Native myelin oligodendrocyte glycoprotein promotes severe chronic neurological disease and demyelination in Biozzi ABH mice

Native myelin oligodendrocyte glycoprotein promotessevere chronic neurological disease and demyelinationin Biozzi ABH mice

Paul A. Smith1,2, Nicole Heijmans1, Boudewijn Ouwerling1, Esther C. Breij3, Nicholas Evans2,Johannes M. van Noort4, Arianne C. Plomp4, C�cile Delarasse5, Bert 't Hart1, Danielle Pham-Dinh5 andSandra Amor1

1 Department of Immunobiology, BPRC, Rijswijk, The Netherlands2 Department of Neuroinflammation, Faculty of Medicine, Imperial College London, London, UK3 Department of Molecular Cell Biology and Immunology, VUmc, Amsterdam, The Netherlands4 Division of Biological Research, Immunological and Infectious Diseases, TNO Prevention and Health, Leiden,The Netherlands

5 Institut National de la Sant� et de la Recherche M�dicale (INSERM), Unit� 546, Universit� Pierre et Marie Curie,H�pital de la SalpÞtri�re, Paris, France

Myelin oligodendrocyte glycoprotein (MOG) is a powerful encephalitogen forexperimental autoimmune demyelination. However, the use of MOG peptides orrecombinant proteins representing part of the protein fails to fully address the possiblepathogenic role of the full-length myelin-derived protein expressing post-translationalmodifications. Immunization of mice with central nervous system tissues from wild-type (WT) and MOG-deficient (MOG–/–) mice demonstrates that MOG in myelin isnecessary for the development of chronic demyelinating experimental autoimmuneencephalomyelitis (EAE) inmice. While immunizationwithWTspinal cord homogenate(SCH) resulted in a progressive EAE phenotype, MOG–/– SCH induced a mild self-limiting acute disease. Following acute EAE with MOG–/– SCH, mice developed T cellresponses to recombinant mouse MOG (rmMOG), indicating that MOG released frommyelin is antigenic; however, the lack of chronic disease indicates that such responseswere not pathogenic. Chronic demyelinating EAE was observed when MOG–/– SCH wasreconstituted with a dose of rmMOG comparable to MOG in myelin (2.5% of total whitematter-derived protein). These data reveal that while immunization with the full-lengthpost-translational modified form of MOG in myelin promotes the development of amore chronic autoimmune demyelinating neurological disease, MOG (and/or othermyelin proteins) released frommyelin during ongoing disease do not induce destructiveautoimmunity.

Introduction

Immunization of susceptible mouse strains with themajor myelin proteins proteolipid protein (PLP) andmyelin basic protein (MBP) or their encephalitogenicpeptide sequences induces an experimental neurologicaldisease of the central nervous system (CNS), experi-mental autoimmune encephalomyelitis (EAE), an ani-mal model of the human disease multiple sclerosis (MS).

Clinical immunology

Correspondence: Sandra Amor, Biomedical Primate ResearchCentre (BPRC), Department of Immunobiology,Lange Kleiweg 139, 2280 GH Rijswijk, The NetherlandsFax: +31-15-284-3999e-mail: [email protected]

Received 10/11/04Revised 22/12/04

Accepted 3/2/05

[DOI 10.1002/eji.200425842]

Key words:EAE

� Multiple sclerosis� Autoimmunity� Demyelination

Abbreviations: MOG: Myelin oligodendrocyte glycoprotein �rmMOG: Recombinant mouse MOG � rhMOG: Recombinanthuman MOG � MS: Multiple sclerosis � SCH: Spinal cordhomogenate � CNS: Central nervous system � PSD: Post-sensitization day � NMS: Normal mouse serum

Eur. J. Immunol. 2005. 35: 1311–1319 Clinical immunology 1311

f 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji.de

More recently, minor myelin antigens such as myelinoligodendrocyte glycoprotein (MOG) [1], myelin-asso-ciated glycoprotein [2], and oligodendrocyte-specificprotein [2], as well as myelin-associated antigens aB-crystallin [3] and other CNS antigens such as amyloid-b[4], have also been shown to be encephalitogenic withvarying degrees of neurological severity and histo-pathology.

