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Induction of antimyelin and antioligodendrocyte antibodies by vaccinia virus An experimental study in the mouse
JouraalqfNelrohr,m~ologl', I (19811 H7-124 ElsevieU North-Holland Biomedical ?r~
Induction
117
of axLtimyelin and antioligodendrocyte antibodies by vaccinia virus
An experimental
study in the iaouse
A.J. Steck, R. T s c h a n n e n and R. ~ h a e f e r Department of NeuJ~O~y, University o/ LauJenne, Latoo~e (Switzerland) (Recifived4 Deccmbea, lOgO) ~Accepled 20 JImui-y, 1981)
Summary The appearance of semm antibodies binding to specific brain antigens was monitored ill mice inoculated intracerebrally with a dermotropic or a nelirotroi,ic strain of va.zcinia virus. Antibodies were measured with a bimtin~ assay ushkg [~llprotein A. Inoculation with the neurotropic strain ca,: sed an induction of sel'a~ antibodies binding to the myedin membrane, the myelin basic protein and olii~> dendrocytes while no induction of binding antibodies to neurons was ~ e d . The dermotropic strain failed ~o elicit the formation of binding antibodies to brnin antigens.
Imre>lon Vaccinia virus belongs to a group of viruses associated with pottinfections ¢ncepi~lomyalitis, a conaition that shows similarities with that of experimental allerlic encephalomyelitis ( H a r ~ and Choppin 1971: Wether et al. 1973), .Both direct viral effects and immunologtcal factors have been discussed as muses of postinfections em:~halomyelitis (Johnson 1980). In the present study we have investi;~ated the appearance of serum antibodies binding to Sl:ecific brain antigms following intracraninl inoculation of mice with 2 strains of vaccinia virus which bay: diffege:tt tropisms and produce contrasting clinical pictures, The v i r t ~ used tu~ a neurotlopic strain of vaccinia virus (strain WR) which is charac:e~.zd by • kigh mortali,'y after intracranial or i n t r ~ infection with concomitant rapid multipli•~ k w u su~.pof~edby IVaet3.~.ff#B tram the S w ~ Na~amd F ~
and ~ • ~
t~
lllililmled in pirl at ~ 3?lid Amimii Meitilill of Ill: Anil'ican A.cldemyol NeululllD,. ~ ml0.
~
:
AJ. Sli~, Depianlm of NeluoloILV0CHUV, l o l l / .
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118
cation of virus in brain and a non-lethal dermoiropic strain of vaccinia virus (strain Elstree) which does not replicate in nervous tissue (Turner 1967). Using a 2-step binding assay with [~2Sl]prutein A we describe the induction of serum antibodies binding 1o the myelin membrane and the oligodendrocytes in mice inoculated with the neurotropic strain of vaccinia virus. The absence of a similar response towards neurons or thymocytes and the ineffectiveness of the dermotropic strain to induce serum antibodi¢.; binding to brain components are discussed in terms of viral tropism and preferential interaction of th,~ neuroiropic strain with the oligo. dendroglia myelin compartment. Materials and Methods Viru.s propagation, purification and inoculation The dermotropic vaccinia virus (strain Elstree) and the nearotropic vaccinia virus (strum WK) were propagated on mouse L-fibroblasts (Tschannen et al. 1979) and purified as described by Dales and Mosbach (1968). Plaque assays were performed according to Ihe method of Valle (1971). Mature mice of the strain NMRI were inoculaled io.ttacerebrally by means of a Hamilton syringe ~ t h a free needle tip of 3 mm for a r,.*producil~ledepth of inoculation. Intracerebral inocul-"tion with the neurotropic strain results in rapid replication of virus within brain (Tschannen et at. 1979) and anir:lals receiving l04 pfu (plaque-forming units) or more died after 3-6 days, while animals receiving 102 pfu or less surviw:~ but appeared ill. On the other hand iuoculation of l0 s pfu of the dermotropic strain did not cause any sign of disease in the mice. In the present study animals received 102 pfu of the neurotropic or the dermotropic strain intracranially and sera drawn on the 4th or the 14th post-inoculation day were kept at - 2 0 ° C anti heated at 56°C for 30 rain before use. [!2~1 ]Protein A microassoy Pr,~cedures for the 2-step binding assay wi~h [12Sl]protein A have been previously descr!bed (Stock and Pcrruisscau 1980). Briefly the cells were incubated in wells of polystyrene microtiter plates with 50 p.l of the: scra to be tested at different dilutions for 45 rain at room temperature followed by 2 washes with PBS containing 2.5~ fetal calf serum. Presence of specific lgG binding to the cell surface was then detected by adding [Z2Sl]prutein A and in,.'ubating for 45 rain at 37°C. After was~aing, the wells were cleaned with cotton tips and radioactivity counted in a Packard Gamma spectrometer. All determirlations were done in duplicate and blanks obtained with cells incubated with medium alone were always subtracted from the experimental value. Antibodies to myelin and myelin basic protein were measured in a solid phase binding test ,vith [J2SI]prot©in A (At.celia and C©lada 197g). Briefly the antigen was incubated in wells of polyvinyl microtiter plates at a concentration of 0.1 mg protein/ml m PBS, 100 ~1 per well. After 2 h the solution was aspirated and 200 p.I of PBS with 1% BSA was added. After I h this solution was removed ar,d 100/d of the sera to be tested was added and incubated for 2 h at room temperature followed by ~- wasaes with PBS. [l:~l]protein A wa-~then added and incubated, for 45 rain at 37°C. After 3 washes with PBS and 7 washes with distilled water the plate was dried, the wells cut out and the pellet counted as described above.
