Coronavirus Infection Induces H-2 Antigen

Expression on Oligodendrocytes and AstrocytesAKio SuzUMuRA, EHUD LAVI, SusAN R. WEISS,DONALD H. SILBERBEIRG

I*ction of the central nervous system by mouse hepatitis virus stain A59, a murineneurotopic coronavirus, induces dass I major histocompatibility complex antigens on

mouse oligodendrocytes and astrocytes, cells that do not normally express theseantigens on their surfaces. This induction, which occurs through soluble factorselaborated by infected glial cells, potentially allows immunocytes to intcract with theglial cells and may play a critical role in the pathogenesis of virus-induced, immune-mediated demyelination in the central nervous system.

N EURAL CELLS DO NOT NORMALLYexpress either class I or class IImajor histocompatibility complex

(MHC) antigens (1). However, dass I anti-gens on most ncural cells (2, 3) and class IIantigens on some astrocytes (2, 4) haverecently been induced by factors secretedfrom activated T cells, or by y-interferon(INF--y). MHC antigens are key elements inthe pathogenesis of viral infection, restrict-ing immunologic resognition of viral anti-gens (5) or functioning as viral receptors(6). Although an autoimmune mechanismhas been implicated for the pathogenesis ofsome viral' infections in the central nervoussystem (CNS) (7), little is known about therelation between viral infection and hostimmune responses to host antigens in theCNS. We have found that CNS infection bymouse hepatitis virus strain A59 (MHV-

A59), a murine neurotropic coronavirus,induces H-2 antigen expression on oligo-dendrocytes and astrocytes in vivo and invitro.The induction of MHC antigen expres:

sion on neural cells by immune factors (2-4)led us to investigate whether viral infectioncould induce MHC antigen expression on

neural cells before their contact with activat-ed T cells or their secreted products. As a

nodel, we selected the infection ofC57BIJ6 mice with MIJV-A59, wipch pro-duces chronic CNS demyelination a,ccompa-nied by lymphocytic cellular infiltration (8);the mechanism of MHV-A59-induced de-myelination is still unknown.We examined the effect of direct viral

infection on the induction ofMHC expres-sion in oligodendrocytes and astrocytes invitro. Oligodendrocytes were isolated from

Fig. 1. In vitro induction ofH-2 antigen expression on oligodendrocytes and astrocytes by supematantfom mixed brain cell cultures infected with MHV-A59. The expression ofMHC antigens was assessedby indirect immunofluorescence ofunfixed viable cells. Monoclonal antibodies against mouse H-2 wereobtained from Bionetics Laboratory (Charleston, SC) (3). Cultures were then double-labeled withantibodies to H-2 and antibodies to GaIC or GFAP (3, 9). Oligodendrocytes (A-C) and astrytes (D-F) stimulated with 10% Sup for 2 days. Astrocyte cultures stimulated with supematant from uninfectedmixed brain cell cultures did not express detectable H-2 antigen (G-I). Viewed with phase-contrast (A,D, and G), fluorescein [GaIC (B), GFAP (E and H)], and rhodamine [H-2Db and H-2Kb (C, F, and I)]optics. Bar, 15 ,um.

23 MAY I986

primary dissociated mixed brain cell culturesofnewborn C57BL/6 mice at 10 days ofagcas described (9). Astrocyte-enriched cultureswere prepared from the same primary cul-tures after oligodendrocytes and otherloosely attached cells in the upper layer hadbeen removed by mechanical agitation (9,10). More than 80% ofthe cells in oligoden-drocyte-enriched cultures reacted wi anti-body to galactocerebroside (anti-GaIC), andmore than 90% in astrocyte-enriched cul-tures reacted with antibody to glial fibrillaryacidic protein (anti-GFAP) 3 days after iso-lation, as assessed by indirect immunofluo-rescence. Contamination ofcultures by mac-rophage-microglia was less than 1%, as de-termined by phagocytosis of latex partides.Fewer than 1% of the cells present at thetime of infection of oligodendrocyte-en-riched cultures were GFAP-positive astro-cytes (9). After 1 day of isolation, cells wereinfected with MHV-A59. Briefly, MHV-A59 (11), prepared as a cell lysate of 17done-i (Cl-i) mouse fibroblasts,' was ap-plied to either oligodendrocyte or astrocytecultures at a multiplicity of infection (MOI)of approximately one plaque-forming unt(PFU) per cell. We have confirmed, by'means of indirect imnmunofluorescence andtitration of infectious virus, that oligoden-drocytes and astrocytes were productivelyinfeed with MHV-A59 in vitro (12). ThisMHV-A59 infection did not induce detect-able MHC antigen (either H-2 or Ia)expression on oligodendrocytes as measumdby indirect immunofluorescence with a sin-gle monoclonal antibody to H-2Db and H-2Kb or antibody to i-A" (3, 4), whenexamined 1, 3, 5, 7, 14, and 21 days aftertinoculation. Astrocytes, however, expressed,H-2 antigen, but not Ia antigen, after 3days. Infecion at different multiplicities ofinfection of MHV-A59 (0.1 or 5.0) gavesimilar results.We sought to determine whether MHV-