MOG is a highly conserved, quantitatively minorconstituent of CNS myelin [5] located on the outermostlamellae of the myelin sheath [6] and on the surface ofmature oligodendrocytes [7], thereby representing ahighly susceptible target for autoimmune attack. Thefailure to detect MOG protein within the thymus hasbeen described to result in an absence of self-toleranceand, as a result, release of a repertoire of MOG-reactivelymphocytes into the periphery [8, 9].

Immunization with MOG induces inflammatoryT cells in both susceptible rodent strains [1, 10, 11]and out-bred non-human primates [12, 13], resulting inan experimental model that resembles many of theclinical and histological features of MS. Furthermore,while antibodies specific for several myelin componentsare able to enhance myelin uptake bymacrophages, onlyantibodies to MOG enhance EAE and demyelination inmice [14, 15].

While the encephalitogenic potential of syntheticpreparations of MOG, i.e. MOG peptides and recombi-nant forms of MOG, has been defined, the pathologicalsignificance relative to the myelin proteins expressedwithin whole spinal cord homogenate (SCH) hasremained unclear. Recently, the generation of MOGnull (MOG–/–) mice that express no overt phenotype hasbeen described [9]. These mice are susceptible tomyelin-induced EAE but are resistant to diseasefollowing immunization with recombinant rat MOG [9].

In the current study, we have used CNS tissuesderived from MOG–/– and WT mice to investigate therole of full-length myelin-derived 'native' MOG, incor-porating post-translational modifications, in chronicneurological demyelinating disease. The clear differencebetween the disease induced with MOG–/– compared toWT tissue demonstrates for the first time that therelatively low levels of MOG in myelin are sufficient tohave a major impact on the course of disease andresultant pathology. Moreover, the fact that Biozzi ABHmice immunized withMOG–/– CNS tissues develop T cellresponses to MOG after acute neurological diseasedemonstrates broadening of themyelin-specific immunerepertoire to MOG during ongoing neurological disease,yet the failure of the mice to develop chronic diseaseindicates that such responses are not pathogenic.

Results

MOG in myelin is necessary for chronicrelapsing EAE

To examine the role of the full-length myelin-derivedMOG for EAE induction, Biozzi ABH (H-2dq1) mice wereimmunized with SCH from C57BL/6 WT mice or frommice lacking the MOG gene (MOG–/–). Immunizationwith SCH from WT mice consistently induced moresevere clinical signs of acute EAE on post-sensitizationday (PSD) 16–17 compared to mice immunized withMOG–/– SCH (p<0.05). Furthermore, while miceimmunized with WT SCH developed chronic disease(Fig. 1), those immunized with MOG–/– SCH recoveredfrom the acute phase and did not show any further signsof clinical disease (PSD 21–45, p<0.05). Similarly, SJLand C57BL/6 mice immunized with WT SCH developedmore severe disease in the acute phase compared tothose immunized with MOG-deficient SCH (data notshown).

To determine whether development of the chronicdisease was merely a function of exhibiting severedisease during the acute phase, we compared WT SCH-and MOG–/– SCH-immunized Biozzi mice that reached ascore �3 for neurological symptoms during the acutephase of EAE (see Materials and methods). It wasobserved that, while this grade of paralysis was observedin both groups, only those in the WT SCH-immunizedgroup developed chronic disease, suggesting that thesignificant differences observed between PSD 21 andPSD 45 were not a direct consequence of the earlier EAEseverity.

Fig. 1. MOG in myelin is necessary for severe chronic EAE inmice. ThemeanEAE group score� SD (n=20) of Biozzi ABHmicefollowing immunization with WT (&) or MOG–/– (*) SCH isshown (*p<0.05).

Paul A. Smith et al. Eur. J. Immunol. 2005. 35: 1311–13191312


MOG in myelin is essential for demyelinationin chronic relapsing EAE

In the spinal cords of Biozzi ABH mice immunized withWT SCH, the histological features during the acute(PSD 10–17), remission (PSD 26–29), and relapse/chronic (PSD 51–56) phases of EAE were essentiallysimilar to that previously reported [16]. Briefly, in acutedisease severe inflammation and minimal demyelina-

tion was observed (Fig. 2A, B), which resolved duringthe remission phase. During the relapse phase, in whichseveral WT SCH-immunized mice developed chronicdisease, significantly higher levels of inflammation(Fig. 2A, C; p<0.05) and extensive demyelination(Fig. 2B, D; p<0.01) were observed as compared toMOG–/– SCH-immunized mice (Fig. 2A, B, E, F).

