DTIC ADA534333: Two Distinct Mechanisms for Induction of Dendritic Cell Apoptosis in Response to Intact Streptococcus pneumoniae

13 Pages·0.21 MB·English

by  Defense Technical Information Center

TheJournalofImmunology
Two Distinct Mechanisms for Induction of Dendritic Cell
Apoptosis in Response to Intact Streptococcus pneumoniae1
JesusColino and Clifford M.Snapper2
Apoptoticdendriticcells(DCs)areineffectiveatinducingimmunity.Thus,parametersthatregulateDCviabilityduringaprimary
infection will help to determine the outcome of the subsequent immune response. In this regard, pathogens have developed
strategies to promote DC apoptosis to counterbalance the nascent primary immune response. We demonstrate, using cultured
bonemarrow-derivedDCs,thatStreptococcuspneumoniaecaninduceDCapoptosisthroughtwodistinctmechanisms:1)arapid,
caspase-independent mechanism of apoptosis induction, critically dependent on bacterial expression of pneumolysin, and 2) a
delayed-onset,caspase-dependentmechanismofapoptosisinductionassociatedwithterminalDCmaturation.Delayed-onsetap-
optosisdoesnotrequirebacterialinternalization,butratheristriggeredbytheinteractionofbacterialsubcapsularcomponents
andbonemarrow-derivedDC(likelyToll-like)receptorsactinginamyeloiddifferentiationfactor88-dependentmanner.Inthis
regard, heavy polysaccharide encapsulation interferes with both DC maturation and apoptosis induction. In contrast, neither
CD95/CD95ligandinteractionsnorTNF-(cid:1)appeartoplayaroleinthedelayedonsetofapoptosis.Thesedataarethefirsttodefine
two mechanistically distinct pathways of DC apoptosis induction in response to an extracellular bacterium that likely have
importantconsequencesfortheestablishmentofantibacterialimmunity. TheJournalofImmunology,2003,171:2354–2365.
P
rogrammed cell death or apoptosis plays a major regula- cobacteria,whichleadstoapoptosis(4),butthisiscorrelatedwith
toryroleinimmuneresponses(1).TNF-(cid:1)releasedearlyin a relative resistance to the experimental infection (14). Macro-
response to bacterial infections (2) and, specifically, sev- phageintracellularkillingofopsonizedStreptococcuspneumoniae
eral bacterial pathogens can directly trigger apoptosis in cells of hasbeenassociatedwiththeinductionofacaspase-dependentbut
themonocyte/macrophagelineage(3–10).However,themolecular Fas-independent apoptosis (11). However, S. pneumoniae pneu-
mechanismsofapoptosisinducedbybacteriaareincompletelyde- molysin, which induces NO production in macrophages (15), is
lineated,particularlyregardingtheinvolvementofdeathreceptors. ineffective at inducing macrophage apoptosis (11). In contrast,
Inthefewcasesinvestigated,theyhavebeenfoundtobeeithernot pneumolysininducesinneuronsandmicrogliaacaspase-indepen-
involvedortobeCD95(Fas)-mediated(3,11).Adirectinteraction dent apoptosis associated with the release of apoptosis-inducing
between bacterial components and cell surface receptors as a factorfrommitochondria(16),whichcontributestoenhancedmor-
trigger for macrophage apoptosis has been reported only for bidity (17). Thus, various stimuli acting on particular cell types
lipoproteins released by Shigella flexneri (12) and, more re- induce apoptosis through distinct pathways leading to different
cently, for the 19-kDa protein of Mycobacterium tuberculosis functionalorpathologicconsequences.
(13), which activate apoptosis through interaction with Toll- TheDCiswidelyregardedasacriticallinkbetweentheinnateand
like receptor-2 (TLR-2)3 (12). adaptiveimmuneresponse.ImmatureDCs,aswellasmacrophages,
Inductionofmacrophageapoptosisbyphagocytosedpathogens detect microbial infections by recognition, via TLRs, of pathogen-
is largely regarded as a microbial strategy to evade the immune associated molecular patterns (18, 19). Signaling through all TLR
response,butinsomeinstancesitcouldbeaneffectivehostmech- members is dependent upon a common adaptor molecule (20), the
anism for pathogen clearance, limiting intracellular replication. myeloiddifferentiationfactor88(MyD88),whichmediatesthepro-
Moreover, apoptotic bodies containing killed bacteria could be ductionofproinflammatorycytokinesand,inpart,thematurationof
taken up by dendritic cells (DCs) and used as a source of Ag to the DC. DC maturation is characterized by the loss of phagocytic
initiate a primary immune response (8). Thus, macrophages in- capacityandtheup-regulationofMHCandmembranecostimulatory
fected with M. tuberculosis exhibit NO-dependent killing of my- molecules.TheseDCresponsestopathogensarekeyelementsinthe
inductionofinnateimmunity,theeffectiveprimingofnaiveTcells,
andtheinitiationofadaptiveresponses(21,22).
