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International Immunology, Vol. 8, No. 11, pp. 1667-1674 © 1996 Oxford University Press

Antibody response in Lewis rats injectedwith myelin oligodendrocyte glycoproteinderived peptides

Motoki Ichikawa, Terrance G. Johns, Martin Adelmann1 and Claude C. A. Bernard

Neuroimmunology Laboratory, Faculty of Science and Technology, La Trobe University, Bundoora,Melbourne, Victoria 3083, Australia1Max-Planck Institute of Psychiatry, Department of Immunology, Am Klopferspitz 18a, 8033 Martinsried,Germany

Keywords: autoimmunity, experimental autoimmune encephalomyelitis, multiple sclerosis, myelin basicprotein, myelin oligodendrocyte glycoprotein

Abstract

Previous studies from our laboratory have demonstrated a predominant response to myelinoligodendrocyte glycoprotein (MOG) in patients with multiple sclerosis (MS) and showed that thismolecule is able to induce in Lewis rats a chronic relapsing MS-like disease with extensivedemyelination. To further study the possibility that MOG is a primary target antigen in MS, we havebegun to investigate the encephalitogenicity and antibody response of different sequences of theextracellular domains of MOG in Lewis rats. We report that none of the synthetic peptidesencompassing the MOG amino acid sequences 1-21, 67-87, 104-117 and 202-218 wereencephalitogenic. In contrast, a single injection of MOG35-55 was able to induce severeneurological signs associated with inflammation and demyelination. All rats injected with MOGpeptides 1-21, 35-55, 67-87 and 202-218 developed a high level of antibodies to their respectiveimmunizing peptides as detected by ELISA and immunoblotting. Although all MOG peptide anti-sera reacted with immunoblots of native MOG separated under reducing conditions, only anti-MOG35-55 and anti-MOG202-218 antibodies reacted to native MOG, when tested under non-reducing conditions. These results indicate that the MOG35-55 peptide, which is found in theextracellular Ig V-like domain of MOG, is not only an encephalitogenic epitope but could also be animportant determinant for initiating antibody-mediated demyelination. As indicated by the absenceof reactivity to the other MOG peptides tested, as well as other central nervous system myelinproteins including myelin basic protein and proteolipid protein, the antibody response produced byMOG peptides is highly restricted.

Introduction

Chronic relapsing experimental autoimmune encephalo-myelitis (CREAE) is a paralytic disease that is observedfollowing immunization with central nervous system (CNS)antigens emulsified in complete Freund's adjuvant (CFA) (1,2).CREAE has long been used as an animal model for thehuman demyelinating disease multiple sclerosis (MS) andhas been extensively studied to analyze the underlying auto-immune mechanisms leading to demyelination (1). Becausemyelin basic protein (MBP) and proteolipid protein (PLP) arethe two most abundant CNS myelin proteins (3), they areconsidered putative target antigens for the pathogenic auto-immune responses occurring in CREAE (2,4). Indeed, many

studies have identified the encephalitogenic peptides of MBPand PLP for various species and have characterized theimmune responses to these myelin peptides (5). In particular,it is well established that the transfer of encephalitogenicCD4+ T cells specific for these MBP or PLP peptides causesEAE in naive syngeneic recipients (4). However, there is nowconsiderable experimental evidence suggesting that otherCNS components may be more relevant target antigensin immune-mediated demyelinating diseases (6-9). Amongthese, myelin oligodendrocyte glycoprotein (MOG) is poten-tially important because it is located on the outermost lamellaof the myelin sheath, is expressed exclusively in CNS myelin

Correspondence to. C. C. A. Bernard

Transmitting editor. M. Taniguchi Received 5 June 1996, accepted 19 July 1996

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1668 Antibody response to MOG peptides

1-2135-55

Table 1 . Amino acid sequence of the synthetic MOG peptidesused in this study

Fig. 1. Proposed MOG model. MOG peptides 1-21, 35-55, 67-87,104-117 and 202-218 are shown as bold lines.