In a further study, Biozzi ABH mice were immunizedwith myelin purified from the spinal cords of WT (n=5)or MOG–/– (n=5) mice (data not shown). Diseaseinduced by WT myelin was similar to that seen with WTSCH (mean score 3.7�0.2, mean onset 17.0�2.1 days).In contrast, MOG–/– myelin induced less severe disease(mean score 1.7�0.5; p<0.02). As with the SCH-immunized mice, inflammation and demyelinationwereprominent in the chronic phase of WT myelin-immu-nized mice (inflammation score 1.3�0.4, demyelinationscore 0.9�0.4), while mice immunized with MOG–/–

myelin exhibited significantly less severe pathology(inflammatory score 0.1�0.1, demyelination score0.0�0.0; p<0.05 and p<0.02, respectively).

Epitope spreading to rmMOG following MOG–/–

SCH-induced EAE

As MOG is a quantitatively minor myelin component, weevaluated whether the MOG in myelin, or that releasedas a consequence of myelin damage, is sufficient to elicitT cell reactivity following acute EAE. Biozzi ABH micewere immunized with WT SCH or MOG–/– SCH, andT cell responses to rmMOG were measured on PSD 10(prior to acute disease) or PSD 26 (after recovery fromacute disease). As expected, mice immunized withMOG–/– SCH failed to generate MOG-specific T cellresponses prior to the onset of neurological disease(Fig. 3), while three out of four mice immunized withWT SCH developed T cell responses to rmMOG at

Fig. 2. MOG in myelin is essential for chronic demyelination inBiozzi ABH mice. Histopathology scores � SEM of (A)inflammation and (B) demyelination in the spinal cords ofBiozzi ABH mice immunized with WT (black bar) or MOG–/–

(white bar) SCH (n=8/group, *p<0.05, **p<0.01). The mice wereanalyzed during both acute EAE (PSD 10–17) and chronic EAE(PSD 51–56). H&E staining (C, E) and LFB/CFV staining (D, F) wasperformed on spinal cords taken on PSD 51 from Biozzi ABHmice immunized with WT SCH (C, D) or MOG–/– SCH (E, F). Thearrowheads indicate large infiltrates (C) and extensive demye-lination (D) in mice immunized with SCH from WT mice.

Fig. 3. T cell proliferative responses to rmMOG in the spleenfollowing immunization of Biozzi ABH mice with WT SCH(black bar) or MOG–/– SCH (diagonal bar) on PSD 10, prior toonset of acute EAE, and on PSD 26, after recovery from acuteEAE. The dashed line represents the upper limit of responses incontrol mice (immunized with CFA only, rechallenged in vitrowith antigen). Stimulation indiceswere calculated as describedin the Materials and methods. The mean � SEM (n=4/group) isshown.



PSD 10. On PSD 26 (after acute EAE), T cell responsesto rmMOG were detectable in both WT and MOG–/–

SCH-immunized mice (Fig. 3). Clearly, these T cellreactivities are directed toward endogenous MOGreleased from myelin during neurological disease.

MOG constitutes 2.5% of the total proteincontent of myelin

To examine whether the amount of MOG in myelin isimmunogenic and/or pathogenic, we first calculated thelevel of MOG in myelin using Western blot analysis(Fig. 4). These studies were performed on humanmyelindue to the quantities of myelin required. To quantifyMOG as a component of myelin, increasing amounts ofrecombinant human MOG (rhMOG) were titrated into afixed amount of whole myelin protein, and the mixtureswere subjected to Western blot analysis using amonoclonal antibody that recognizes an epitope foundon both the recombinant protein and MOG in myelin. Ineach sample the resulting two major bands representingthe shorter recombinant version and full-length MOGwere quantified by densitometric scanning. The amountof recombinant protein required to produce a signalequal to that of full-length MOG was used to define theabsolute amount of the latter. Using the known amountof total myelin protein used to prepare each sample as areference, the weight percentage of MOG in myelin wascalculated.