Department of Pathology, Uniformed Services University of the Health Sciences,
Bethesda,MD20814 Despite the critical importance of the DC in the response to
primary infections, very little is known regarding the ability of
ReceivedforpublicationApril21,2003.AcceptedforpublicationJune25,2003.
bacteriatoinduceapoptosisinDCs.OnlyListeriamonocytogenes
Thecostsofpublicationofthisarticleweredefrayedinpartbythepaymentofpage
charges.Thisarticlemustthereforebeherebymarkedadvertisementinaccordance hasbeendemonstratedtoinduceapoptosisinDCsviatheactionof
with18U.S.C.Section1734solelytoindicatethisfact. hemolysin(23).DCs,however,appeartoexhibitmechanismsthat
1ThisworkwassupportedbyNationalInstitutesofHealthGrants1R01AI49192and counterbalance apoptotic stimuli that otherwise efficiently induce
1R01AI46551.
apoptosisinmacrophages.Thus,matureDCsarerelativelyresis-
2AddresscorrespondenceandreprintrequeststoDr.CliffordM.Snapper,Depart- tant to the proapoptotic action of TNF-(cid:1)(24, 25) and CD95-me-
mentofPathology,UniformedServicesUniversityoftheHealthSciences,4301Jones
BridgeRoad,Bethesda,MD20814.E-mailaddress:[email protected] diated apoptosis (26), and this resistance is associated with the
3Abbreviations used in this paper: TLR, Toll-like receptor; DC, dendritic cell; up-regulation of FLIP (26) and Bcl-xL (24). A relative resistance
MyD88,myeloiddifferentiationfactor88;BMDC,bonemarrow-deriveddendritic ofDCsforapoptosisinductioninresponsetobacteriawouldpo-
cell;PLN,pneumolysin-deficientmutant;PG,peptidoglycan;PI,propidiumiodide;
tentiallyplayanimportantroleinoptimizingtheirabilitytoactas
FMK,fluoromethylketone;CD95L,CD95ligand;CM-DiI,chloromethylbenzamido
derivativeofDiI;Ct,cytochalasin;MFI,meanfluorescenceintensity. thekeyAPCduringaprimaryimmuneresponse.Inthisreport,we
Copyright©2003byTheAmericanAssociationofImmunologists,Inc. 0022-1767/03/$02.00
Report Documentation Page Form Approved
OMB No. 0704-0188
Public reporting burden for the collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and
maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information,
including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington
VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to a penalty for failing to comply with a collection of information if it

does not display a currently valid OMB control number.
1. REPORT DATE 3. DATES COVERED
APR 2003 2. REPORT TYPE 00-00-2003 to 00-00-2003
4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
Two Distinct Mechanisms for Induction of Dendritic Cell Apoptosis in
5b. GRANT NUMBER
Response to Intact Streptococcus pneumoniae
5c. PROGRAM ELEMENT NUMBER
6. AUTHOR(S) 5d. PROJECT NUMBER
5e. TASK NUMBER
5f. WORK UNIT NUMBER
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION
Uniformed Services University of the Health Sciences,Department of REPORT NUMBER
Pathology,Bethesda,MD,20814
9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S)
11. SPONSOR/MONITOR’S REPORT
NUMBER(S)
12. DISTRIBUTION/AVAILABILITY STATEMENT
Approved for public release; distribution unlimited
13. SUPPLEMENTARY NOTES
14. ABSTRACT

15. SUBJECT TERMS
16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18. NUMBER 19a. NAME OF
ABSTRACT OF PAGES RESPONSIBLE PERSON
a. REPORT b. ABSTRACT c. THIS PAGE Same as 12
unclassified unclassified unclassified Report (SAR)
Standard Form 298 (Rev. 8-98)
Prescribed by ANSI Std Z39-18
TheJournalofImmunology 2355
wished to determine the susceptibility of DCs to apoptosis in re- pleted of T cells, B cells, and other MHC class II-expressing cells by
sponse to the Gram-positive extracellular bacterium S. pneu- incubationfor30minat4°CwithamAbcocktailcomposedofAF6-120.1
