were lysedwithmammalian protein lysis solution (Thermo Fisher Scientific). Celllysates were heat denatured at 75 °C for 15 min in the presence of areducing agent containing 500 mM dithiothreitol (DTT) (Invitrogen).Cell lysates were then diluted to 30 µg/ml in 100 mM sodiumcarbonate buffer (pH 9.6) and 100 µl of this solution was added to96-well Nunc immobilizer™ amino plates (Nalge Nunc Inc.). Plateswere again heated in water bath at 75 °C for 15 min and left forcoating at room temperature for 2 h. ELISA procedure was performedas described earlier. Specific antibody titer (ΔOD) was determined bythe OD obtained with the lysate of LN18OSP cell line subtracted by theOD obtained with the lysate of LN18CTR cell line. Sensitivity andreproducibility of the assay were evaluated by titration experimentsusing the anti-OSP/claudin-11 polyclonal antibody (Santa CruzBiotechnology).

2.5. Statistical data analysis

The Mann–Whitney U test was performed for the comparison ofOSP reactivity among patients and controls. The analysis wasperformed by GraphPad Prism 5.00. Threshold for OSP reactivity inELISA assays was calculated as mean delta OD±3 standard deviations

Fig. 1. Recombinant expression of native OSP to determine antibody binding. Immunofluorhuman OSP/claudin-11 monoconal antibody is shown (a). Flow cytometry histogram showline) and LN18OSP (green line) cells (b). Titration curve showing ΔMFI (difference between mLN18CTR cells) at various concentrations of anti-human OSP/claudin-11 monoclonal antibodyOSP/claudin-11 monoclonal antibody in native ELISA is shown (d). Delta OD indicates the difIn western blot (e) 22-kDa and 44-kDa bands corresponding to OSP/claudin-11 are only visi

of control group. Threshold for native OSP reactivitywas calculated forflow cytometry assay as mean delta MFI±3 standard deviations ofcontrol group.

3. Results

3.1. Validation of OSP expression and efficacy of screening methods

The LN18 cell line was transduced with OSP cDNA containingvector and an empty lentiviral vector to obtain LN18OSP and anappropriate control cell line, LN18CTR respectively. Both cell lines weregrown under the same conditions and solely differed from each otherby the expression of OSP. Surface expression of OSP was confirmed byImmunofluorescence and flow cytometry with a monoclonal antibodyspecific for OSP/claudin-11 (Fig. 1a–b). A highly reproducible surfacestaining was observed at different concentrations of the monoclonalantibody (Fig. 1c) demonstrating that the LN18OSP cell line can beused to screen serum and CSF for antibody responses to native OSP ina cell based flow cytometry assay. Similar results with the sameantibody were obtained in a cell based ELISA that preserves themembrane bound antigens in its native form (Fig. 1d). Specificexpression of OSP in LN18OSPcell lysate was confirmed by westernblotting (Fig. 1e) using anti-OSP polyclonal antibody. Approximately22-kDa band corresponding to claudin-11monomer and 44-kDa bandcorresponding to dimer were only visible with LN18OSP cell lysate. Thesame lysate was denatured and used for the coating of ELISA plates indenatured antigen ELISA. A titration curve (Fig. 1f) with polyclonalanti-OSP antibody represents validity of assay to determine specificantibody responses to OSP in denatured condition.

escence staining of LN18CTR [Left (×200)] and LN18OSP cells [Right (×200)] with anti-ing anti-human OSP/claudin-11 monoclonal antibody staining with the LN18CTR (blueedian fluorescence intensity: MFI obtained with LN18OSP minus the MFI obtained withis given (c). Titration curve showing delta OD at various concentrations of anti-human

ference between OD obtained with the LN18OSP and the OD obtained with LN18CTR cells.ble in LN18OSP cell lysate. Titration curve for denatured antigen ELISA (f) is also shown.

84 M. Aslam et al. / Journal of Neuroimmunology 221 (2010) 81–86

3.2. MS serum and CSF show elevated anti-OSP reactivity in denaturedconditions

We measured the serum and CSF reactivity of MS patients andcontrols towards linear/denatured epitopes of OSP protein. Allsera and CSF samples were adjusted to a total IgG concentration of20 mg/l in each assay. In our screening experiment we observedhigher reactivity in sera (mean ΔOD 0.45±0.16) of MS patientsas compared to control sera (mean ΔOD 0.32±0.08), (P≤0.05,Fig. 2a). A similar trendwas also observed in the CSF analysis (P≤0.05,Fig. 2b).

We also observed elevated anti-OSP 114–120 reactivity in serumand CSF from our patients and controls. In subgroup analysisstatistically significant difference in anti-OSP 114–120 reactivity wasachieved in sera and CSF from RRMS patients compared to controls.(Fig. 2c–d).

3.3. MS sera and CSF are not reactive towards native OSP/claudin-11

We applied two different assay systems to test MS serum or CSFreactivity towards native OSP. In our cell based ELISA assay the serum(n=50) andCSF (n=40) samples fromMSpatientswere tested againstcontrol serum (n=28) and CSF (n=18) for native OSP reactivity. Themean ΔOD values of MS serum (0.29±0.10) and CSF (0.18±0.06) didnot differ significantly to that of control serum (0.31±0.12) and CSF(0.20±0.07) in cell based ELISA assay (Fig. 3a–b). Stratified analysis ofdifferent disease groups and controls did not show elevated reactivitytowards native OSP.

Absence of MS serum and CSF reactivity towards native OSPwas confirmed in a cell based flow cytometry assay. The mean ΔMFIvalues from MS serum (7.8±3.2) and CSF (3.6±2.1) did not differsignificantly from control serum (7.4±3.8) and CSF (3.8±1.9)(Fig. 3c–d).