'Physiological' doses of rmMOG induce EAE

Based on the level of MOG in myelin, it was estimatedthat 1 mg SCH, the dose used to immunize mice,

contains between 3 and 4 lg MOG. This dose wascalculated from a) the amount of myelin isolated fromspinal cord tissue (weight/weight), i.e. 40–50%, b)amino acid analysis of myelin showing that it contains20–25% protein [17], and c) data in the present studywhereby 2.5% of the protein in myelin is MOG. Todetermine if this level of MOG is encephalitogenic,Biozzi ABH mice were immunized with between 1 and500 lg rmMOG (the higher doses represent dosesgenerally used in EAE studies). It was observed thatat doses higher than 10 lg rmMOG, the majority of miceexhibited chronic relapsing EAE (Table 1). Immuniza-tion with 5 lg rmMOG also induced EAE in 3/5 mice, ofwhich 3/3 developed relapses. Likewise, while only 1/5mice immunized with 2 lg rmMOG developed EAE, thismouse also exhibited a relapse at a comparable timepoint to mice immunized with higher doses (Table 1).

Fig. 4. Western blot analysis demonstrating the concentrationof MOG in myelin. rhMOG (1–125) was added in amountsranging from 6.25 ng to 1.6 lg in twofold increments to a fixedsample (20 lg) of total myelin-derived protein from MS brains.Western blotting was performed using the MOG-specificmonoclonal antibody Z12. Myelin-derived MOG is visible as amore diffuse band than recombinant protein due to glycosyla-tion of the natural full-length protein.

Table 1. rmMOG-induced chronic relapsing EAE in Biozzi ABH micea)

rmMOGdose(lg)

Acute Relapse

No. EAE Group score Mean EAEscoreb)

Onset dayc) No. EAE Group score Mean EAEscoreb)

Onset dayc)

1 0/5 0.0�N/A N/A N/A 0/5 N/A N/A N/A

2 1/5 0.4�0.4 2.0�N/A 15.0�N/A 1/1 1.0�N/A 1.0�N/A 32.0�N/A

5 3/5 1.7�0.8 2.8�0.7 11.0�0.6 3/3 0.8�0.4 1.3�0.3 29.0�3.0

10 5/5 3.5�0.4 3.5�0.4 16.4�3.0 5/5 3.1�0.6 3.1�0.6 26.0�4.0

25 6/6 4.1�0.1 4.1�0.1 12.0�0.7 3/3 4.0�0.0 4.0�0.0 20.3�1.5

50 7/8 2.8�0.5 3.1�0.4 12.3�1.1 5/6 3.3�0.7 3.9�0.1 19.6�0.6

100 7/7 3.9�0.1 3.9�0.1 11.1�0.6 6/7 2.9�0.6 3.3�0.5 24.3�2.5

200 8/8 3.5�0.3 3.5�0.3 12.3�0.4 7/7 3.7�0.5 3.7�0.5 25.6�1.5

500 8/8 3.1�0.4 3.1�0.4 12.9�1.8 5/6 2.8�0.7 3.4�0.5 28.6�3.4

a) Animals were immunized with different doses of rmMOG (corresponding to residues 1–116) in CFA on days 0 and 7. Afterimmunization and again at 24 h, mice were injected with 200 ng pertussis toxin.

b) Mean � SEM maximum clinical score from animals exhibiting EAE in a group.c) Mean � SEM day of onset of neurological signs in mice developing EAE in a group.



Histological examination of mice immunized with 2 or5 lg rmMOG that developed EAE revealed inflamma-tion and demyelination in the spinal cord. As controls,immunization with varying doses (3–500 lg) of anunrelated recombinant protein, rMRF-4, did not inducedisease or pathology in the CNS of Biozzi mice. This isthe first report showing that low doses of MOG,equivalent to the levels in myelin, induce both clinicaland histopathological signs of EAE.

Antibodies to MOG peptides and rmMOG areabsent in WT and MOG–/– SCH-immunized mice

To determine if mice immunized with SCH or low dosesof rmMOG develop serum antibody responses tormMOG or overlapping MOG peptides, ELISA wereperformed. While antibody responses to MOG peptidesand rmMOG were observed in mice immunized withhigh doses (100 lg) of rmMOG (16/23), only 1/10 miceimmunized with 2–5 lg developed antibody responses.However, immunization with either WT (n=22) orMOG–/– (n=24) myelin or SCH did not evoke antibodyresponses to either rmMOG or MOG peptides in anymouse.