(mouseIgG2aanti-I-Ab),RA3-6B2(ratIgG2aanti-CD45R/B220),53-5.8
moniae.Usingbonemarrow-derivedDCs(BMDCs),weshowfor
(ratIgG1anti-CD8b.2),andRB6-8C5(ratIgG2bratanti-Ly-6G)andwere
the first time that bacteria can induce DC apoptosis through two
negatively selected by magnetic bead cell sorting using a mixture of af-
distinct mechanisms: 1) a rapid, caspase-independent mechanism finity-purifiedpolyclonalgoatanti-ratIgGandgoatanti-mouseIgGcon-
ofapoptosisinduction,criticallydependentonbacterialexpression jugatedbeads(BioSpheres;Biosource,Camarillo,CA).AllofthemAbs
ofpneumolysin,and2)adelayed-onset,caspase-dependentmech- werepurchasedfromBDPharMingen(SanDiego,CA).Thebonemarrow
cellsobtainedwereculturedat1.25(cid:2)106cells/mlincellculturemedium
anism of apoptosis induction associated with terminal DC matu-
supplemented with 10 ng/ml murine recombinant GM-CSF, kindly pro-
ration.Delayed-onsetapoptosiswasindependentofFas/Fasligand videdbyDr.L.Grinberg(Biosynexus).Ondays3and5thesupernatant
interactions and TNF-(cid:1)and did not require bacterial internaliza- was removed and replaced with fresh medium containing GM-CSF. Ex-
tion,butinsteadwasdependentuponinteractionbetweenbacterial perimentswereperformedwiththenonadherentandlooselyadherentcell
populationfromculturesatdays6–8.
subcapsularcomponentswithacellsurfaceBMDCreceptor(like-
lyaTLR)actinginaMyD88-dependentmanner.
BacterialpulsingandstimulationofBMDCsforinvitro
Materials and Methods
experiments
Mice
BMDCscollectedafter6–8daysofculturewerewashedtwotimesincell
C57BL/6NmicewereobtainedfromtheNationalCancerInstitute(Gaith- culturemediumandplatedat106BMDCs/mlofmediumwithoutGM-CSF
ersburg,MD).MyD88(cid:1)/(cid:1)mice(27)werekindlyprovidedbyDr.Douglas in24-or48-wellcellcultureplates(Costar,Corning,NY).After30min,
Golenbock(DepartmentofMedicineandMicrobiology,BostonUniversity toallowcellstosettle,peptidoglycan(PG)(100–0.1(cid:2)g/ml)orabacterial
SchoolofMedicine,Boston,MA)withpermissionfromDr.S.Akira(Re- suspensionincellculturemediumcontainingthedesiredamountofinac-
searchInstituteforMicrobialDiseases,OsakaUniversity,Osaka,Japan) tivatedS.pneumoniaewasaddedtotheculturesandmaintainedduringthe
and were bred and genotyped in our facility. C3.MRL-Faslpr (MRL/lpr) pulse. Unless indicated otherwise, S. pneumoniae type 14 was used at a
mice, genetically deficient in CD95 expression, C3H/HeJ-Faslgld (C3H/ ratioof800bacteriaperBMDCthroughouttheexperiments.Insomeex-
gld)mice,geneticallydeficientinCD95Lexpression,andtheircorrespond- periments,BMDCswerestimulatedwith10(cid:2)g/mloflowendotoxinanti-
ingbackgroundcontrols,C3H/HeJmice,werepurchasedfromTheJack- CD95proapoptoticmAb(cloneJo2,hamsterIgG;BDPharMingen)inthe
sonLaboratory(BarHarbor,ME).Micewereusedat8–10wkofageand absenceofbacteriaor1hbeforebacteriawereaddedtotheculturetotest
weremaintainedinapathogen-freeenvironmentattheUniformedServices BMDC susceptibility to CD95-mediated apoptosis. Alternatively, for
UniversityoftheHealthSciences(Bethesda,MD).Theexperimentsinthis pulse-chaseexperiments,BMDCswerecollectedafterthepulse,theexcess
studywereconductedaccordingtotheprinciplessetfourthintheGuidefor of bacteria were extensively washed, and the BMDCs were replated in
theCareandUseofLaboratoryAnimals(InstituteofAnimalResources, mediumwithoutGM-CSFandculturedforthedesiredchaseperiod.For
NationalResearchCouncil,DepartmentofHealth,EducationandWelfare, experiments with live bacteria, the pulse was conducted in cell culture
NationalInstitutesofHealth,78-23). mediumwithoutantibioticsandwaschasedinculturemediumcontaining
S.pneumoniaestrainsandbacterialAgs 20(cid:2)g/mlgentamicin.