and is highly immunogenic (10,11). Furthermore, a numberof studies have demonstrated that anti-MOG antibodies areable to cause demyelination both in vivo and in vitro (12-18). The mechanisms by which such antibodies producedemyelination are varied, but include the activation of myelinproteases leading to MBP degradation (9), the activation ofcomplement (17) and the enhanced phagocytosis of myelinby macrophages (17). Recently, we produced a chronicrelapsing MS-like disease with extensive demyelination inLewis rats by a single injection of MOG or the derived peptide,MOG35-55 (9). The clinical course and the pathology of theCNS lesions in this new animal model were very similar tothose of the human disease MS. Interestingly, sera from suchaffected rats reacted specifically with MOG, but not with anyother myelin or brain proteins (9), suggesting that as observedin the cerebral spinal fluid of MS patients (19), the autoanti-body response is of restricted specificity. In order to furthercharacterize the regions of MOG which may be encephalito-genic and able to stimulate autoreactive B cells, we immunizedrats with various peptides encompassing different sequencesof the extracellular domains of MOG (Fig. 1). We report herethat four out of five MOG peptides tested were able tostimulate a strong humoral response. Among those, onlyMOG35-55 was able to induce a chronic relapsing de-myelinating disease. Further, we show that the antibodyresponse generated against these peptides is of restrictedspecificity.

Methods

Animals and reagents

Lewis rats were purchased from the Walter and Eliza HallInstitute of Medical Research (Melbourne, Victoria, Australia).

Residues Amino acid sequence

1-2135-5567-87104-117202-218

GQFRVIGPGHPIRALVGDEAEMEVGWYRSPFSRWHLYRNGKGRTELLKESIGEGKVALRIQNSYQEEAAVELKVEDLHRRLAGQFLEELRNPF

Peptides were synthesized according to the rat MOG sequence ofGardiner et al. (36).

The synthetic rat MOG peptides 1-21, 67-87, 104-117 and202-218 were gifts from Professor A. Ben Nun (The WeizmannInstitute of Science, Rehovot, Israel). MOG peptide 35-55,obtained from Auspep (Melbourne, Victoria, Australia), wassynthesized by standard F-moc chemistry and the purity(>95% pure) determined by reverse-phase HPLC. This panelof five MOG peptides was selected as they represent potentialimmunodominant sites of MOG as predicted by the 'T-site'computer program (Medlmmune, Gaithersburg, MD) (20).The sequence of these peptides is shown in Table 1. MOGwas purified from rat brain by affinity chromatography andthe purity determined by immunoblotting with highly specificantibodies to other myelin proteins as described elsewhere(21). The non-glycosylated histidine fusion protein represent-ing the Ig extracellular domain (amino acids 1-125) of mouseMOG (rMOG) was prepared as reported in Amor et al. (22).Myelin was purified from human brain white matter by themethod of Norton and Poduslo (23). The mouse monoclonalanti-MOG antibody, clone 8-18C5 (24), was purified fromascites fluid by protein affinity chromatography on a ProteinG-Sepharose 4 Fast Flow column (Pharmacia LKB, Uppsala,Sweden) according to the manufacturer's specifications. Themouse monoclonal anti-human MBP antibody, clone 65(2-1),was a gift from Professor P. R. Carnegie (Murdoch University,Western Australia) (25). The rat anti-PLP mAb, clone AB3(26), was purchased from AGMED (Bedford, MA).

Immunization

Lewis rats were injected with either whole rat MOG (100 ng),MOG-derived peptides (200 ng) or guinea-pig MBP (50 ng)dissolved in saline and emulsified with an equal volume of CFA,supplemented with 4 mg/ml of Mycobacterium tuberculosis(Difco H37 RA) in the hind footpads (50 nl per footpad) aspreviously described (9). Control rats received CFA supple-mented with Mycobacterium tuberculosis only. Rats wereassessed daily for clinical signs of disease such as weightloss and hindleg and foreleg paralysis (9). Unless otherwisestated, blood samples were obtained by tail vein puncture 8weeks after injection and sera were stored at -20°C. Animalswere cared for in accordance with La Trobe University andthe National Health and Medical Research Council of Australiaguidelines.