119
Cell isolation and culture Oilgodendrocytes we~ isolated from calf brain according to the procedure of Poduslo et al,(1978) with some modifications(Steck and Pen'uisseau 1980). For the isolation of neurons from rat brains we followed the procedure of Farooq and Norton (Farooq and Norton 1978) except that sucrose was used instead of Ficoil. Sucrose gradients consisted of 15 ral 0.9 M sucrose, 5 ml 1.35 M sucrose. 5 ml 1.55 M sucrose and 5 ml 2.0 M sucro.~e. The tube~; were centrifuged 15 rain at 3500 rpm and neurons were collected front the 1.55 M-2.0 M sucrose interphase. Ofigodendrocytes and neurons were maintained for 24-48 h in ti~ue culture medium (Dulbeoco's modified Eagle's medium) before u~ed in the [r2Sl]I:rotein A microassay. Strain G 26-24 which is a line isolatot from a mouse oligodendrogiloma and mouse neuroblastoma strain NB 2A were grown as monolayer culture in Dnlheeco's modified Eagle's medium containing I0% fetal calf serum. Thymecytes were isolated from fresh mouse thymus. Some immunological characteristics of these cells have been recently published (Sleek and Perruisseau 1980). Myelin isolation Myelin was prepared from bovine brain according to the method of Norton (1971). Myelin basic protein was isolated from purified bovine myelin following the procedure of Golds and Braun (1978). Myelin basic protein extracted by this method was essentially homogeneous with minor amounts of lower molecular weight peptides which were likely degradation products of myelin basic protein zq observed by SDS polyacrylamide gel electrophoresis.
Figure IA illustrates the presence of myelin binding antibodies in sera obtained 14 days after intracraniai inoculation of mice with the neurotropic strain. Binding values averaged 7.500 cpm at a I : 10 dilution, which represents a 15-fold increase over the values for sera obtained 4 days after inoculation with the neurotropi¢ strain or for sera obtained from mice inoculated with the dermotropic strain, With this 14th day post-inoculation neurotropic serum binding values were stiff significantly higher, at a dilution of 1:640, than for the other sera, giving a liter of I : 10240. The binding of the 14th day neuroti'opic sera to myelin could be most completely removed by absorption with myelin, while absorption with virus caused only a slight decrease of binding (Table I). To analyze further the spocificity of the immup.e response toward.q myelin induc.~l by the nenrotropio strain, sera were ~nalyzed for 'he presence of mitibodies against the myelin basic protein'(Fig, IB). 8¢ra obtained 14 days after inoculation with the neurotropic strain had an average tiler of 1:1280 and binding values at I:10 dilution averaged 6000 opm, which rcpresent~ a 10-fold increase over the values for sets from mice inoculatad with the dermotropic strain. The slight increase in binding at higher dilutions observed with sela that have no significant letel of bindiug antibodies reflects non-.q~ocific bindhag to the myelin basic protein, a strongly positively chetSed molocul¢. This non-specific interactioncould I~ a b o ~ by
120
.f .s. 20
Ill
l/wm Va~o I / ~ ~,~o 1/4w J ~
dt~t~n
l/w
Fi~. I. ~'rum ant/b~ii~:s to ~pecific brain tnt{$c'ns mc~ured with (t~[]protcin A in vtcciaJa inocula|cd mice. O - - - O 4~h post-incculat/on day, g - - - - O 14tb post-inoculalioa day ncurotwpic strain; D ~ 4th post-inoculation dqy, • "' |I 14th pc~l-inocuiation day dermotropic s~r~dr,. 4: Myelin binding antibodies; B: Myelin basic protein binding antibodies. Mice were ivo~ulated intraocrebrally with Ill 2 plKlUes forming units vaccinia ~irus WR or FAstre¢.Sera f;'om 3-4 animals wcsc tested ;n each group. Results o~laJned at a ~o dilut/on ]tad a .qm~dard dcvial:on of no $n.'ater thsm I.