The S. pneumoniae capsular type 14 was generously provided by Sam
Wilson(UniformedServicesUniversityoftheHealthSciences,Bethesda, Propidiumiodide(PI)uptakeassayandtrypanblueexclusion
MD).Thecapsulartype3(strainWU-2)andthestablenonencapsulated
assay
variantJD6/11ofthisstrain(28)werekindlyprovidedbyDr.J.Yother
(UniversityofAlabama,Birmingham).Thecapsulartype2(strainD39),its CellmembranepermeabilityandviabilityweretestedbyPIuptakeassay
isogenicpneumolysin-negativemutant(29),andthenonencapsulatedvari- or trypan blue exclusion assay. PI uptake for BMDCs was analyzed by
antofD39(strainR36A)werekindlyprovidedbyDr.D.E.Briles(Uni- placing 1 (cid:2) 106 cells/ml in cold PBS containing 5 (cid:2)g/ml PI (Sigma-
versityofAlabama).LPS-free,purifiedpeptidoglycanfromStaphylococ- Aldrich,St.Louis,MO).After5minofincubationatroomtemperaturein
cusaureuswasprovidedbyDr.R.Schuman(Biosynexus,Rockville,MD). thedark,theredfluorescenceemittedbythePIintercalatedintheDNAof
cellsthathavelostmembraneintegritywasanalyzedonaflowcytometer
Bacterialcultureandpreparationofthebacterialinoculum
(EPICSXL-MCL;BeckmanCoulter,Miami,FL).Thetrypanblueexclu-
IsolatedcolonieswereselectedfromColumbiabloodagarplatesandwere sion assay was conducted by diluting a cell suspension by 1/5 v/v with
grown in Todd Hewitt broth to mid-log phase, collected, heat killed by 0.4% trypan blue (Sigma-Aldrich). Viable cells were counted using a
incubationat60°Cfor1h,andextensivelywashedinPBS.Sterilitywas hemocytometer.
confirmedbysubcultureonbloodagarplates.Thepneumolysin-deficient
mutant(PLN)wasgrowninthesamemedium,butsupplementedwith0.3
(cid:2)g/ml erythromycin. The bacterial suspension of the type 14 strain was AnnexinV-FITCstaining
adjustedwithPBSto109CFU/mlbyanabsorbancereadingof0.6at650
nm. This bacterial suspension was used as a standard to normalize, by Bacteria-pulsedBMDCsandnon-pulsedBMDCswerecollectedatdiffer-
FACSanalysis,thedensityoftheotherheat-killedS.pneumoniaestrains. enttimepoints,washed,andadjustedto106BMDCs/mlincold10mM
Specifically,wedeterminedthenumberofeventsacquiredintheappro- HEPES/NaOH(pH7.4)containing140mMNaCl,2.5mMCaCl2,and5
priateforwardsidescattergateduring1minusingserial10-folddilutions (cid:2)g/mlPIandAnnexinV-FITC(BDPharMingen).BMDCswereincubated
ofthebacterialsuspensions.Thisapproachavoidserrorsintheestimation for15minatroomtemperatureinthedarkandwereimmediatelyanalyzed
ofbacterialdensitybyphotometricturbidityduetoheavyencapsulation. byflowcytometry.BMDCstreatedfor4hinculturewith6 (cid:2)Mcampto-
Bacteriawerethenaliquotedat1010CFU/mlandfrozenat(cid:1)80°Cuntil thecin(Sigma-Aldrich),apotentinhibitoroftopoisomeraseI,wereusedas
theirusetopulseBMDCsinculture.Whenlivebacteriawereused,cul- a positive control for induction of apoptosis. BMDCs treated with 0.2%
turesinmid-logphasewerecollected,washedinPBS,and,afteradjust- saponin(Sigma-Aldrich)wereusedasapositivecontrolforinductionof
menttothesameabsorbanceasthecorrespondingheat-killedsuspension, necrosis.AnnexinVavidlybindstocellsexpressingcellsurfacephospha-
wereuseddirectlyintheexperiment. tidylserine,aphospholipidtranslocatedfromtheinnerleafletoftheplasma
membrane to the outer leaflet very early during the apoptosis program,
Cellculturemedium whencellsarestillnonpermeabletoPI.CellspositiveforAnnexinVand
negativeforPIareundergoingapoptosis,whereascellsintheendstageof
ThecellculturemediumconsistedofRPMI1640supplementedwith5%
apoptosisarepositiveforbothAnnexinVandPI(31).
FCS (BioWhittaker, Walkersville, MD), 50 (cid:2)M 2-ME, 20 (cid:2)g/ml genta-
micin,1mMsodiumpyruvate,2mML-glutamine,0.1mMnonessential
aminoacids,and25mMHEPES.