A59 infection could induce MHC expres-'sion on oligodendrocytes through solublefactors elaborated' by infected CNS cells.Primary dissociated mixed brain cell cultureswere prepared from newborn C57BI/6mice and grown in 75an2 culture flask(Falcon, two brains per flask) for 10 daThen cultures were infected with 0.5 ml ofstock MHV-A59 at 1 x 107 PFU per flask.Supematant media from infected cultures,(Sup) was collected at days 1, 3, and 5 afterinoculation and twice a week thereafr.,Supernatant media from parallel uninfcultures and cultures inoculated with unin-

A. Suzumura and D. H. SilbaberF, Department ofNurology, University of Pennsylvama School ofMd--.ane, Phlahdel a, PA 19104.E.Lavi and S.R. Weiss, Deparat of Microbiology,Univcrsity ofPennsylvania Scol ofMedicine,Phila&phia, PA 19104.

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Fig. 2. H-2 antigen expression on glial cells from infected live mice. C57BL/6 mice at 5 to 7 days ofage(Charles River) were inoculated intracerebrally with 20 p,l ofstock virus at 400 PFU per inoculum (8).Two days after inoculation, brains were dissected out, minced, and digested with trypsin (Gibco, 2.5mg/mI) and deoxyribonuclease I (Sigma, 10 ,ug/ml) for 30 minutes in a shaking water bath at 37°C. Thecell suspensions were plated on poly-L-lysine-coated cover slips at a density of 5 x 104 per squarecentimeter. Oligodendrocytes were isolated from the enzymatically dissociated cell suspension byPercoll density gradient (19) and plated in the same manner. At 1 or 3 days in vitro, they were examinedfor MHC expression by indirect immunofluorescence (3). (A to C) GalC-positive oligodendrocytes inisolated oligodendrocyte cultures; (D to F) GFAP-positive astrytes in dissociated brain cell cultures.(A and D) Phase-contrast optics; (B) GaIC and (E) GFAP, fluorescein optics; and (C and F) H-2,rhodamine optics.

fected 17Cl-1 cell lysate were collected in thesame manner and served as controls. Oligo-dendrocyte- and astrocyte-enriched cultureswere treated with 1% to 50% (in finalconcentration) Sup for 2 days starting 1 dayafter isolation, and examined by indirectimmunofluorescence for their MHC (H-2and Ia) antigen expression at day 3 (3). TheSup induced H-2 antigen expression of thecorresponding haplotype on oligodendro-cytes and astrocytes (Fig. 1), but did notinduce detectable Ia antigen expression onthese cells. Monoclonal antibodies againstnoncorresponding H-2 haplotypes, normalmouse serum, or supematant of SP2/0 (3)did not stain these cells. Thus, the H-2expression on these cells is unrelated to theinduction of Fc receptors that may occur incertain viral infections (13). Although fluo-rescence after treatment with antibodies toH-2 on oligodendrocytes was weaker thanthat on astrocytes, more than 80% of oligo-dendrocytes and almost all astocytes ex-pressed detectable H-2 antigen. The mini-mum requirement for stimulation of H-2induction was 1% Sup for 2 days or 5% Supfor 1 day. Sup obtained 1, 3, 5, and 17 daysafter inoculation induced H-2 antigenexpression on glial cells with no obviousdifference noted from each other.

Since direct viral inoculation in vitro didnot induce MHC expression on oligoden-drocytes, this induction of H-2 expressionby Sup was probably mediated by factorsfrom MHV-A59-infected cells in the pri-mary cultures ofmixed brain cells and not byinfectious virus. To study this, we appliedSup after inactivation of virus. The ultravio-let (UV) light irradiation ofMHV-A59 forcither 3 hours or overnight inactivated in-

fectious virus but did not alter the ability ofSup to induce H-2 activity. We also exam-ined UV-inactivated MHV-A59 to see if itcould induce primary cultures of mixedbrain cells to elaborate the H-2-inducingfactors. The supematant media from cul-tures inoculated with inactivated MHV-A59had no stimulatory effect on MHC induc-tion. Thus, the H-2-inducing factors in theSup were soluble factors from MHV-A59-infected cells in primary mixed brain cellcultures, but not MHV-A59 itself.To identify the cells responsible for the