~ for e#c'h group. Binding curvc:, shown arc from a typical cxpchment. diluting the sera in albumin-contalnie`g buffer instead o f PBS (data not shown). T o address the qucstiun of w h e t h e r this i m m u n e response is restricted to the myelin m e m b r a n e or wheu%er it is directed towards o t h e r b r a i n structt.res as well, several cell :ypes were tested in the [tZ~l|proteln A b i n d i n g assay (Fig. 2). W h e n oligndendrocyles isolated from c a l f b r a i n were used as target cells (Fig. 2A), a significant Slier of binding antibodies was ~,j:~in found only in sera o b t a i n e d on the 14th post-inoculation de,;' with the neurotropic strain. Binding values with this sera averaged 17,000 c p m at. 1 : 2 0 dilution, which represen(s a n 8-fold increase TABLE ] EFFECT OF ABSO~.PT[ON ON THE BINDING OF 14th DAY NEUROTROPIC SF.RA TO MYELII-,I 0.8 ml of serum diluted I : 10 in PBS was abmrbad with either 6 × 10~ pfu vaccinia WR o r 5 mg lyophil/scd mye!~n for I h at 370C and overnight at 4oC. After cestriful~tio~ for 45 rain st 20000×&, supernal;mrs were recovered and used in the binding atsty with ['2Sl~rotein A, Treatlncm oi" anti, rum
[ J~l]Prolcin A binding (cpm)
Inhibit/on (~)
Non absol?k'd Virus (WRi Myelin
5~03 5478 807
6 86
121
A
1"
/
s e~
~ o 1/~o 1pro
Vm 1Ao l?m
Filg.2. Serum antibodies Io isolated cellsmeasured with [n~l]pmtein A in vaccinia-inoct~ted mio:. 0 0 4th post-inoculation day, @ - - - - I t 14th post-inoculation day neurolropic strain; D ~ 4th pmt-inocula~o~ day. m - - - m 14th post-inoculation day dermotroplc strain. A: Bovine olia~lendracy~ (0.2× 10s cells/well); tr: Rat neurons (0.2X 104 cells) well~; C: Mot~ t 11 cytes (0.2XI0 cells/we .). Sera from 3-4 animals were lested in each [o'o~. Results 0bu6ned ~tt • i dihttieu had • standard deviation of no ~.ater d~m ~0% for each group. Binding ¢Ltrvessho~n are from •
over the otber sera. In contrasi when n c ~ o n s isolated from rat b l a i n were used (Fi s. 2B), n o difference in binding was observed between the sera o f a~imeds infected with the nenrotropic o r the dermotropic str~dns, Binding values averesed 87~00 c p m at a 1 : 20 dilation J.nd about 4000 c p m at a 1 : 80 dilution, Net~ro~s were much more difficult to manipulate than oligodendrocyle$ in the b i n d i n 8 assay and 8ave high oindin s values with different non-immtme ~ r a . This rcpre~,mts probably n o n specific binding and it could result from the fact thnt neurons are larger, have p ~ and thus are much more heteroseneous than ofigodendrocyles. I n an additional experiment (Table ~;) ~ r a obtained on th~ 14th post-inuculation TAIDLE2 B I N D I N G O F 14th D A Y N E U R O T R O P l C
$FJ:A T O D[FFF.REN~' C E L L LINES
In this e01oedme~l,cells (0,75 X 106 ol/sodendn-,cyte~or thymocytes and 0.45 × 106 G26-24 c~ NB2.A well) ~ incubated with 2~,000 cpm [12sl~p~1>telnP_ Otherwise assay coeditlons were M dek-~bed in Matet~ls and ]¢kqbod~. Each number re~n~cntt the avera~ of 3-4 determinatio~ wilh • staadmd deviation 1of all measm'em~ls of n0t IXeate~thno 20f3. Ceil
[~]]Protein A bindinS (cpm × l0 -3) at sensm d ~ S o n 1:20
Ol~$ode,~umcy~ f~ "~.6.24 NB2h Th~,n~e,/ta
113.9 15.8 B.7 2.9
1:40 71.8 10.1 6.4 t.t,
122
day with the neurotrnpic strain ~,ere tested simultaneously against bovine oligodendrocytes, 2 murine clonal cell lines, a neuroblastoma cell line NB 2A and a glioblaston~a cell line G 26-24 a~ well as mouse thymocytes. A significant binding was obtained only with the oligodendro~.ytes. The glioblastoma cell line and the neuroblastoma line gave only weak binding vaiu¢,~, and binding values to the thymocyles were in the background range. In addition, thymocytes were tested against the different sera obtained from inoculated mice (Fig. 2C). No difference in binding was observed be~wcen animals inoculated with the dermotropic or the neurutropic strain. Binding values averag'~l 1400 cpm at a 1:20 dilution and 100 at a I : 80 dilution.