Determinationofhypodiploidnucleibyflowcytometry
Bonemarrowcellculture
Thepercentageofapoptoticnuclei(i.e.,hypodiploid)wasmeasuredafter
DCswereculturedfrombonemarrow,aspreviouslydescribed(30),with PIstaininginhypotonicbufferbyflowcytometry,essentiallyasdescribed
slightmodifications(22).Briefly,bonemarrowcellsuspensionswerede- byNicolettietal.(32).Briefly,BMDCswerecollectedatdifferenttime
2356 BACTERIALINDUCEDDCAPOPTOSIS
pointsduringthepulsewithbacteriaordrugtreatmentandwashedexten- treatmentmarkedlyinhibitedtheinternalizationofCM-DiI-labeledbacte-
sively,andthecellpelletcontaining1(cid:2)106BMDCswasgentlyresus- ria ((cid:4)4% of the mean fluorescence intensity (MFI) uptake by untreated
pendedin1mlofhypotonicfluorochromesolution(50(cid:2)g/mlPIin0.1% cells).DMSOwasusedasdiluentfortheCt,butitsconcentrationinthe
sodiumcitrateplus0.1%TritonX-100).Thetubeswereplacedat4°Cin culturewas(cid:4)0.2%.IdenticalvolumesofDMSO,asfoundinCt-treated
thedarkovernight,andthePIfluorescenceofindividualnucleiwasmea- cultures,wereaddedasacontrol.
suredbyflowcytometricanalysis(32).BMDCstreatedincultureduring
12–24hwith1(cid:2)g/mlactinomycinD(Sigma-Aldrich)wereusedaspos-
itivecontrolsofapoptosis.BMDCstreatedwith0.2%saponinwereusedas CytokineELISA
positivecontrolsofnecrosis.
The concentrations of specific cytokines released into the medium of
Determinationofcellularcaspaseactivity BMDC cultures were measured using optimized standard sandwich
ELISA.Recombinantcytokinesusedasstandards,aswellasthecapture
Caspase-8andcaspase-3activityinBMDCextractsobtainedatdifferent
mAbs, biotinylated mAbs used for detection, and streptavidin-alkaline
time points during the pulse with bacteria or drug treatment were deter-
phosphatasewerepurchasedfromBDPharMingen.Streptavidin-alkaline
mined using the corresponding colorimetric assay kits, following the in-
phosphatasewasusedincombinationwithp-nitrophenylphosphatediso-
structions of the manufacturer (Calbiochem, San Diego, CA). The
dium(Sigma-Aldrich)assubstratetodetectthespecificbinding.Standards
caspase-3kitusesAc-DEVD-p-nitroanilineassubstrate,andthecaspase-8
wereincludedineveryplateandthesamplesweretestedinduplicate.The
kitusesIETD-p-nitroaniline.Thespecificinhibitorsandpurifiedenzymes
detectionlimitsoftherespectiveELISAswere4pg/mlforIL-6,70pg/ml
providedaccurateandspecificquantitationofthecaspaseactivity.
forIL-10,6pg/mlforIL-12(p40/p70),125pg/mlforIL-12(p70),and12
Inhibitionofcaspaseactivity pg/mlforTNF-(cid:1).
CaspaseactivitywasirreversiblyinhibitedbytreatmentofBMDCswith50
(cid:2)Mofthecorrespondingcell-permeablecaspaseinhibitor(R&DSystems, Statistics
Minneapolis, MN). The caspase-specific inhibitors used were Z-WEHD-
fluoromethylketone(FMK)(caspase-1inhibitor),Z-DEVD-FMK(caspase-3 Datawereexpressedasarithmeticmean(cid:5)SEMoftheindividualvalues.
inhibitor), Z-YVAD-FMK (caspase-4 inhibitor), Z-YVAD-FMK (caspase-6 Levelsofsignificanceofthedifferencesbetweengroupsweredetermined
inhibitor), Z-IETD (caspase-8 inhibitor), Z-LEHD (caspase-9 inhibitor), Z- by the Student t test. Any p values (cid:4)0.05 were considered statistically
AEVD(caspase10inhibitor),andZ-LEED-FMK(caspase-13inhibitor).The significant.
inhibitorwasaddedtothecultures30minbeforethepulsewithbacteriaand
wasmaintainedintheculturemediumduringthepulse.ThepeptideZ-F-A-
FMK, was added to the cultures at the same concentration as the caspase
Results
inhibitorstocontrolfortheeffectsofthefluoromethyl-ketonederivativeson
caspaseactivity.BMDCculturesincubatedwithDMSOwereusedtomonitor Heat-killedS.pneumoniaeinduceapoptosisofBMDCs
anypotentialcytotoxiceffectsofthediluents.
