Mechanism of interferon therapy of Multiple Sclerosis : studies in an animal model

88 Pages·1999·2.9 MB·English

by  Mujtaba, Mustafa Ghulam

MECHANISMOFINTERFERONTHERAPYOFMULTIPLESCLEROSIS:
STUDIESINANANIMALMODEL
By
MUSTAFAGHULAMMUJTABA
ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOLOFTHE
UNIVERSITYOFFLORIDAINPARTIALFULFILLMENTOFTHE
REQUIREMENTSFORTHEDEGREEOF
DOCTOROFPHILOSOPHY
UNIVERSITYOFFLORIDA
1999
ACKNOWLEDGMENTS
Iwouldliketothankmymentor,Dr.HowardM.Johnson,fortakingme
intohislaboratoryandguidingmethroughmystudies. Hehasalwaysbeen
knowledgeableandlogical,whichmakehimagreatmentor. Imustalsoexpress
mythankstomycommitteemembers-Dr.EdwardHoffman,Dr.JakeStreit,Dr.
JanetYamamoto,andDr.JulieMaupin-fortheirtime,patience,andeffort. Many
thankstomyfellowgraduatestudentsandlabmatesbothpastandpresent,
includingBarbara.Jeanne,Brian,Prem,Taishi,Martez,Pedro,Amy,George,
Joe,Kendra,Scott,Karrie,Wiggins,andTim. Theymakethelabmore
"interesting"andfun,atleastatcertaintimes.
Finally,Ithankmyfamilyfortheirsupportthroughmygraduateschool
studies. Myparents,GhulamandUzraMujtaba,havealwaysbeenthereforme. I
thankmybrotherandsistersandtheirfamiliesfortheirhelpandencouragement.
ManythanksgotomyAfghancousinswhomarealwaysagreatbreakfrommy
graduatestudies. Ioweagreatdebtofgratitudetomywonderfulfamilyand
friends.
ii
TABLEOFCONTENTS
ACKNOWLEDGMENTS
ii
LISTOFTABLES v
LISTOFFIGURES vi
ABSTRACTvii
CHAPTERS
1 INTRODUCTION 1
Overview 1
MultipleSclerosis 2
ExperimentalAllergicEncephalomyelitis 5
Interferons 8
2 IFNtINDUCESSTABLEREMISSIONINCHRONICEAE 11
Introduction 11
MaterialsandMethods 12
IFNt 12
InductionofEAE 12
AdministrationofIFNt 13
HistologicalEvaluation 13
ProliferationAssayandIsolationofTCellsandBCells 14
ELISAforMBP-SpecificAntibodies 15
Results 15
IFNtBlocksFurtherRelapsesintoParalysisofEAE-
AfflictedMice ". 15
RedEuActEioMnicaendbPyrIevFeNnttionofLymphocyticInfiltratesin 17
DeactivationofMicrogliaFollowingIFNtTreatment 17
IFNtTreatedMiceHaveaLowerAntibodyLevelAgainst
MBPthanControlMice 22
IFNtInhibitedMBP-SpecificBCellProliferation 22
IFNtInhibitedMBP-SpecificTCellProliferation 29
Discussion 34
3 CD4TSUPPRESSORCELLSMEDIATEIFNtPROTECTION
AGAINSTEAE 35
iii
Introduction 35
MaterialsandMethods 36
IFNs 36
AntibodiesandCytokines 36
InterferonInductionofSuppressorCells 37
InductionofEAECells 37
CD4TCellIsolationandDepletion 38
ProductionofSuppressorFactor 38
ProliferationAssay 38
Results 39
IFNc--TreatedSpleenCellsInhibitMBP-SpecificTCell
Proliferation 39
IFNtInductionofSuppressorCellsisDose-Dependent 39
EFN-cSuppressorCellsProtectMiceAgainstEAE 44
IFNc-InducedSuppressorCellsareCD4Tcells 44
SuppressorCellsProduceSolubleSuppressorFactor(s) 49
IFNt-InducedSuppressorCellsProduceIL-10andTGFp 49
IL-10andTGFpActSynergisticallytoInhibitMBP-
SpecificTCellResponses 54
Discussion 54
4 CONCLUSION 58
REFERENCES 66
BIOGRAPHICALSKETCH 78
iv
LISTOFTABLES
Table Page
1. TreatmentofSJLmicewithIFNxafterinductionofEAE 16
2. IFNinhibitionofMBP-sensitizedspleencells 65