production of the H-2-inducing factors, weinfcted different glial cell cultures and as-sayed thenm, with UV-irradiated supema-tant, for their ability to induceMHC expres-sion on oligodendrocytes and astrocytes.Astrocyte-enriched cultures and mixed glialcell cultures from which oligodendrocytes orfibroblasts were deleted (14) produced H-2-inducing factors. However, supernatantfrom oligodendrocytes, meningeal fibro-blasts, or spleen cell cultures of the samestrain ofmouse, infected with MHV-A59 inthe same manner, did not induce MHCantigen expression on glial cells. These ob-servations indicated that astrocytes are morelikely to play a predominant role in theproduction of the factors, although wecould not rule out the possible participationof macrophage-microglia in the productionof the H-2-inducing factors.

It is possible that MHV-A59-infectedbrain cells produce IFN's that may stimulateinduction of H-2 expression, although IFNproduction by MHV-infected brain cells isreportedly low (15). The addition of anti-bodies to mouse interferon [anti-IFN-oa/1(16)] in various concentrations (10 to 106

neutralization units per milliliter) into cul-tures with 10% Sup did not abolish theability of Sup to induce H-2. Thus, the H-2-inducing factors in Sup were probablyunrelated to IFN-a/13. Since our brain cellcultures do not usually contain lymphocytes,and since Sup from spleen lymphocytes in-oculated with MHV-A59 did not induceMHC antigens on glial cells, it is also unlike-ly that the factors are related to IFN--y.However, the precise characterization ofthefactors awaits fiurther elucidation.To confirm whether the H-2 induction by

MHV-A59 infection occurs in vivo, weinoculated mice intracerebrally with MHV-A59 (8) and examined brain cell cultures forMHC expression. MHV-A59 inducedexpression ofH-2 antigen on GalC-positiveoligodendrocytes and GFAP-positive astro-cytes (Fig. 2). We did not detect H-2 anti-gen expression on oligodendrocytes and as-trocytes prepared from uninfected mice orfrom the mice inoculated with uninfectedcell lysate of 17 Cl-i. These H-2-positivecells were still observed at 28 days in vitrowithout any additional stimulation, but oli-godendrocytes and astrocytes did not ex-press detectable amounts of Ia antigenthroughout the culture period.Our results, combined with the results of

previous studies on H-2 induction by thefactors from activated T cells (2, 3), indicateat least two ways for neural cells to expressH-2 antigen in certain pathological condi-tions-immune-mediated and virus-mediat-ed mechanisms. Schrier et al. (17) haveshown that expression ofMHC dass I anti-gen in rat kidney cells is switched off whenthe cells are transformed by a highly onco-genic adenovirus. More recently, however,Rosenthal et al. (18) have shown that infec-tion of mouse embryo cells with eitheroncogenic or nononcogenic adenovirus re-sults in transcriptional activation of the H-2K gene. Despite the large increase in cyto-plasmic H-2K messenger RNA, they couldfind only a marginal increase in surface H-2K antigen by radioimmunoassay. Howev-er, as we have shown, expression of detect-able surface H-2 antigen on oligodendro-cytes and astrocytes is induced during neu-rrpic MHV-A59 incton. This H-2 induc-tion may play a critical role in the interactionbetween virus-infected cells and immuno-cytes to intiate virus-induced, immune-me-diated demyelination in the CNS, a possibleanimal model of multiple sderosis.

REFERENCES AND NOTES1. J. Schnitzer and M. Schachner,J. Neurommund. 1,

429 (1981); U. Tihaugott, E. L. Reinherz, C. S.Rainc, Scienc 219, 308 (1983); S. L. Hauser et al.,J. Neuroimumnol. 5, 197 (1983); RP P. Lisak at al.,Brain Res. 289, 285 (1083); L. A. Lampson, C. A.Fisher, J. P. Whelan,J. Immuno. 130,2471 (1983).

2. L. A. Lampson and C. A. Fisher, Proc. Natd. Acad.

SCIENCE, VOL. 232992

SO. USA. 81,6476(-(1981); G NH. W. WO,P. F.Bardt, L. Chark-Lewis, F. Ba J. W. Scrader,Natr (ZLdm) 310, 688 (1984).

3. A. Stiuma anid D. H. S i l- Rss. 336,171 (1985).

4. M. RL Hrs, J. Weieblin, M. Pierres, C. Gois,Nusa. Lat. 41, 199 (1983); A. 1ontaa, W.Fieaz, -I. Wekerle, Nature (Ld) 307, 273(1984); A Suzumua, D. H. Silbe R. P.IJ.Nk :, i press.