Histologic examination of tissues

Brains and spinal cords were dissected and immersion-fixedin 4% neutral buffered formalin. Sections of 5 urn thickness



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Antibody response to MOG peptides 1669

were cut from the forebrain, cerebellum, hindbrain and spinalcord, and stained with hematoxylin & eosin or Luxol fast bluefor evidence of inflammation and/or demyelination. Sectionswere examined without knowledge of the injection regime bytwo investigators (C. C. A. B. and A. Slavin) for perivascularinfiltrations and demyelination.

ELISA

Synthetic MOG peptides or purified rat MOG were diluted toa concentration of 1 (ig/ml in carbonate buffer, pH 9.6, and50 ^I/well coated onto 96-well microtiter plates (DisposableProducts, SA, Australia) pretreated with 0.2% glutaraldehyde.Recombinant mouse MOG was used at a concentration of10 (xg/ml with 100 |il of the dilution being applied to eachwell. After incubation at 37°C for 3 h, the plates were washedthree times with (PBS) containing 0.1% Tween 20 and blockedwith 2% BSA (Boehringer, Mannheim, Germany) in PBS at4°C overnight. The plates were then incubated with 50 \i\ ofa 1/1,000 dilution of rat serum for 1 h at 37°C. After washingthree times, appropriately diluted biotinylated goat anti-ratIgG (Sigma, St Louis, MO) was added to the plates andincubated for 1 h at 37CC. After incubation with streptavidin-horseradish peroxidase (Amersham, Sydney, Australia) for 30min at 37°C, the reaction products were visualized using 2,2'-azino-bis[3-ethylbenz-thiazoline-6 sulfonic acid] as a sub-strate and read at 405 nm with a microplate reader (Flow,McLean, VA).

SDS-PAGE

Tricine-SDS-PAGE was performed according to the methoddescribed by Schagger and Von Jagow (27) with minormodifications. The gel consisted of a stacking gel (4%T,3%C), a spacer gel (10%T, 3%C) and a separating gel(16.5%T, 6%C), all prepared in 1 M Tris and 0.1% SDS,pH 8.45. Electrophoresis was performed using 0.2 M Tris,pH 8.9, as an anode buffer and 0.1 M Tris, 0.1 M Tricine and0.1 % SDS, pH 8.25, as a cathode buffer. After electrophoresis,the gels were stained with Coomassie blue R250 or transferredto a PVDF membrane (Immobilon-P; Millipore, Bedford, MA)for immunoblotting. Standard SDS-PAGE of purified rat MOGor human myelin was carried out according to the method ofLaemmli using 13.5% polyacrylamide gels (28).

Immunoblotting

After transfer, PVDF membranes were blocked with 3% BSAin Tris-buffered saline (TBS). Dilutions of rat sera and themouse monoclonal anti-MOG antibody (8-18C5) were madein TBS containing 0.05% Tween 20 and 0.25% BSA and wereincubated with the membrane for 90 min. After washing threetimes, biotinylated goat anti-rat IgG (Sigma) or biotinylatedgoat anti-mouse IgG (Sigma) was added and incubatedfor 1 h as above. Following incubation with streptavidin-horseradish peroxidase for 20 min, reactivity was detectedusing ECL and autoradiography according to the manufac-turer's instructions (Amersham, Sydney, Australia).

Purification of anti-MOG35-55 peptide antibody

An affinity column containing MOG35-55 coupled to immobil-ized diaminodipropylamine (Pierce, Rockford, IL) was usedto purify MOG35-55 antibodies. Before use, the column was

washed extensively with 1 M NaCI containing 0.1% sodiumazide. Serum samples from immunized rats (0.2 ml diluted1:10 with PBS) were applied to the column, the flow throughcollected and bound antibodies eluted with 0.1 M glycine-HCI, pH 2.5. The fractions were neutralized with 1:10 thevolume of 1 M Tris, pH 9, and extensively dialysed against0.15 M NaCI. Before testing for their antibody activity, eachfraction was diluted so that the final concentration corres-ponded to the initial serum dilution of 1:1000.