Discussion Whefeas Ihe neurotropic vaccinia virus (strain WR) can replicate in the CNS,the dermotropic vaccinia virus (strain Elstree) replicates in dermal tissue, not in neuronal tissue. We have shown previously that highly purified vaccinia virus of strain WR, which was grown for several passag~ in mouse brain integrated in its structure myelin basic protein from the CNS and had a different polypeptide composition when compared to the same strain which v,as grown ill mouse L-fibroblasts (Tschannen et aL 1979). These findings prompted us to investigate the immune response to specific brain antigens after intracranial inoculation of mice with the neurotropic or the dermotropic strain of vaccinia virus. In this report we present evidence for the ability of the neurntropic strain to elicit an immunological reaction that appears directed essentially towards the myelinoligodandrocyte compartment. The neurotropic strain is known not to infect neurons and replicates primarily in mesenchymal cell or glial cells though the exact cell type is not known (Kristensson et al. 1970). Preliminary work suggests that the neurotropic strain preferentiat:y infects ol~godcndroglia in vitro (R. Schaefer, unpublished results). Sensitization could thus result from replication of virus within oligodendroglia wltL subsequent release o[ hidden immunogens such as the myelin basic protein. Other mechanisms could however be also involved. Infection wi!h the neurotroplc strain induces an increase in vascular permeabilit,, permitting p~oteins to penetrate into the brain parenchyma, though the dermotropic strain is also ~:nown to induce a slight brain edema (K.ristensson and Sourander 1969). From these data it is not possible to decide whether sensibilizafion to myelin and ofigodendrocytes is solely a direct effect of virus multiplication within these cells or whether it involves also deterioration of the blood-braln barrier. Another possibility to be considered is thaf there may exist a crns~ reactivity between viral proteins and polypeptides from the myelin membrane. Such a cross reactivity has been suggested for oligoclanal antibodies to measles and the myelin basic protein (Panitch et aL 1980). This does not seem to be the case here since the myeh~n binding activity is not removed by absorption with vaccinia virus. The absence of an induction of serum antibodies with binding activity to neurons by the neurotropic strain could be explained by the fact that neurons are not infected and therefore no immanogen is released. Alternatively neurons may not carry such strong immunogens as gaiactocerehroside, which is known to be pre,~nt in purified oligodendrocytes (Raft et aL 1978) and in mydin, or the myelin b ~ i c
123
p;otein, a major component of the myefin membrane (Carnegie and Duakley 1975). Thy-I. a cell-surface glycoprotein that is mainly displayed by thymic lymphocytes and neurons, could be a potential antigen (Morris et aL 1980). In this respect it is interesting to point that alloantigens or tumor-specific antigens may differ fundamentally from tlssue-gpecific antigens capable of inducing autoimmu!rtity as to the nature of the immunological response they evade. "Die C N S is immunologically privileged due to its lack of lymphative drainage. Antigens present solely there as is the case for the myelin basic protein are beyond the reach o f the" recognizing mechanisms of the immunologic~l system. When introduced to the org,mism outside the C N ~ as is the case in the stadias of E A E or when presentation to the i ~ m u n e system occurs in the course o f a viral infec~.ion as is probably the cau" in this study with the inoculation of a neurotropic strain o f vaceinia virus, sens~tzation ~o the myelin bazic protein takes place. Hopefully a more precise anderstan,|ing o f virusinduced a u t o i m m u n i t y to the myelin membrane or the oligodeodrocyt ~ will pr~Jvide some insight in the pathogrnesis of demyelinating diseases.
Acknowledgement The authors thank Mrs. A.-M. Vo0oz for secretarial help. Re~ren¢¢s Acco!la, R.S. and F. Celada, Immune,response agaJnsLthe ~-galactosides¢ enzyme of E, coli at pr~utsor cell level, Europ. J. Immunnl., 8 0978) 688-692. Carnegie, P.R. and P~. Dunklcy. Basic proteins of central and per/pheral nervo~ system myelin. In: B.W. Agranoff a~
124 Sleck. AJ. and C. Perruir,xeam,Characterization ol nl~mbrane r0,~'kez~of i.soluted o]/Eodcndr~leS and c]or~l lines of th.- nervous system. J. Nvurol. Sci., 47 (1980) 135-144.
T~channen, R,, A.J. Steck and R. Schaefer. Mechanisms in the patbo~enes/s of post.mfectiotts vaccinia vin~s enc~ha]omyc~ in the mov.~, N~'uros~. r~tt., 15 (1979) 29~-~0. Tum'.r.G.S., Respiratory ip~octlon of mice w~th vaccinia virus, J. ~ Virol,, I {1967) 399-402. Valle. ,~l., Faclors affecting plaque array of animal viruses. Acza Pa~. Micr~olol. Stand.. Suppl. 219. Sec' • (1971) "]-69. W~,incr. L.P, R.T. Johnson and R.M. He,orlon, Viral ~nfeclion~ and demyelinating disca.~, N, ,Vngl,J, Mcd., 2~8 (1973) 1103-1110.