5. . Zimk,= I and P. C. Dobrt, Nauw(t ) 2"; M (1974)- R.- M. dieragr andM. B.A. Oldsone, Ncadt.Ad Sci. U.SA. ,733666 (1976); S. Japobson at at.,J. Ixmwmu. 133,754 (1984).

6A. eH*iiU a.., Prew. Nad. Aad. Si U.SA. 76,3846 (1978); T. 1alad and C. A Minis, Na(nd..) 30, 59 (1984).

7. J. 0. Fkming and L. P. Weiner, inImne efNcWs Sy,Wm Infees, P. O. Bhlam, V. ter Mcu-

iCf, F. Clifford 1ow, Eds. (Elsvier m,1983), pp. 91-96.

8. E. Lavi, D. H. Gikden, Z. Wrobkwak, L. B. Ro0k,S. R Weia,N 34,597 (194); E. Lavi, D.H. Gil, M,. S. R. W VW.Y S1563 ( 4.

9. A. S. Bhat, P. A.Ei, L P. isakD. H. S i Brest Ra. 324, 37 (1984).

10. K. D. McCarthy and J. de Veilus,J. B 85890(1980).

11. Ihe stock biW-g59wa obtained frzJ. L Iawitz U. A. RobbTC t 'BadY LI4bowitz *,Vien~ 94385 (1979)].

12. E. ai S in p ton.13. A A. R M Treschner, K K. Sbi, H.

Brands,J. I 117, 253 (19);-J, CostA,AS. Rabson, C. Yee, T. T N26, 251 (1977).

14. J. E MerriD S aI., 224,1428(1984).15. A. R. Cols ad., Isfea. Im1_. 40, 1192(1983).

16. P was ptbyD. M,Medical llge of Kornol,

and Di. M Mur U. I . 130,2}i198 )

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18. 'A. R a, -ibi. 315, 579 (1985).D9. B. JD. H R. P. Li4k

20.' 9; Hs NS1037 And NS2195put & I -fro the National Muk

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30 Sepber 1986; accepted 24 January 1986

Dispersal of a

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.MEREDIm BLACKWELL, J. ROBERTBEiuRIES, JOHN C MOSE.,THELMA J. PERRY

It bh e -w s_7: "Udmoiae withahrods :4:ve phyIln rFbtodhpsws th*e LalmibeiacHowv .direct dcirelo'imcth oftetaiso fwtaeaf an ascosoe- olide,uwive obeea fe wiedfordimr by mis

assmlaerel riaiyonathtol;oby

PRVIOUSLY UNKNOWN MERHODof fiul ( s )ervelopoenthwit antreme special-

ization for arthropod dispersal has beediscovered. The new iformaton providestie only eidnceofthe affini-ty of any of the inu c, nto-mogenous funi f repor iy in thiscentury (1).The p cil ame 1 p

had been lknwn dfiomtin amdf asubstrates (2, 3). We report h an undc-scribed s p eeIss Sscie pecies that iasoiedwith thesouthem pine beete symbioicLunqvs (2) fii ;-notce s ;:;:laibeween ascospores of .a a epreumed hhomyrces, Th i spceand Acanniola speces fromm in bbeetle habitas in Poa and i2). Howcver, conidia were nt prodedInthe specimens, and tey wme rImerely as ascospores (2).The anamorph is charactezed by a no-

mycelial taus consistig oftwo cells andadarkened hoklfast in linear ar t(1).Altffoug T batrida was first reported in1914 (1), itsdevelopmenths notobsrved until now. We foud that theascospores of Pyridio differentiate intoaThaiola state while still within the ascUsand ascocarp (Fig. lA).Mo gl -ifferentiation of the cospore be withearly development of a seum to dividc dtc

spore into two unequal cel,followed bygradua k)s of ew

darkened holdfist (Fig. l,AahdB)atthend of the distal ceias it s pswidtin the asp; and attenu.io£fdIprximal ccll tip into a spine (i IC) atadditional septum may be fore in idisl cell (4). The as p are r-Mklased to the eri al oio oitfurst,i a gn usmass.-M hav

been obsved crawing over te peiccks, where the a

ad by the holdfast (FigP 10); enitconidium formation ocacrs later in the t-minal --ll (Fig. 0C). PMunibly, cxdoc

ifXnidiag:byageuwevntoprdsthe tclomorphic (seual) thal(.Thus the ariystery of the y

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teleotnorph could not have boenpfor Thaxipeida becaue this-mane Of- ammorph~produ-ctio by direci: developmnt~.~

the ascspobas otbeenobser ed"b*Previo'Usly decrfibedacmcr.nmrareinge-celed(yeats) or mycelial In MI

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