Reconstitution of MOG–/– SCH with 5 lg rmMOGinduces chronic EAE and demyelination

To investigate whether reconstitution of MOG–/– SCHwith rmMOG at a dose equivalent to that in myelin couldrestore the chronic neurological deficits seen in WTSCH-induced EAE, mice were immunized with MOG–/–

SCH to which either 3 lg or 5 lg rmMOGwas added. Asbefore, WT SCH-immunized mice developed severechronic EAE, while mice immunized with MOG–/– SCHdeveloped a significantly milder disease betweenPSD 13 and 17 (p<0.05). Addition of 3 lg rmMOG toMOG–/– SCH induced disease similar to MOG–/– SCH,but the peak severity was comparable to EAE induced byWT SCH. When MOG–/– SCH was reconstituted with5 lg rmMOG, the disease was similar to that ofWT SCH-immunized Biozzi ABHmice, in which the severity of theclinical disease in the acute phase was significantlyhigher (p<0.05) on PSD 13 and 17 as compared toimmunization with MOG–/– SCH alone (Fig. 5A). Duringchronic EAE (PSD 22–30), the severity of diseasefollowing immunization with WT SCH or MOG–/–

SCH plus 5 lg rmMOG was significantly higher thanthat observed with either MOG–/– SCH alone or MOG–/–

SCH supplemented with 3 lg rmMOG (p<0.05).Histological signs of EAE following immunization withMOG–/– SCH plus 5 lg rmMOG were similar to those inmice immunized with WT SCH (Fig. 5B), while the CNSpathology in mice immunized with MOG–/– SCH plus3 lg rmMOG was similar to that in mice immunized

with MOG–/– SCH only (Fig. 5B). T cell proliferativeresponses to rmMOG were absent on PSD 10 followingimmunization with MOG–/– SCH alone or MOG–/– SCHplus 3 lg rmMOG (Fig. 5C). In contrast, such responseswere observed in mice immunized with WT SCH or

Fig. 5. Reconstitution of MOG–/– SCH with rmMOG restoreschronic neurological deficits and demyelination in Biozzi ABHmice. (A) Mean maximum clinical score of mice in a groupexhibiting EAE following immunizationwithWT SCH (n=15/15)(&), MOG–/– SCH supplemented with 5 lg rmMOG (n=6/6) (^),MOG–/– SCH supplemented with 3 lg rmMOG (n=14/15) (~), orMOG–/– SCH (n=16/17) (*) (*p<0.05). (B) Inflammation (blackbar) and demyelination (white bar) scores � SEM in the spinalcords of Biozzi ABH mice on PSD 30 following immunizationwith WT SCH(n=8), MOG–/– SCH supplemented with 5 lgrmMOG (n=6), MOG–/– SCH supplemented with 3 lg rmMOG(n=8), or MOG–/– SCH (n=8). (C) T cell proliferative responses tormMOG on PSD 10 (before acute EAE) and PSD 30 (afterrecovery) in Biozzi ABH mice immunized with WT SCH (blackbar), MOG–/– SCH supplemented with 5 lg rmMOG (gray bar),MOG–/– SCH supplemented with 3 lg rmMOG (checkered bar),or MOG–/– SCH (diagonal bar). The mean � SEM (n=4/group) isshown. The dashed line represents the upper limit ofresponses in control mice.



MOG–/– SCH plus 5 lg rmMOG. Following recoveryfrom acute EAE (PSD 30), mice immunized with eitherMOG–/– SCH alone or MOG–/– SCH plus 3 lg rmMOGdeveloped proliferative response to rmMOG, while theresponses in the other two groups increased comparedto PSD 10.

Phagocytosis is affected by the absence of MOG

MOG-specific monoclonal antibodies have previouslybeen shown to enhance myelin phagocytosis [14] andaugment EAE severity [15]. To determine if thechronicity of disease observed in mice immunized withWT SCH or myelin is a function of enhanced macro-phage phagocytosis of myelin, we performed a macro-phage phagocytosis assay using the macrophage cell lineJ774.2. It was observed that in the presence of eithernormal mouse serum (NMS) or heat-inactivated serumas well as under serum-free conditions, the phagocytosisof WT myelin was consistently higher as compared tophagocytosis of MOG–/– myelin (***p<0.001) (Fig. 6).Similar data were obtained using bone marrow-derivedmacrophages (data not shown).