ThelengthoftimeduringwhichDCsmaintaintheirviabilityafter
QuantitationofsolubleCD95ligand(CD95L) contactwithabacterialpathogenlikelyplaysacriticalroleintheir
ability to promote anti-bacterial Ig responses (22). Thus, initial
The concentration of soluble CD95L released into the culture medium,
duringthepulsewithbacteria,wasquantifiedbyELISA(QuantikineM), studies tested the ability of heat-killed S. pneumoniae to induce
followingtheinstructionsofthemanufacturer(R&DSystems). apoptosis in immature in vitro-derived bone marrow dendritic
cells. The inability of BMDCs to exclude trypan blue, which re-
FlowcytometricanalysisofBMDCsurfaceAgexpression
flects their loss of cell membrane integrity and which occurs rel-
Allstepswereperformedonice.ForstainingBMDCsforflowcytometry, ativelylateduringtheprocessofapoptosis,wasobservedlargely
theFcreceptorswerespecificallyblockedwith2.5(cid:2)g/ml/106BMDCsof
between48and72hafteradditionofheat-killedbacteriatoculture
anti-CD16/CD32mAb(clone2.4G2;BDPharMingen)inPBScontaining
1%FCS(stainingbuffer)45minbeforeandduringthestaining.Cellswere (Fig.1A).Thus,by72h,(cid:4)35%ofBMDCsexcludedtrypanblue
stainedbyincubationfor30minwithbiotinylatedorPE-conjugatedmAbs comparedwith(cid:3)90%atinitiationofcultureorafter72hinme-
(BD PharMingen) specific for CD11c (clone HL3), CD8 (clone 53-6.7), diumalone.ThekineticsandpercentagesofBMDCsshowingPI
CD11b(M1/70),I-Ab(cloneAF6-120.1),I-Ad(cloneAMS-32.1),H2-Kb
uptakeafterculturewithS.pneumoniaeweresimilartothoseob-
(AF6-88.5), CD40 (clone 3/23), CD80 (16-10A1), CD86 (clone GL1),
CD25 (clone PC61), CD54 (clone 3E2), CD95 (clone Jo2), and CD95L servedusingtrypanblue(datanotshown).
(cloneKay-10).TheincubationwithbiotinylatedmAbswasfollowedby To determine whether loss of trypan blue exclusion after S.
stainingwithPE-streptavidinconjugatefor15min.FordetectionofCD16/ pneumoniaeactivationreflectedinductionofBMDCapoptosis,we
CD32expressionwiththespecificAb(clone2.4G2),theFcblockingstep
measured both the percentage of cells staining positive for An-
wasomitted.Irrelevantisotypeandspecies-matchedmAbswereusedas
nexinV(whichresultsfromtranslocationofphosphatidylserineto
stainingcontrols.CellswereanalyzedonanEPICSXL-MCL(Beckman
Coulter).Deadcellsanddebriswereexcludedfromanalysisbygatingon the outer leaflet of the plasma membrane) and the percentage of
theappropriateforwardandsidescatterprofile. hypodiploid nuclei, as determined by PI staining (which reflects
nuclearfragmentation)duringthe72hcultureperiodwithS.pneu-
PhagocyticcapacityofBMDCs
moniae.AprogressiveincreaseinthepercentageofBMDCseither
The time course of bacterial internalization was tracked by pulsing the staining for Annexin V (Fig. 1B) or showing hypodiploid nuclei
BMDCs with S. pneumoniae labeled with the fluorescent lipophilic cell
(Fig.1C)wasobservedbeginning12hafteradditionofS.pneu-
tracker chloromethylbenzamido derivative of DiI (CM-DiI; Molecular
Probes,Eugene,OR),aspreviouslyreported(22).Briefly,atdifferenttime moniaeandcontinuinguntil72h,bywhichtime(cid:3)80%apoptotic
pointsofthepulse,BMDCswerecollectedandwashedinPBS,andviable cells were observed. In contrast, (cid:4)10% of BMDCs cultured in
BMDCs were analyzed by flow cytometry, gated so as to exclude free mediumalonewereapoptoticafter72h.InductionofBMDCap-
bacteriaonthebasisofcellsize.BMDCspulsedwithunlabeledbacteriaat
optosis after S. pneumoniae addition was also closely correlated,
thesameratioswereusedtodeterminenonspecificfluorescence.
beginningafter12h,withaprogressiveincreaseintheenzymatic
Cytochalasin(Ct)treatment activityoftheapoptosis-initiatorcaspase-8(Fig.1D).