Induction
117
of axLtimyelin and antioligodendrocyte antibodies by vaccinia virus
An experimental
study in the iaouse
A.J. Steck, R. T s c h a n n e n and R. ~ h a e f e r Department of NeuJ~O~y, University o/ LauJenne, Latoo~e (Switzerland) (Recifived4 Deccmbea, lOgO) ~Accepled 20 JImui-y, 1981)
Summary The appearance of semm antibodies binding to specific brain antigens was monitored ill mice inoculated intracerebrally with a dermotropic or a nelirotroi,ic strain of va.zcinia virus. Antibodies were measured with a bimtin~ assay ushkg [~llprotein A. Inoculation with the neurotropic strain ca,: sed an induction of sel'a~ antibodies binding to the myedin membrane, the myelin basic protein and olii~> dendrocytes while no induction of binding antibodies to neurons was ~ e d . The dermotropic strain failed ~o elicit the formation of binding antibodies to brnin antigens.
Imre>lon Vaccinia virus belongs to a group of viruses associated with pottinfections ¢ncepi~lomyalitis, a conaition that shows similarities with that of experimental allerlic encephalomyelitis ( H a r ~ and Choppin 1971: Wether et al. 1973), .Both direct viral effects and immunologtcal factors have been discussed as muses of postinfections em:~halomyelitis (Johnson 1980). In the present study we have investi;~ated the appearance of serum antibodies binding to Sl:ecific brain antigms following intracraninl inoculation of mice with 2 strains of vaccinia virus which bay: diffege:tt tropisms and produce contrasting clinical pictures, The v i r t ~ used tu~ a neurotlopic strain of vaccinia virus (strain WR) which is charac:e~.zd by • kigh mortali,'y after intracranial or i n t r ~ infection with concomitant rapid multipli•~ k w u su~.pof~edby IVaet3.~.ff#B tram the S w ~ Na~amd F ~
and ~ • ~
t~
lllililmled in pirl at ~ 3?lid Amimii Meitilill of Ill: Anil'ican A.cldemyol NeululllD,. ~ ml0.
~
:
AJ. Sli~, Depianlm of NeluoloILV0CHUV, l o l l / .
016.1-~7111/11/11110-0000/II)l.~© I~lllvili/Noilh-Hollliid B k ~ d ] ,'-t
~'t4~'loDd.
e~
118
cation of virus in brain and a non-lethal dermoiropic strain of vaccinia virus (strain Elstree) which does not replicate in nervous tissue (Turner 1967). Using a 2-step binding assay with [~2Sl]prutein A we describe the induction of serum antibodies binding 1o the myelin membrane and the oligodendrocytes in mice inoculated with the neurotropic strain of vaccinia virus. The absence of a similar response towards neurons or thymocytes and the ineffectiveness of the dermotropic strain to induce serum antibodi¢.; binding to brain components are discussed in terms of viral tropism and preferential interaction of th,~ neuroiropic strain with the oligo. dendroglia myelin compartment. Materials and Methods Viru.s propagation, purification and inoculation The dermotropic vaccinia virus (strain Elstree) and the nearotropic vaccinia virus (strum WK) were propagated on mouse L-fibroblasts (Tschannen et al. 1979) and purified as described by Dales and Mosbach (1968). Plaque assays were performed according to Ihe method of Valle (1971). Mature mice of the strain NMRI were inoculaled io.ttacerebrally by means of a Hamilton syringe ~ t h a free needle tip of 3 mm for a r,.*producil~ledepth of inoculation. Intracerebral inocul-"tion with the neurotropic strain results in rapid replication of virus within brain (Tschannen et at. 1979) and anir:lals receiving l04 pfu (plaque-forming units) or more died after 3-6 days, while animals receiving 102 pfu or less surviw:~ but appeared ill. On the other hand iuoculation of l0 s pfu of the dermotropic strain did not cause any sign of disease in the mice. In the present study animals received 102 pfu of the neurotropic or the dermotropic strain intracranially and sera drawn on the 4th or the 14th post-inoculation day were kept at - 2 0 ° C anti heated at 56°C for 30 rain before use. [!2~1 ]Protein A microassoy Pr,~cedures for the 2-step binding assay wi~h [12Sl]protein A have been previously descr!bed (Stock and Pcrruisscau 1980). Briefly the cells were incubated in wells of polystyrene microtiter plates with 50 p.l of the: scra to be tested at different dilutions for 45 rain at room temperature followed by 2 washes with PBS containing 2.5~ fetal calf serum. Presence of specific lgG binding to the cell surface was then detected by adding [Z2Sl]prutein A and in,.'ubating for 45 rain at 37°C. After was~aing, the wells were cleaned with cotton tips and radioactivity counted in a Packard Gamma spectrometer. All determirlations were done in duplicate and blanks obtained with cells incubated with medium alone were always subtracted from the experimental value. Antibodies to myelin and myelin basic protein were measured in a solid phase binding test ,vith [J2SI]prot©in A (At.celia and C©lada 197g). Briefly the antigen was incubated in wells of polyvinyl microtiter plates at a concentration of 0.1 mg protein/ml m PBS, 100 ~1 per well. After 2 h the solution was aspirated and 200 p.I of PBS with 1% BSA was added. After I h this solution was removed ar,d 100/d of the sera to be tested was added and incubated for 2 h at room temperature followed by ~- wasaes with PBS. [l:~l]protein A wa-~then added and incubated, for 45 rain at 37°C. After 3 washes with PBS and 7 washes with distilled water the plate was dried, the wells cut out and the pellet counted as described above.