Discussion

Using CNS tissues from MOG-deficient and WT mice forthe induction of EAE, we demonstrate an important roleof myelin-derived full-length 'native' MOG during thedevelopment of chronic disease and demyelination inmice. Biozzi ABH (H-2dq1) mice immunized with SCH ormyelin developed less severe neurological signs of acuteEAE and CNS pathology whenMOGwas absent from thecomplex CNS antigen mixture. Furthermore, the miceimmunized with MOG–/– SCH developed T cell re-sponses to rmMOG following acute EAE, indicating thatMOG released from myelin was antigenic but failed to

induce chronic disease. This is the first report thatdefinitively demonstrates broadening of the myelin-specific immune repertoire to MOG during ongoingneurological disease.

The heterogeneity of the clinical and pathologicalfeatures in chronic relapsing EAE and MS may resultfrom determinant spreading, where CNS antigensreleased through myelin damage induce a broadeningof the (auto)immune repertoire. While determinantspreading has yet to be convincingly demonstrated inMS, spreading of the responses has been reported inchronic relapsing EAE [18–25]. Such demonstration iscrucially important to examine the impact of determi-nant spreading on strategies for antigen-specific inter-vention in chronic demyelinating disease. The genera-tion of MOG-specific T cell responses following MOG–/–

SCH-induced neurological damage provides definitiveevidence that myelin damage leads to release of MOGand that this 'native' MOG is immunogenic. However, inthis model, it appears that the immunogenicity is notpathogenic, since mice do not develop progressivedisease. In line with this finding, Biozzi ABH miceinfected with Semliki Forest virus develop T cellresponses to myelin proteins known to induce chronicrelapsing EAE [1, 2, 26, 27], but these animals fail todevelop clinical disease (S. Amor, unpublished data).Furthermore, while Biozzi ABH mice with chronicrelapsing EAE induced by MOG peptide 8–21 developedT cell responses to other encephalitogenic myelinepitopes, 'tolerance' to MOG 8–21 prevented furtherrelapses (P. Smith, unpublished data), suggesting thatimmune responses to other myelin epitopes do notsignificantly contribute to further episodes of disease. Itis probable that while myelin-specific T cells aregenerated, they are kept under control either by theCNS micro-environment or by conditions in local lymphnodes [28, 29].

In contrast to the study by Linares et al. [30], our datademonstrate that Biozzi ABH mice show a markeddifference in EAE severity following immunization withWT compared to MOG-deficient myelin. This discre-pancy is likely due to differences in the immunizationprotocol and adjuvant composition. The significantdifferences observed between WT and MOG–/– SCH-induced EAE in Biozzi ABH mice indicate that immuneresponses to myelin-derived MOG play a major role notonly in disease onset but also in determining the severityof chronic neurological deficits.

While robust T cell responses to rmMOG wereobserved following immunization with WT SCH and,after acute EAE, with MOG-deficient SCH, we wereunable to demonstrate humoral responses to rmMOG.The fact that we previously detected antibody responsesto recombinant rat MOG in Biozzi ABH mice [1]following immunization with SCH but could not detect

Fig. 6. Enhanced opsonization of WT spinal cord-derivedmyelin compared to MOG–/– spinal cord-derived myelinfollowing incubation with amousemacrophage cell line underserum-free conditions (black bar) or in the presence of NMS(diagonal bar) or heat-inactivated serum (white bar) in vitro(***p<0.001). The mean � SEM is shown.



antibodies specific for rmMOG (this study) emphasizesthe importance of using syngeneic proteins as autoanti-gens. Moreover, glycosylation of a MOG peptideimproved the detection of specific autoantibodies inthe sera of MS patients [31, 32]; thus, the failure todetect antibodies to rmMOG or MOG peptides in ourstudy may be due to influences of post-translationalmodifications present on MOG in myelin within ourspinal cord preparations. The role of the glycosylation ofMOG in EAE is currently being addressed usingglycosylated preparations of MOG. These data suggesta return to more immunologically complex antigenssuch as SCH andmyelin to understand the role of myelinproteins in the MS and/or EAE process of myelindamage.