Fig. 2. Serum and CSF antibodies to denatured OSP and OSP peptide. Serum and CSF reactivitwas compared between MS patients and controls using an ELISA assay based on denatured Otop for each group with standard deviation). Mann–Whitney U test is used to compare groagainst denatured OSP (a, b; right panels) and OSP 114–120 peptide (c; d; right panels) in sonset. P values are only shown for significant difference between patients and controls in th(Threshold=mean delta OD of control group±3 standard deviations).

4. Discussion

OSP is an interesting candidate autoantigen in MS due to itsexpression and relevance for myelin structure and function. Becauseof its similarities with PMP-22, it is considered to be involved inoligodendrocyte proliferation, differentiation and migration (Tiwari-Woodruff et al., 2006). Immunization with claudin-11 peptide (103–123) induces optic neuritis in Rhesus monkeys. The animals alsodevelop an antibody response to claudin-11 peptide (103–123)(Bajramovic et al., 2008). In another study in SJL/J mice; immunizationwith soluble recombinant OSP/claudin-11 (lacking hydrophobic trans-membrane domains of the native molecule) induced experimentalautoimmune encephalomyelitis and optic neuritis. The IgG antibodiesin these mice were directed against OSP 22–46 (Kaushansky et al.,2006). Bronstein et al. usedwestern blot assays to identify CSF reactivityof MS patients to recombinant human OSP expressed in E. coli andhuman brain homogenate. They also reported binding of pooled CSFfrom six RRMS patients to OSP 114–120 peptide. Anti-OSP antibodies inCSF of RRMS patients were tested for their ability to influenceoligodendrocyte progenitor cell (OPC)migration, however a convincinglink could not be established (Tiwari-Woodruff et al., 2004).

It is important to note that the conventional assays applied toidentify autoantibody reactivity in MS e.g. ELISA, western blot etc.mostly fail to provide native folding conditions for the underlyingtargets and therefore only determine reactivities to the denaturedand unfolded epitopes or cannot discriminate between native andnon-native reactivities (O'Connor et al., 2007). Such antibodies aremore likely to be originated as a result of ongoing pathogenesis andrepresent an epiphenomenon in MS pathology that might be the casein anti-OSP reactivity.

In the current study we applied different assays to investigate theantibody response to OSP in MS. Sera and CSF samples of patients andcontrols were investigated by ELISA based on peptide (OSP114–120),

y towards denatured OSP (a, b; left panels) and OSP 114–120 peptide (c, d; left panels)SP or OSP 114–120 respectively. Horizontal bars indicate median (also indicated at theups. P values are indicated (ns = non-significant). Comparison of antibody reactivityerum and CSF between controls and MS patients stratified for disease course and age ofe subgroup analysis. Dotted lines indicate threshold for anti-OSP serum/CSF reactivity

Fig. 3. Serum and CSF antibody response to native OSP. Serum and CSF reactivity towards native OSPwas compared betweenMS patients and control individuals in ELISA assay basedon native OSP (a, b) and cell based flow cytometry assay (c, d). Horizontal bar indicates median (also shown at the top of each group with standard deviation). Mann–Whitney U testwas used to compare groups. Stratified analysis of different subgroups is also shown (right panels). Threshold for native OSP serum/CSF reactivity is indicated by dotted line(Threshold=mean delta OD/delta MFI of control group±3 standard deviations).

85M. Aslam et al. / Journal of Neuroimmunology 221 (2010) 81–86

full length denatured and native OSP. We also applied a cell basedflow cytometry assay that allowed us to detect antibodies that wouldbind to OSP expressed on the surface of glial cells. We confirmed aprevious report of higher antibody titers in MS patients to denaturedOSP protein and the OSP peptide (Bronstein et al., 1999). This higherreactivity was found particularly in RRMS patients. By contrast wedid not observe significant reactivity towards native OSP in any MSsubgroup.

This finding contrasts the results of studies on myelin oligoden-drocyte protein (MOG) another candidate protein in autoimmunediseases of the CNS. While antibodies to non-conformational andconformational epitopes are found in MOG induced EAE models, onlyantibodies binding native MOG can mediate demylination in vivo(von Büdingen et al., 2002; Breithaupt et al., 2008). In humans alsoantibodies to denatured and native MOG are found but littlecrossreactivity is seen between antibodies binding denatured andnative protein. High titer antibodies to native MOG seem to occurpredominantly in children affected by inflammatory demyelinatingdisease, while these titers are only rarely observed in adult MSpatients (McLaughlin et al., 2009; Brilot et al., 2009). Interestinglythese antibodies bind to myelin and seem to induce demylinationupon transfer in EAE rats (Zhou et al., 2006).

Based on our findings in the current and previous studies weconclude that the antibody response to OSP predominantly targetsnon-conformational epitopes of the protein. Since these antibodies donot bind the native protein expressed on glial cells, it is likely that theantibody does not contribute to antibody mediated demyelination.Since antibody titers are predominantly higher in RRMS than CIS,SPMS and other disease groups we believe that the occurrence of theantibodies is a result and not the cause of the autoimmune attack tothe CNS. Most likely the antibodies represent an epiphenomenon inMS pathology, an effect that may originate after initial demyelinationevent, when degraded myelin proteins are released to the peripheryand generate polyspecific T and B cell responses.

Acknowledgements

The studywas supported by the Deutsche Forschungsgemeinschaft(He2386/7-1), and the Bundesministerium für Bildung und Forschung(krankheitsbezogenes Kompetenznetz MS, Control-MS). MohammadAslam was supported by the DAAD.

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