Results

Encephalitogenic activity of MOG peptides

Consistent with our previous study (9), Lewis rats injectedwith whole rat MOG or with MOG35-55 developed a severerelapsing-remitting neurologic disease associated with CNSinflammation and demyelination (Table 2). The first symptomsof disease, involving weight loss and weakness of hind limbs,were observed 2 weeks after immunization. Rats recoveredfully within 3 or 4 days but half of them experienced a relapsecharacterized by severe hind and forelimb paralysis 6-7weeks after immunization. Histological examination at thetime of severe disease revealed multifocal demyelinationassociated with perivascular mononuclear cell infiltrationswhich were most prominent in the dorsal columns of thespinal cord (data not shown). In contrast, none of the otherMOG peptides tested, i.e. MOG peptides 1-21, 67-87, 104-117 and 202-218, were able to produce clinical or histologicaldisease (Table 2).

Antibody activity to whole MOG and MOG peptides

The serum antibody reactivity of rats injected with whole ratMOG or its peptides was tested 8 weeks after immunizationby ELISA and immunoblotting against the panel of immunizingpeptides and whole MOG. The monoclonal anti-MOG anti-body, 8-18C5, was used as a positive control for anti-MOGantibody activity.

ELISA

All rats immunized with MOG peptides 1-21, 35-55, 67-87and 202-218 developed high levels of antibody against theirimmunizing peptide but did not show cross-reactivity to anyof the other MOG peptides tested (Table 2). None of the seraobtained from the rats injected with MOG 104-117 or from thecontrol rats injected with adjuvant alone reacted to the MOGpeptides. Likewise, none of the sera from rats injected withrat MOG had significant levels of antibody activity to any ofthe MOG peptides tested. Interestingly, the monoclonal8-18C5 anti-MOG antibody did not react with any of thesepeptides. Each of the anti-peptide antisera was subsequentlytested against the whole MOG molecule. As illustrated inTable 2, antibodies raised against MOG35-55 and MOG202-218 peptides showed high reactivity to native MOG, whilstanti-MOG1-21 antibodies showed low reactivity. Sera fromanimals injected with MOG67-87, guinea pig MBP or CFAalone displayed no reactivity. As expected, the 8-18C5 anti-body and the sera from rats injected with whole rat MOG hadhigh reactivity to rat MOG (Table 2). In a series of separateexperiments it was found that sera from animals immunized



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Table 2. MS-like disease and antibody response in Lewis rats immunized with MOG peptides

lmmunogena

MOG 1-21MOG 35-55MOG 67-87MOG 104-117MOG 202-218

Whole MOGAdjuvant alone

No. of rats

Clinicalsigns

0/35/50/30/40/4

4/50/3

with

Histologicalsigns

0/35/50/30/40/4

4/50/3

Serum

1-21

1.330.040.060.080.10

0.110.05

antibody reactivity

35-55

0.091.970.060.090.15

0.160.04

tob

67-87

0.050.041.060.160.13

0.120.05

104-117

0.070.040.060.130.11

0.190.04

202-218

0.040.040.060.091.72

0.190.04

MOG

0.321.600.100.121.80

1.400.12

BSA

0.040.040.060.070.12

0.070.05

aAnimals were injected with 100 ng of whole rat MOG or with 200 ng of rat MOG peptides emulsified with CFA, supplemented withMycobactehum tuberculosis. Animals were observed up to 100 days post-injection.

%era obtained 8 weeks after immunizations were tested by ELISA at 1/1000 dilution. Results are expressed as the mean optical density(OD) units at 405 nm of duplicate determinations. Bold numbers indicate reactivities that were 3 SD or more above background. Themonoclonal anti-MOG antibody (8-18C5) used as positive control reacted only with MOG (OD = 1.450) but not with any of the MOGpeptides tested.

kDa

66.2-

45.0-

31.0-

21 .5- '

14.4-

(a)

•I*MWS 1 2 3 4 5

1

Fig. 2. Autoradiograms of immunoblots of purified rat MOG. (a) MOG was electrophoresed under reducing conditions, (b) MOG waselectrophoresed under non-reducing conditions and probed with: 1, monoclonal anti-MOG antibody (8-18C5); 2, anti-MOG1-21 antibody; 3,anti-MOG35-55 antibody; 4, anti-MOG67-87 antibody; 5, anti-MOG202-218 antibody. MWS, molecular weight standard. Each antibodyobtained 8 weeks after immunizations was tested at 1/400 dilution.

with whole MOG or with MOG35-55 also reacted with therecombinant mouse MOG, the optical density being 1.66 and0.32 respectively at 1/1000 dilution.