119
Cell isolation and culture Oilgodendrocytes we~ isolated from calf brain according to the procedure of Poduslo et al,(1978) with some modifications(Steck and Pen'uisseau 1980). For the isolation of neurons from rat brains we followed the procedure of Farooq and Norton (Farooq and Norton 1978) except that sucrose was used instead of Ficoil. Sucrose gradients consisted of 15 ral 0.9 M sucrose, 5 ml 1.35 M sucrose. 5 ml 1.55 M sucrose and 5 ml 2.0 M sucro.~e. The tube~; were centrifuged 15 rain at 3500 rpm and neurons were collected front the 1.55 M-2.0 M sucrose interphase. Ofigodendrocytes and neurons were maintained for 24-48 h in ti~ue culture medium (Dulbeoco's modified Eagle's medium) before u~ed in the [r2Sl]I:rotein A microassay. Strain G 26-24 which is a line isolatot from a mouse oligodendrogiloma and mouse neuroblastoma strain NB 2A were grown as monolayer culture in Dnlheeco's modified Eagle's medium containing I0% fetal calf serum. Thymecytes were isolated from fresh mouse thymus. Some immunological characteristics of these cells have been recently published (Sleek and Perruisseau 1980). Myelin isolation Myelin was prepared from bovine brain according to the method of Norton (1971). Myelin basic protein was isolated from purified bovine myelin following the procedure of Golds and Braun (1978). Myelin basic protein extracted by this method was essentially homogeneous with minor amounts of lower molecular weight peptides which were likely degradation products of myelin basic protein zq observed by SDS polyacrylamide gel electrophoresis.
Figure IA illustrates the presence of myelin binding antibodies in sera obtained 14 days after intracraniai inoculation of mice with the neurotropic strain. Binding values averaged 7.500 cpm at a I : 10 dilution, which represents a 15-fold increase over the values for sera obtained 4 days after inoculation with the neurotropi¢ strain or for sera obtained from mice inoculated with the dermotropic strain, With this 14th day post-inoculation neurotropic serum binding values were stiff significantly higher, at a dilution of 1:640, than for the other sera, giving a liter of I : 10240. The binding of the 14th day neuroti'opic sera to myelin could be most completely removed by absorption with myelin, while absorption with virus caused only a slight decrease of binding (Table I). To analyze further the spocificity of the immup.e response toward.q myelin induc.~l by the nenrotropio strain, sera were ~nalyzed for 'he presence of mitibodies against the myelin basic protein'(Fig, IB). 8¢ra obtained 14 days after inoculation with the neurotropic strain had an average tiler of 1:1280 and binding values at I:10 dilution averaged 6000 opm, which rcpresent~ a 10-fold increase over the values for sets from mice inoculatad with the dermotropic strain. The slight increase in binding at higher dilutions observed with sela that have no significant letel of bindiug antibodies reflects non-.q~ocific bindhag to the myelin basic protein, a strongly positively chetSed molocul¢. This non-specific interactioncould I~ a b o ~ by
120
.f .s. 20
Ill
l/wm Va~o I / ~ ~,~o 1/4w J ~
dt~t~n
l/w
Fi~. I. ~'rum ant/b~ii~:s to ~pecific brain tnt{$c'ns mc~ured with (t~[]protcin A in vtcciaJa inocula|cd mice. O - - - O 4~h post-incculat/on day, g - - - - O 14tb post-inoculalioa day ncurotwpic strain; D ~ 4th post-inoculation dqy, • "' |I 14th pc~l-inocuiation day dermotropic s~r~dr,. 4: Myelin binding antibodies; B: Myelin basic protein binding antibodies. Mice were ivo~ulated intraocrebrally with Ill 2 plKlUes forming units vaccinia ~irus WR or FAstre¢.Sera f;'om 3-4 animals wcsc tested ;n each group. Results o~laJned at a ~o dilut/on ]tad a .qm~dard dcvial:on of no $n.'ater thsm I.