We have recalculated MOG to constitute approxi-mately 2.5% of total white matter-derived protein,indicating that mice immunizedwith 1 mg SCH received3–5 lg MOG. At this level rmMOG alone or added toMOG-deficient SCH induced chronic relapsing EAE andgenerated a similar T cell response to rmMOG as seen inWT SCH-immunized mice. Although the encephalito-genic potential of recombinant MOG has been estab-lished in several EAEmodels [1, 33–37], this report is thefirst to describe that rmMOG at equivalent levels to thatin myelin is sufficient to induce chronic relapsing EAE.

We previously described how an antibody directed toMOG, but not antibodies specific for other myelinantigens, augmented phagocytosis of myelin as well asexacerbated clinical EAE, possibly by complement-dependent mechanisms [14]. In this study macrophageuptake of MOG–/– myelin was clearly reduced, suggest-ing that the low clinical and histological signs of EAE inMOG–/– SCH- or myelin-induced EAE may be due in partto decreased macrophage activity, possibly via comple-ment-dependent mechanisms. However, the discre-pancy between in vivo and in vitro data indicates thatother processes may also be relevant.

Taken together, these data confirm a disproportion-ate pathogenic role for MOG in the initiation andchronicity of EAE due to an augmented inflammatoryand demyelinating immune response. Importantly, therole of MOG in inducing chronic relapsing disease doesnot appear to result from determinant spreading but islikely to involve other mechanisms yet to be unraveled.

Materials and methods

Animals

Biozzi ABH (H-2dq1) and C57BL/6 (H-2b) were bred from stockat the BPRC, The Netherlands and Imperial College London,UK. Mice with a null mutation in the MOG gene (MOG–/–) onthe C57BL/6 background were obtained from Equip� deNeurog�n�tique Mol�culaire, Universit� de Paris [9] and bred

at Imperial College London or BPRC. According to laws onanimal experimentation in both the UK and the Netherlands,the procedures of this study have been reviewed and approvedby the respective committees. The housing, care, andbiotechnical handlings were in conformity with guidelinesset by the committees.

Recombinant MOG production

E. coli strain JM109was transfectedwith the cDNA encoding N-terminal amino acids 1–116 of mouse MOG [38] or N-terminalamino acids 1–125 of human MOG and a 31-amino acid fusionprotein sequence ligated to the pRSET A (Invitrogen, UK)expression vector, as previously described [39]. A 56-aminoacid recombinant protein, Xenopus myogenic regulatoryfactor-4 (MRF-4), expressed in an identical bacterial vectorsystem, was used as a control protein [40].

Antigens

SCH was lyophilized and reconstituted in PBS prior to use aspreviously described [1, 16]. Myelin was purified from thespinal cords of WT and MOG–/– mice as previously described[41], and protein concentrations were determined using theBradford technique. Overlapping MOG 22-mer peptides with7-amino acid overlaps (based on the entire mouse sequence)were purchased from ABC Biotechnology (UK).

Calculation of MOG in white matter

The concentration of MOG in normal appearing white matterfrom human myelin isolated from the brains of MS donors wasdetermined by Western blot analysis. Total protein wasobtained by solubilization of the samples in 80% tetrahy-drofurane: 20% water: 0.1% trifluoroacetic acid, and sub-sequent delipidation was performed by repeated etherprecipitation. Defined amounts of rhMOG (1–125) weretitrated into a fixed sample of white matter-derived totalprotein and subjected to SDS-PAGE. Western blotting wasperformed using monoclonal antibody Z12, which recognizesboth normal full-length MOG and rhMOG. Since rhMOGdiffers in molecular weight from the natural forms of MOG, thedifferent versions of MOG appear as separate bands on aWestern blot. Protein signals were evaluated using densito-metric scanning, and the point of signal equivalence for eachversion of MOGwas used to calculate the amount of full-lengthMOG in myelin.