Immunoblotting

Anti-peptide antisera with positive antibody titers were sub-sequently investigated for their reactivity to whole MOGby immunoblotting. Figure 2 shows that under reducingconditions, antibodies against MOG peptides 1-21, 35-55,67-87 and 202-218 reacted to whole MOG, with the anti-MOG35-55 antibody showing the strongest reactivity. How-ever, under non-reducing conditions, anti-MOG35-55 andanti-MOG202-218 antibodies reacted strongly with wholeMOG, while anti-MOG 1-21 and MOG67-87 antibodies didnot recognize whole MOG (Fig. 2) even when such antiserawere tested at 1/200 dilutions (data not shown).

To further determine if the anti-MOG peptide antibodiesgenerated in Lewis rats were only reactive to immunizingpeptides or could also cross-react with the other MOGpeptides, reactivity to all five peptides was tested by immuno-

blotting following separation by Tricine-SDS-PAGE. Thiselectrophoretic system was used as it allows a superiorresolution of small proteins (27). Figure 3 shows the Tricine-SDS-PAGE analysis of MOG peptides stained with Coomassieblue. MOG1-21 was detected as a band at 4.0-4.8 kDa,MOG35-55 at 3.7-4.4 kDa, MOG67-87 at 3.1-3.7 kDa,MOG104-117 at 1.1-1.5 kDa and MOG202-218 at 3.7-4.4 kDa. These peptides were then transferred to PVDFmembranes and probed with the antisera generated againsteach of the five MOG peptides studied here. As illustratedby the example shown in Fig. 4, reactivity to MOG peptides35-55, 67-87 and 202-218 was clearly demonstrated in theserum from rats injected with each of the three correspondingMOG peptides. However, none of these specific antibodieswere cross-reactive with any of the other MOG peptidestested. None of the sera obtained from the control rats (CFAalone) reacted with any of the blots containing the separatedpeptides. No reactivity to the MOG peptides was detectedwith the 8-18C5 antibody and the sera from rats immunizedwith whole MOG (data not shown).



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Reactivity of affinity purified anti-M0G35-55 antibody

To confirm that the reactivity of the anti-MOG35-55 antibodiesto native MOG was not due to diverse antibody populations,serum samples taken 6 and 10 weeks post-injection ofMOG35-55 were purified using a MOG35-55 affinity column.As exemplified by the results shown in Fig. 5, flow-throughfractions tested by ELISA had little reactivity against nativeMOG or MOG35-55. By contrast, bound antibodies reactedstrongly to both preparations. Interestingly, affinity-purifiedanti-MOG35-55 antibodies from the 10 weeks sample reactedmore strongly to native MOG than did antibodies from the 6weeks sample (Fig. 5).

Reactivity of anti-MOG peptide antisera to human myelin

In order to ascertain if the anti-MOG-specific peptide antiseracould recognize native MOG in myelin, and perhaps alsoreact with other myelin components, human myelin proteinswere separated under non-reducing conditions, transferredto membrane and probed with the anti-MOG peptide antisera.Among the four anti-MOG peptide antibodies tested, onlythose directed to MOG35-55 and to MOG202-218 reactedstrongly with the 28 kDa form of MOG and, to a lesser extent,

kDa

-3.5

1 2 -» a 5 MWS

Fig. 3. Tricine-SDS-PAGE analysis of MOG peptides after Coomassieblue staining. MWS, molecular weight standard; lane 1, MOG202-218; lane 2, MOG104-117; lane 3, MOG67-87; lane 4, MOG35-55;lane 5, M0G1-21.

with the 55-58 kDa form of MOG (Fig. 6). No reactivity toother myelin components could be observed with these sera.(Fig. 6). Both MOG bands were also detected with themonoclonal 8-18C5 antibody.