~ for e#c'h group. Binding curvc:, shown arc from a typical cxpchment. diluting the sera in albumin-contalnie`g buffer instead o f PBS (data not shown). T o address the qucstiun of w h e t h e r this i m m u n e response is restricted to the myelin m e m b r a n e or wheu%er it is directed towards o t h e r b r a i n structt.res as well, several cell :ypes were tested in the [tZ~l|proteln A b i n d i n g assay (Fig. 2). W h e n oligndendrocyles isolated from c a l f b r a i n were used as target cells (Fig. 2A), a significant Slier of binding antibodies was ~,j:~in found only in sera o b t a i n e d on the 14th post-inoculation de,;' with the neurotropic strain. Binding values with this sera averaged 17,000 c p m at. 1 : 2 0 dilution, which represen(s a n 8-fold increase TABLE ] EFFECT OF ABSO~.PT[ON ON THE BINDING OF 14th DAY NEUROTROPIC SF.RA TO MYELII-,I 0.8 ml of serum diluted I : 10 in PBS was abmrbad with either 6 × 10~ pfu vaccinia WR o r 5 mg lyophil/scd mye!~n for I h at 370C and overnight at 4oC. After cestriful~tio~ for 45 rain st 20000×&, supernal;mrs were recovered and used in the binding atsty with ['2Sl~rotein A, Treatlncm oi" anti, rum
[ J~l]Prolcin A binding (cpm)
Inhibit/on (~)
Non absol?k'd Virus (WRi Myelin
5~03 5478 807
6 86
121
A
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Filg.2. Serum antibodies Io isolated cellsmeasured with [n~l]pmtein A in vaccinia-inoct~ted mio:. 0 0 4th post-inoculation day, @ - - - - I t 14th post-inoculation day neurolropic strain; D ~ 4th pmt-inocula~o~ day. m - - - m 14th post-inoculation day dermotroplc strain. A: Bovine olia~lendracy~ (0.2× 10s cells/well); tr: Rat neurons (0.2X 104 cells) well~; C: Mot~ t 11 cytes (0.2XI0 cells/we .). Sera from 3-4 animals were lested in each [o'o~. Results 0bu6ned ~tt • i dihttieu had • standard deviation of no ~.ater d~m ~0% for each group. Binding ¢Ltrvessho~n are from •
over the otber sera. In contrasi when n c ~ o n s isolated from rat b l a i n were used (Fi s. 2B), n o difference in binding was observed between the sera o f a~imeds infected with the nenrotropic o r the dermotropic str~dns, Binding values averesed 87~00 c p m at a 1 : 20 dilation J.nd about 4000 c p m at a 1 : 80 dilution, Net~ro~s were much more difficult to manipulate than oligodendrocyle$ in the b i n d i n 8 assay and 8ave high oindin s values with different non-immtme ~ r a . This rcpre~,mts probably n o n specific binding and it could result from the fact thnt neurons are larger, have p ~ and thus are much more heteroseneous than ofigodendrocyles. I n an additional experiment (Table ~;) ~ r a obtained on th~ 14th post-inuculation TAIDLE2 B I N D I N G O F 14th D A Y N E U R O T R O P l C
$FJ:A T O D[FFF.REN~' C E L L LINES
In this e01oedme~l,cells (0,75 X 106 ol/sodendn-,cyte~or thymocytes and 0.45 × 106 G26-24 c~ NB2.A well) ~ incubated with 2~,000 cpm [12sl~p~1>telnP_ Otherwise assay coeditlons were M dek-~bed in Matet~ls and ]¢kqbod~. Each number re~n~cntt the avera~ of 3-4 determinatio~ wilh • staadmd deviation 1of all measm'em~ls of n0t IXeate~thno 20f3. Ceil
[~]]Protein A bindinS (cpm × l0 -3) at sensm d ~ S o n 1:20
Ol~$ode,~umcy~ f~ "~.6.24 NB2h Th~,n~e,/ta
113.9 15.8 B.7 2.9
1:40 71.8 10.1 6.4 t.t,
122
day with the neurotrnpic strain ~,ere tested simultaneously against bovine oligodendrocytes, 2 murine clonal cell lines, a neuroblastoma cell line NB 2A and a glioblaston~a cell line G 26-24 a~ well as mouse thymocytes. A significant binding was obtained only with the oligodendro~.ytes. The glioblastoma cell line and the neuroblastoma line gave only weak binding vaiu¢,~, and binding values to the thymocyles were in the background range. In addition, thymocytes were tested against the different sera obtained from inoculated mice (Fig. 2C). No difference in binding was observed be~wcen animals inoculated with the dermotropic or the neurutropic strain. Binding values averag'~l 1400 cpm at a 1:20 dilution and 100 at a I : 80 dilution.