v
1
LISTOFFIGURES
Figures Page
1. HiswtoiltohgiPcBalSeavanlduaItFiNotnofEAEmicebeforeandaftertreatment 19
2. IFNxcausesdeactivationofmicroglia 21
3. InhiobfiEtiAonEobfyanItFiN-tMBPantibodyproductioninthechronicform 24
4. Inhibitionofanti-MBPantibodyproductionintheacuteformof
EAEbyIFNt 26
5. IFNEtAiEnhmibiictesMBP-specificproliferationofBcellsfromchronic 28
6. IFNEtAiEnhmibiictesMBP-specificproliferationofBcellsfromacute 31
7. IFNtinhibitsMBP-specificTcellproliferation 33
8. IFNc-treatedspleencellsinhibitMBP-specificTcellproliferation....4
9. IFNtinductionofsuppressorcellsisdose-dependent 43
10. IFNt-inducedsuppressorcellscandelaytheonsetofEAEinmice...46
11. IFNt-inducedsuppressorcellsareCD4Tcells 48
12. IFNt-inducedCD4Tsuppressorcellsproducesolublesuppressor
factors(s) 51
13. BloecfkfeacgtesoofnIMFBNtP-isntdiumculeadtisounpporfessesnosritciezieldaEndAEsumpporuessesosrplseuepnercnealltsant
withmonoclonalantibodiestoIL-10andTGFp 53
14. IL-10andTGFpactsynergisticallytoinhibitMBP-specificTcell
responses 56
15. oIfFNntaiivnedsupclteieonncoefllCsD4suppressorTcellsinhibitSEAstimulation 61
16. IFNitn-htirbeiattSeEdBCDst4imTulcaetlilosnporfodnuaicveessupplpereenssceolrlsfactor(s)which 63
vi
AbstractofDissertationPresentedtotheGraduateSchoolofthe
UniversityofFloridainPartialFulfillmentoftheRequirementsfor
theDegreeofDoctorofPhilosophy
MECHANISMOFINTERFERONTHERAPYOFMULTIPLESCLEROSIS:
STUDIESINANANIMALMODEL
By
MustafaGhulamMujtaba
August1999
Chairperson: HowardM.Johnson
MajorDepartment: MicrobiologyandCellScience
Interferon(JFN)tauisatypeIIFNthatwasoriginallyidentifiedasa
pregnancyrecognitionhormoneproducedbytrophoblastcells. Itisaspotentan
antiviralagentasIFNccandIFNp,butlacksthetoxicityassociatedwithhigh
concentrationsoftheseIFNsintissuecultureandinanimalstudies. Previouslyit
hasbeenshownthatinterferonxpretreatmentinhibitsthedevelopmentofbothacute
andchronicmouseexperimentalallergicencephalomyelitis(EAE),ananimalmodel
forthehumandemyelinatingdiseasemultiplesclerosis(MS). Here,weshowthat
IFNtinducedremissioninSJL/JmicethathadongoingchronicactiveEAE
disease,andprotectedmiceagainstsecondaryrelapses.IFNttreatmentreversed
lymphocyteinfiltrationandmicroglialactivationinthecentralnervoussystem.
MicethatweretreatedwithIFNthadlowerlevelsofanti-MBP(myelinbasic
protein)antibodiesthanuntreatedmiceinbothchronicandacuteformsofEAE.
vii
MBPinducedproliferationinBcellsfromEAEmice,buttreatmentwithIFNt
eitherinvivoorinvitroblockedthisactivation. Furthermore,IFNtinhibitedMBP
activationofTcellsfromEAEmice. Thus,IFNtinhibitsthehumoralarmaswell
asthecellulararmoftheautoimmunediseaseEAE. IFNtpreventsEAEinmiceby
inductionofsuppressorcellsandsuppressorfactors. Suppressorcellscanbe
inducedbyIFNtintissuecultureandinvivobyeitherintraperitonealinjectionor
byoraladministrationtomice. IncubationofsuppressorcellswithMBP-sensitized
TcellsblockedordelayedtheMBP-inducedproliferation. Further,intraperitoneal
injectionofsuppressorcellsintomiceblockedinductionofEAEbyMBP.