Discussion

Although considerable attention has been given to the pos-sible pathogenic role played by autoreactive CD4+ T cells inMS, it should be emphasized that primary demyelinationcould also be mediated by autoantibodies (8,11,29). Indeed,one of the important immunopathological features of thisdisease is the intrathecal synthesis of excessive amounts ofIg. These Ig are restricted in their heterogeneity (19) withsome showing autoreactivity to CNS antigens (30,31). Whilstthe role of such autoantibodies in the pathogenesis of MS isstill undefined, there is increasing evidence suggesting thatthe extensive immune-mediated demyelination seen in theCNS of such patients is probably caused by a synergybetween autoreactive T and B cells (14,15,29).

As part of our ongoing efforts aimed at identifying theputative CNS components responsible for triggering the auto-immune responses leading to demyelination, we recentlyreported that MOG, an exclusive and quantitatively minorcomponent of CNS myelin, could induce in Lewis rats arelapsing-remitting neurologic disease with extensive plaque-like demyelination (9). The experiments described in thispaper verify the encephalitogenic potential of rat MOG inLewis rats and demonstrate its ability to stimulate a vigorousB cell response. Moreover, by using synthetic peptidesencompassing five different sequences of the extracellulardomains of rat MOG (amino acid sequences 1-21, 35-55,67-87, 104-117 and 202-218) we confirm that one of thepredominant encephalitogenic epitopes of MOG for the Lewisrat is located in the Ig-like domain, between amino acidresidues 35 and 55 (9). Indeed, none of the other four peptidesrepresenting other extracellular regions of MOG were able toinduce neurological signs or histological lesions within theCNS. These results are consistent with those of Liningtonet al. (29) who showed that none of the T cell lines specific

14.4-i(a) (b)

MWS 1 1

Fig. 4. Autoradiograms of immunoblots of MOG peptides. MOG peptides (2.5 ug) were electrophoresed using Tricine-SDS-PAGE and probedwith (a) anti-MOG35-55 antibody, (b) anti-MOG67-87 antibody and (c) anti-MOG202-218 antibody. MWS, molecular weight standard; lane 1,M0G1-21; lane 2, MOG35-55; lane 3, MOG67-87; lane 4, MOG104-117; lane 5, MOG202-218. Each antibody obtained 8 weeks afterimmunizations was tested at 1/400 dilution.



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Antibody titers againstE3 MOG35.55D whole MOGB BSA

0.06W 10W

Eluate

6W 10W

Flow through

Fig. 5. Reactivity of affinity-purified MOG35-55 antibody to nativeMOG and MOG35-55 peptide. Serum samples (0.2 ml) taken 6 and10 weeks after sensitization were passed over a MOG35-55 affinitycolumn, the unbound protein was washed out with PBS (flow through)and bound Ig eluted with glycine-HCL buffer (eluate). Antibodyreactivity of each fraction was tested by ELISA as described in Fig.2. Bars represent the mean OD values (at 405 nm) of triplicatedeterminations.

kDa66.2-

45.0

31.0

21.5

1 -

14.4-

MWS 1

Fig. 6. Autoradiograms of immunoblots of human myelin. Myelin (2.5ng) was electrophoresed under non-reducing conditions and probedwith: 1, anti-MOG antibody (8-18C5); 2, anti-MOG35-55 antibody;3, anti-MOG202-218 antibody; 4, anti-MBP antibody; 5, anti-PLPantibody. Anti-peptide antibodies obtained 8 weeks afterimmunizations were tested at 1/400 dilution and the mAb were usedat 0.1 jig/ml.

for MOG peptides 1-20, 61-80, 98-117 and 198-218 wereencephalitogenic in Lewis rats, but a T cell line directed toMOG peptide 35-55 was able to transfer histological EAEwhich was clinically silent (29). However, clinical signs anddemyelination could be produced in these clinically silentanimals, when 8-18C5, the MOG-specific mAb, was injectedi.v. following cell transfer of the MOG35-55 T cell line (29).