Induction of EAE

Mice were injected s.c. into two sites on the flanks with asonicated emulsion consisting of either 1 mg SCH, 500 lgmyelin, or doses of 1 lg to 500 lg rmMOG, dissolved in PBSand emulsified in complete Freund's adjuvant (CFA) on PSD 0and 7 [1]. Pertussis toxin (200 ng) derived from Bordetellapertussis (Sigma-Aldrich, UK) was dissolved in PBS wasadministered i.p. on PSD 0, 1, 7, and 8 as previously described[26]. As controls for the EAE and in vitro studies, mice wereimmunized with various doses of the MRF-4 protein or withCFA only. Mice were weighed and graded daily for neurological



symptoms: 0, normal; 1, limp tail; 2, impaired righting reflex;3, partial hind limb paralysis; 4, complete hind limb paralysis;5, complete forelimb paralysis/moribund. A grade of 0.5 wasgiven for signs of lesser severity. Statistical analysis of clinicalscores was carried out using the Mann-Whitney Rank Sum test(SigmaStat).

T cell proliferation assays

Spleens were removed and a single-cell suspension preparedusing Lympholyte-M (Cedar Lane Laboratories, USA). Cells(1�106/ml) were cultured in RPMI medium supplementedwith 2% NMS, 2 mM L-glutamine, 100 IU/ml penicillin,100 lg/mL streptomycin, 5 mM Hepes, and 5�10–5 M 2-mercaptoethanol with 20 lg/well mouse MOG peptides orrmMOG for 72 h. Proliferation was measured by theincorporation of [3H]-thymidine (Amersham Biosciences)following addition of 1 lCi/well during the last 18 h ofculture and is expressed as mean counts per minute (cpm) �SD of triplicate cultures. Stimulation indices (SI) werecalculated as the proliferative response in the presence ofantigen divided by the proliferative response in the absence ofantigen.

ELISA

Microlon plates (Greiner Bio-one, Germany) were coatedovernight at 4�C with 10 lg/ml mouse MOG peptides,rmMOG, or MRF-4 protein in PBS. Plates were washed twicein PBS-Tween (PBS-T) and blocked for 1 h at 37�C with 2%BSA/PBS. After blocking, 100 lL diluted plasma (1:100) in 1%BSA/PBS were added and incubated for 2 h at 37�C. Plasmafrom naive mice (NMS) was used as a negative control. Afterwashing in PBS-T, the plates were incubated for 1 h at 37�Cwith alkaline phosphatase-conjugated rabbit anti-mouse Ig(Dako, Denmark). The reaction product was visualized using p-nitrophenyl phosphate-Tris buffer (Sigma-Aldrich, UK) and theabsorbance read at 405 nm. An absorbance above the meanplus three SD of the NMS reactivity against the peptides wastaken as positive.

Flow cytometry analysis of myelin phagocytosis

Myelin prepared from the spinal cords of WTand MOG–/– micewas labeled with DiI [14] and washed three times in PBS. FreshNMS was prepared from clotted peripheral blood, and heat-inactivated (Hi) serum was prepared by incubating it for30 min at 56�C to inactivate the complement system. Themouse macrophage cell line J774.2 (ECACC, UK) wassuspended in DMEM containing 5% Hi fetal calf serum, and5�105 cells were incubated in 24-well plates overnight to allowadherence. Cells were incubated with DMEM only or DMEMcontaining either 5% non-Hi or Hi NMS. To each well, 20 lgmyelin was added, and the macrophages were allowed tophagocytose for 1.5 h at 37�C. After incubation, cells werewashed three times with DMEM to remove free myelin.Macrophages were detached from the plates using 4 mg/mllidocaine (Sigma) in PBS (10 min, 37�C). Fluorescenceintensity (FL2 channel), a measurement for binding anduptake of DiI-labeled myelin, was determined using a FACScan

cytometer. The mean fluorescence intensity of phagocytosis inserum-free medium was set to 100%, and the effects of NMSand Hi NMS were calculated from this baseline. Significancewas determined by the Student's t-Test (SigmaStat).

Histology

Brain and spinal cords fixed in 5% formal saline wereprocessed for routine histology and 5-lm sections cut. Thesections were stained with hematoxylin and eosin (H&E) toevaluate inflammatory infiltrates or Luxol fast blue/cresyl fastviolet (LFB/CFV) to assess the degree of demyelination [27].An observer unaware of the treatment groups performed lightmicroscopy to qualitatively examine the degree of inflamma-tion and demyelination [15]. Statistical analysis of histologicalscores was carried out using the Mann-Whitney Rank Sum test(SigmaStat).

Acknowledgements: This research was supported bygrants from the Multiple Sclerosis Society of GreatBritain and Northern Ireland and Stichting MS Re-search, The Netherlands.

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