Toblockparticleinternalization,BMDCsweretreatedfor2–3hwithCtA Activity of the effector caspase, caspase-3, although rapidly
(5(cid:2)g/ml)orCtD(5(cid:2)g/ml)beforethepulsewithbacteria.Forpulsetimes spikinginitially,declinedthereafterduringthefirst24htosignif-
shorterthan5h,theCtwasmaintainedduringthepulse,otherwiseCtwas icantlybelowthelevelsfoundinunstimulatedBMDCs(Fig.1D).
washed out and 1 (cid:2)g/ml fresh Ct was added and maintained during the
Caspase-3activityreturnedtonormallevelsby48handwasagain
pulse. Unpulsed BMDCs treated under these conditions remain viable
((cid:3)82%)after24hofculture,undergoingminimalCt-inducedapoptosis found at reduced levels at 72 h. BMDCs treated with saponin, an
(5%inBMDCstreatedwithCtA;12%inBMDCstreatedwithCtD).The inducerofnecrosis,didnotup-regulateeithercaspase-8(Fig.1D)or
TheJournalofImmunology 2357
FIGURE 1. Heat-killed S. pneu-
moniae induced BMDC apoptosis.
BMDCs pulsed with heat-killed S.
pneumoniaewereharvestedatdifferent
timepoints,andtheprogressionofap-
optosis was determined by measuring
the percentage of cells permeable to
trypan blue (A), the percentage of PI-
negative BMDCs binding a conjugate
ofAnnexinV-FITC(B),thepercentage
ofhypodiploidnucleideterminedbyPI
staining(C),andthecaspase-8(D)and
caspase-3(E)activitypresentwithinthe
cell extracts. BMDCs treated with 1
(cid:2)g/mlactinomycinDor6(cid:2)Mcampto-
thecinwereusedasapositivecontrolof
apoptosis.BMDCsculturedin0.2%sa-
poninwereusedaspositivecontrolsof
necrosis.Thedatashownarerepresen-
tative of two independent experiments
induplicate.
caspase-3(Fig.1E)activity,asexpected.Bacterialinterferenceinthe onsetofapoptosis((cid:3)24h)comparedwithBMDCsthatwereex-
enzymatic assay was excluded by demonstrating that high bacterial posed continuously to bacteria. No increase in apoptosis was ob-
densitiesdidnotinhibittheenzymaticactivityofpurifiedcaspase-3 served when BMDCs were in contact with bacteria for only 30
andthatcaspase-3activitywasnotdetectedinthebacterialprepara- min.Nevertheless,aprogressiveincreaseinthepercentageofhy-
tionitself(datanotshown). podiploid nuclei at a given time point (Fig. 2A) and extension of
BMDCsexhibitedminimalapoptosiswithinthefirst20hafter DNAfragmentation(datanotshown)wereobservedasthedura-
bacterial exposure. In contrast, treatment with actinomyn D or tionoftheinitialbacterialpulsewasincreasedfrom3to6hand
camptothecin, which are primary inducers of apoptosis, led to a againfrom6htocontinuousexposure.Theseresultsindicatethat
rapid increase in percentages of hypodiploid nuclei and both thesignalsrequiredtoinducedelayedapoptosisareprovidedtothe
caspase-3 and caspase-8 activity in BMDCs. Thus, (cid:3)70% of BMDCswithinthefirsthoursofbacterialcontact,withenhanced
BMDCswereapoptotic10haftertreatment,alevelofapoptosis levels of apoptosis occurring as bacterial exposure is prolonged.
observedonly72hafterbacterialexposure(Fig.1).Collectively, Weobservedthatasimilartimecourseofexposuretobacteriawas
thesedatasuggestthatheat-killedS.pneumoniaeinduceadelayed- required for the subsequent induction of BMDC phenotypic mat-
onset, caspase-dependent apoptosis of BMDCs that is likely not uration(22).
secondary to the direct action of an apoptosis inducer, such as a
Liveandheat-killedS.pneumoniaeinduceapoptosisthrough
bacterialtoxin.
differentmechanisms
Continuousexposuretoheat-killedbacteriaisnotcriticalfor
The induction of BMDC apoptosis by heat-killed S. pneumoniae
theinductionofapoptosis
indicatedthatheat-sensitivemetabolicproducts,includingtoxins,
Wenextperformedbacterialpulse-chaseexperimentstodetermine werenotnecessaryforapoptosisinduction,butitdidnotruleout
thetimerequiredforBMDCstobeincontactwithbacteriatoelicit anadditionalrolefortheseproductswhenBMDCsareexposedto
apoptosis.AsshowninFig.2A(opensymbols),BMDCsexposed live bacteria. To determine this, we directly compared the effi-
foreither3or6htoheat-killedbacteriashowedasimilartimeof ciency of apoptosis induction in response to live vs heat-killed
FIGURE 2. KineticsofDNAfragmentation
inducedinBMDCsbyliveorheat-killedwild-
typeandpneumolysin-defectivemutantsofS.