The strong encephalitogenic potential of the 35-55sequence of MOG is further emphasized by its ability toinduce a relapsing-remitting neurological disease in some

strains of mice (32,33). The finding that MOG35-55 caninduce immunopathogenic responses in different species aswell as in animals of the same species having a differentMHC is at variance with what is observed with MBP and PLPwhere animals with different MHC haplotypes respond todistinct epitopes of MBP and PLP (2,4,5). Investigations aretherefore currently underway in our laboratory to ascertain ifthe susceptibility of various animals species to develop theMOG-induced MS-like disease is controlled by gene(s)associated with or outside the MHC.

In a recent study, Potter et al. (34) reported that when micewere immunized with the encephalitogenic PLP peptide 178-191, the anti-PLP peptide antibodies in the serum of thesemice were cross-reactive with three other PLP peptides (97-110, 209-217 and 215-228) and that none of the anti-PLPpeptide antibodies reacted with native PLP. Our findings areat variance with those obtained with PLP peptides, in thatnone of the antibodies raised against a specific peptide,including MOG peptide 35-55, were cross-reactive with anyof the other MOG peptides. Some of these anti-MOG peptideantisera were also able to react with the native MOG. Asremoval of MOG35-55 antibodies by immunoabsorptioneliminated the reactivity to native MOG, it is likely that thereactivity was restricted to this peptide, although we cannotcompletely rule out the possibility of other B cell epitopeswithin the MOG molecule. Given the strong antibody responsedirected to some of the MOG peptides, it is somewhatsurprising that no antibodies to other myelin antigens couldbe detected. This is particularly so in animals injected withMOG35-55 as these rats had extensive plaque-like demyelin-ation. Whether or not antibodies to other myelin antigens areproduced, but remain localized in the brain, needs furtherinvestigation. The reason for the difference between thefindings obtained with PLP peptides and the current resultswith MOG peptides is unclear but may be related to theirimmunogenic properties and/or their respective amount inmyelin. While PLP is the major structural protein of myelin,representing some 50% of total protein, MOG represents only-0.05% of total protein (3,21,35). However, despite thesequantitative differences, MOG must have a high immunogenicpotential since immunization with CNS tissue homogenategenerates high levels of anti-MOG antibodies (11). On theother hand, one would expect that following demyelinationthe level of MOG available for antigen presentation would berelatively low. Accordingly, the number and types of antigen-presenting cells effectively presenting MOG may be highlyrestricted, a situation which is unlikely to occur in the case ofthe quantitatively more abundant PLP molecule.

MOG is a protein of 218 amino acids which is predicted tohave two transmembrane domains and an Ig-like extracellulardomain (36-38). It is also thought that residues 200-218form an extracellular 'tail' (36). In the present investigation,antibodies were produced against three peptides derivedfrom the Ig-like domain, i.e. M0G1-21, MOG35-55 andMOG67-87, and to the MOG peptide 202-218, correspondingto the extracellular tail. All antisera reacted to whole MOGunder reducing conditions in Western blots; however, undernon-reducing conditions, only antisera to MOG35-55 andMOG202-218 reacted with whole MOG. The reason why someof our anti-MOG peptide antibodies are able to recognize the



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native MOG whilst others are not is uncertain, but the failureof some anti-peptide antibodies to recognize their counter-parts in the native protein is a well known observation.While the three-dimensional structure for MOG has not beenreported, a recent analysis performed by Linsley et al. (39)reveals a similar overall structural homology between theV-like Ig domain of MOG and that of the co-stimulatorymolecule B7. Using the B7 model, we found no obviousstructural reasons which could explain the difference inreactivity of the anti-MOG peptides to the native MOG.Peptides 1-21, 35-55 and 67-87 have similar charge andhydrophilicity, are found in similar locations within the Igdomain, containing sequences that form part of both the(3-sheet and loop regions, and all should have significantportions exposed to the surface of MOG. Therefore, and assuggested by the absorption study described in Fig. 5, thestronger binding of the anti-MOG35-55 antibody to the nativeMOG may simply be due to a maturing of the antibodyresponse resulting in higher affinity antibodies. In view of thefact that the MOG peptide 35-55 represents an immunodomin-ant epitope of MOG and is encephalitogenic, this is perhapsnot unexpected since an effective threshold of high-affinityantibodies bound to oligodendrocytes would be requiredto initiate the cascade of events leading to demyelination.However, as described elsewhere (40), it is unlikely that theability of a MOG peptide to produce antibodies capable ofrecognizing native MOG is the only prerequisite for develop-ment of neurological signs.