SuppressorcellspossessedtheCD4Tcellphenotype,andproducedsoluble
suppressorfactorsthatinhibitedMBPactivationofTcellsfromEAEmice. The
suppressorfactorswerefoundtobeIL-10andTGFp,whichactedsynergistically
toinhibittheMBPactivationofTcellsfromEAEmice.Thesefindingsare
importantforunderstandingthemechanism(s)bywhichtypeIIFNsprotectagainst
autoimmunedisease.
viii
CHAPTER1
INTRODUCTION
Overview
Interferons(IFNs)areagroupofglycoproteinsthatplayimportantmodulatory
rolesinthevertebrateimmunesystem. Originallydiscoveredin1957fortheirabilityto
interferewithviralreplicationincells(IsaacandLindenmann,1957),theyarenowknown
toalsohaveantiproliferativeeffectsonavarietyofcelltypes. Forthisreason,IFNshave
beenusedforthetreatmentofautoimmunediseases,viralinfections,andseveraltypesof
cancers(Guttermanetal,1994).Interferon-tau(IFNx)isatypeIIFNthatwasoriginally
identifiedasapregnancyrecognitionhormoneproducedbytrophoblastcellsinsheep. Itis
aspotentanantiviralagentasIFNocandIFNp,butlacksthetoxicityassociatedwithhigh
concentrationsoftheseIFNsintissuecultureandinvivo(Bazeretal,1989;Pontzeretal,
1991;Soosetal,1995a;Soosetal,1995b).
MultipleSclerosis(MS)isoneofthemostcommondiseaseofthecentralnervous
system(CNS). InMS,thelossofmyelinisaccompaniedbyadisruptionintheabilityof
thenervestoconductelectricalimpulsestoandfromthebrain,andthisproducesthe
variousdebilitatingsymptomsofMS.Experimentalallergicencephalomyelitis(EAE)isa
murinemodelusefulforstudyingthedemyelinatingdiseaseMS. Myelinbasicprotein
(MBP)hasbeenshowntobeoneoftheprimaryCNSantigensresponsibleforinductionof
autoimmunityintheEAEmodel(Zamviletal,1990). ImmunizationofmicewithMBP
resultsintailandlimbparalysisduetolymphocyticinfiltrationanddemyelinationinthe
CNS(Zamviletal,1990). MBP-specificantibodiesandautoreactiveMBP-specificTcells
arealsothoughttocontributetotheexacerbationofEAEandMS(Sun,1993;Warrenet
1
al,1993;Gerritseetal,1994;Saoudietal,1995;Wangetal,1995). Various
inflammatorycytokinesliketumornecrosisfactor-alpha(TNFa)andIFNycontributetothe
breakdownofmyelinonnervecellsintheCNS. Furthermore,microglia,theresident
macrophageoftheCNS,arethoughttoplayakeyroleinrecruitinglymphocytesand
functionasantigen-presentingcellinMSandEAE(Benveniste,1997;Streitetal,1995;
Hickeyetal,1988).
Currently,thetypeIIFN,IFNp,istheonlyFDAapprovedcytokinetreatmentof
MS. Previously,ithasbeenshownthatadministrationofIFNttomiceatthetimeof
immunizationwithMBPblockedthedevelopmentofEAEinmicewithoutassociated
toxicity;howeverthemechanismofsuchactionhasnotbeenfullyelucidated(Soosetal,
1995a). Thus,thepurposeofthisstudyistodeterminethemechanismbywhichIFNt
suppressesautoimmuneresponsesinEAE.
MultipleSclerosis
MultipleSclerosis(MS)isachronic,demyelinating,inflammatorydiseaseofthe
CNS. MScommonlyaffectsyoungadultsandmostlywomen(Ebersetal.1986). MSis
commonlyfoundinCanada,theUnitedStates,SouthAmerica,andEurope;nearthe
equatorMSisunknown(Ebersetal,1986). Inthisdiseasethereisaninflammationof
myelin,whichisfattyinsulationorcoveringofnervecellextensions,knownasaxons
(Lassmann,1998). Messagesaresentalongaxonstoothernervecellsinakindof
electricalsignals. Myelininsulatestheaxonstohelpgettheseelectricalimpulsesthrough.
Itstopscurrentsfromflowingbetweentheindividualaxons. Italsohelpstospeedthe
conductionoftheelectricalsignal. Thisenablespeopletomovealmostwithoutthinking.
WhenmyelinisaffectedinMS,impulsestravelovertheaxonsveryslowly,ifatall,and
thereissomeelectricalinterferencebetweenaxons. Messagesarenotsentefficiendyand
canfailtogetthroughsothatapersonlosestheabilitytomakesmooth,rapid,and