Discussion Whefeas Ihe neurotropic vaccinia virus (strain WR) can replicate in the CNS,the dermotropic vaccinia virus (strain Elstree) replicates in dermal tissue, not in neuronal tissue. We have shown previously that highly purified vaccinia virus of strain WR, which was grown for several passag~ in mouse brain integrated in its structure myelin basic protein from the CNS and had a different polypeptide composition when compared to the same strain which v,as grown ill mouse L-fibroblasts (Tschannen et aL 1979). These findings prompted us to investigate the immune response to specific brain antigens after intracranial inoculation of mice with the neurotropic or the dermotropic strain of vaccinia virus. In this report we present evidence for the ability of the neurntropic strain to elicit an immunological reaction that appears directed essentially towards the myelinoligodandrocyte compartment. The neurotropic strain is known not to infect neurons and replicates primarily in mesenchymal cell or glial cells though the exact cell type is not known (Kristensson et al. 1970). Preliminary work suggests that the neurotropic strain preferentiat:y infects ol~godcndroglia in vitro (R. Schaefer, unpublished results). Sensitization could thus result from replication of virus within oligodendroglia wltL subsequent release o[ hidden immunogens such as the myelin basic protein. Other mechanisms could however be also involved. Infection wi!h the neurotroplc strain induces an increase in vascular permeabilit,, permitting p~oteins to penetrate into the brain parenchyma, though the dermotropic strain is also ~:nown to induce a slight brain edema (K.ristensson and Sourander 1969). From these data it is not possible to decide whether sensibilizafion to myelin and ofigodendrocytes is solely a direct effect of virus multiplication within these cells or whether it involves also deterioration of the blood-braln barrier. Another possibility to be considered is thaf there may exist a crns~ reactivity between viral proteins and polypeptides from the myelin membrane. Such a cross reactivity has been suggested for oligoclanal antibodies to measles and the myelin basic protein (Panitch et aL 1980). This does not seem to be the case here since the myeh~n binding activity is not removed by absorption with vaccinia virus. The absence of an induction of serum antibodies with binding activity to neurons by the neurotropic strain could be explained by the fact that neurons are not infected and therefore no immanogen is released. Alternatively neurons may not carry such strong immunogens as gaiactocerehroside, which is known to be pre,~nt in purified oligodendrocytes (Raft et aL 1978) and in mydin, or the myelin b ~ i c
123
p;otein, a major component of the myefin membrane (Carnegie and Duakley 1975). Thy-I. a cell-surface glycoprotein that is mainly displayed by thymic lymphocytes and neurons, could be a potential antigen (Morris et aL 1980). In this respect it is interesting to point that alloantigens or tumor-specific antigens may differ fundamentally from tlssue-gpecific antigens capable of inducing autoimmu!rtity as to the nature of the immunological response they evade. "Die C N S is immunologically privileged due to its lack of lymphative drainage. Antigens present solely there as is the case for the myelin basic protein are beyond the reach o f the" recognizing mechanisms of the immunologic~l system. When introduced to the org,mism outside the C N ~ as is the case in the stadias of E A E or when presentation to the i ~ m u n e system occurs in the course o f a viral infec~.ion as is probably the cau" in this study with the inoculation of a neurotropic strain o f vaceinia virus, sens~tzation ~o the myelin bazic protein takes place. Hopefully a more precise anderstan,|ing o f virusinduced a u t o i m m u n i t y to the myelin membrane or the oligodeodrocyt ~ will pr~Jvide some insight in the pathogrnesis of demyelinating diseases.
Acknowledgement The authors thank Mrs. A.-M. Vo0oz for secretarial help. Re~ren¢¢s Acco!la, R.S. and F. Celada, Immune,response agaJnsLthe ~-galactosides¢ enzyme of E, coli at pr~utsor cell level, Europ. J. Immunnl., 8 0978) 688-692. Carnegie, P.R. and P~. Dunklcy. Basic proteins of central and per/pheral nervo~ system myelin. In: B.W. Agranoff a~
124 Sleck. AJ. and C. Perruir,xeam,Characterization ol nl~mbrane r0,~'kez~of i.soluted o]/Eodcndr~leS and c]or~l lines of th.- nervous system. J. Nvurol. Sci., 47 (1980) 135-144.
T~channen, R,, A.J. Steck and R. Schaefer. Mechanisms in the patbo~enes/s of post.mfectiotts vaccinia vin~s enc~ha]omyc~ in the mov.~, N~'uros~. r~tt., 15 (1979) 29~-~0. Tum'.r.G.S., Respiratory ip~octlon of mice w~th vaccinia virus, J. ~ Virol,, I {1967) 399-402. Valle. ,~l., Faclors affecting plaque array of animal viruses. Acza Pa~. Micr~olol. Stand.. Suppl. 219. Sec' • (1971) "]-69. W~,incr. L.P, R.T. Johnson and R.M. He,orlon, Viral ~nfeclion~ and demyelinating disca.~, N, ,Vngl,J, Mcd., 2~8 (1973) 1103-1110.