Given the strong antibody response seen following immuniz-ation with MOG202-218 peptide, the observation that theseantibodies are able to recognize native MOG, plus the pre-vious report that this overall sequence is able to generate apeptide-specific T cell response in Lewis rats (29), it is unclearwhy this peptide failed to induce clinical or histologicaldisease. However, several possibilities can be contemplated.In a study aimed at assessing the in vivo pathogenic potentialof a panel of anti-MOG mAb, Piddlesden et al. (17) reporteda striking variation in the ability of these antibodies to initiatedemyelination in Lewis rats with MBP-induced EAE. Thisdifference in demyelinating activity was not related to speci-ficity for a given antibody, since all were shown to be CNSmyelin specific, all recognized MOG epitopes on oligodendro-cytes and all stained oligodendrocytes in a similar manner(17). Thus, like some of these mAb, the anti-MOG202-218antibodies may not be pathogenic. Alternatively, this peptidemay not be accessible to the pathogenic humoral immuneresponse. Indeed, it has been presumed that the C-terminalregion of native MOG forms an extracellular 'tail' but, assuggested by Gardinier et al. (36), it is possible that thisregion is not exposed at the outer surface of the myelin sheathbut is in fact intracellular. If this is the case, it is unlikely thatthis sequence of MOG would act as a target for primaryantibody-mediated demyelination and thus explains theinability of MOG202-218 to induce disease.

Several papers have reported that the 8-18C5 anti-MOGmouse mAb, raised against rat cerebellar glycoproteins,possesses demyelinating activity both in vivo and in vitro (12-17). We clearly demonstrate here that 8-18C5 does not reactto any of the extracellular peptides of MOG tested in thisstudy but did react with the native MOG. Thus, although

8-18C5 mediates demyelination, the site of antibody bindingof 8-18C5 is either different from that of the anti-MOG35-55antibody or directed against a conformational determinantlocalized within this region. The possibility that antibodies toMOG35-55 are able to initiate demyelination in Lewis rats iscurrently under investigation in our laboratory, using anti-MOG35-55 antibodies and T cells sensitized to MBP (14).

In summary, the results of this study corroborates andextends our (9) and other reports (29) showing that MOG, aminor CNS myelin component, is capable of inducing immune-mediated demyelination and a highly restricted B cellresponse in Lewis rats. These findings implicate MOG as animportant primary target autoantigen for immune-mediateddemyelination in MS, a contention supported by our recentdemonstration of a predominant T cell response to MOG inthe peripheral blood of MS patients, the suggested presenceof anti-MOG antibody in the CSF of such patients (41) andthe well documented demyelinating activity of MOG reactiveantibody both in vivo and in vitro (12-17). Taken together,these results emphasize that a synergism between a T cellresponse and anti-MOG antibodies recognizing native MOGor some of its determinants, is critical for the development ofsevere demyelinating disease. Further investigations into theautoimmune-mediated demyelination pathways generated byMOG peptides may not only provide important insights intothe pathogenic mechanisms of MS but also have distinctimplications for the development of future specific therapeuticapproaches to MS.

Acknowledgements

We would like to thank Professor Atsushi Komiyama, Department ofPediatrics, Shinshu University School of Medicine, for his continuoussupport and interest, as well as Professor Avi Ben-Nun for providingus with some of the MOG peptides used in this study. We are gratefulto Sharon Harris for typing this manuscript. This work was supportedby the National Health and Medical Research Council (NH&MRC)and the Multiple Sclerosis Societies of Australia. T. G. J. is a recipientof an NH&MRC post-doctoral fellowship.

Abbreviations

CFACNSCREAE

EAEMBPMOGMSPLP

complete Freund's adjuvantcentral nervous systemchronic relapsing experimental autoimmuneencephalomyelitisexperimental autoimmune encephalomyelitismyelin basic proteinmyelin oligodendrocyte glycoproteinmultiple sclerosisproteolipid protein

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