pneumoniae. A, BMDCs were pulsed with
heat-killedorliveS.pneumoniaecapsulartype
14ataratioof800bacteriaperBMDC.Dur-
ingtheperiodsofpulseindicated,freebacteria
wereremovedbywashing,andBMDCswere
recultured for the indicated periods of chase.
The percentages of hypodiploid nuclei were
determinedafterthepulseandafter20,48,or
72hofchase.B,Comparisonofthepercentage
ofBMDChypodiploidnucleiafterpulsewith
liveorheat-killedS.pneumoniaecapsulartype
2 (strain D39) and its isogenic pneumolysin-
defectivemutant(strainPLN).Thepercentage
of hypodiploid nuclei was determined at 24,
48,and/or72h.Oneexperimentrepresentative
oftwoisshown.
2358 BACTERIALINDUCEDDCAPOPTOSIS
FIGURE 3. Bacterialdosedependenceofcytokinesecretion,phenotypicmaturation,andDNAfragmentationinducedbyheat-killedS.pneumoniae.
ConcentrationsofTNF-(cid:1)andIL-6secretedintotheculturemedium(A)andMFIofBMDCsstainedwithmAbsspecificforMHCclassIIandCD86after
12and24h,respectively,afterpulsewithbacteriaatvariablebacteria:BMDCratios(B),expressedasthepercentageoftheconcentrationorMFIobtained
afterpulsewithbacteriaata1:800ratio(100%).C,Thepercentageofhypodiploidnucleiwasdeterminedaftera72-hpulsewiththeindicatedbacteria:
BMDCratios.Oneexperimentrepresentativeofthreeisshown.
bacteria. Live S. pneumoniae induced BMDC apoptosis after a apoptosisthroughreleaseofatoxin(pneumolysin),whereasnonvia-
much shorter exposure time and with significantly faster kinetics ble(orlivepneumolysin-deficient)bacteriaarecapableofstimulating
than was observed in response to heat-killed bacteria (Fig. 2A). BMDCstoundergoaprimaryprocessthatleadstoadelayedonsetof
The presence of live, but not heat-killed, bacteria in BMDC cul- apoptosis.
tures for as little as 30 min was sufficient to induce a significant
increase in apoptosis by 3 h ((cid:3)15% hypodiploid nuclei) with Theextentofapoptosisinducedbyheat-killedS.pneumoniaeis
(cid:3)80% apoptosis by 24 h. No further changes occurred in the ki- directlycorrelatedwiththelevelofBMDCactivationand
maturation
netics or extent of apoptosis induction with greater bacterial ex-
posuretimesof3or6h.Toeliminateanycontinuedproliferation Apoptosis induced by heat-killed S. pneumoniae begins to occur
of residual bacteria, possibly remaining after washing and recul- soonafterthephenotypicmaturationofBMDCsiscomplete(i.e.,
ture of BMDCs in medium alone, we added either gentamicin (cid:6)24h),suggestingthatdelayedonsetapoptosisisadirectreflec-
(whichinactivateextracellularbacteria)oranantibioticcocktailof tionofterminalmaturation.AsshowninFig.3,boththelevelof
gentamicin,penicillin,andstreptomycin(whichlikelyalsoinacti- secretionofIL-6andTNF-(cid:1)(Fig.3A)andespeciallythemagni-
vateintracellularbacteria).SupernatantsfromfreshculturesofS. tudeofphenotypicmaturation(i.e.,up-regulationofMHCclassII
pneumoniae grown in medium (RPMI 1640 supplemented with and CD86; Fig. 3B) in response to varying ratios of bacteria to
5% FCS without antibiotics), added to BMDC cultures supple- BMDCdirectlycorrelatedwiththepeakpercentagesofhypodip-
mentedwithantibiotics,inducedBMDCapoptosisatsimilarlevels loidnucleiobservedat72h(Fig.3C).BacteriatoBMDCsratios
and following similar kinetics as in BMDC cultures pulsed with under 50:1, which were ineffective at inducing apoptosis, also
liveS.pneumoniae(datanotshown). failedtoinducesignificantsecretionofcytokinesandphenotypic
Therapidkineticsofinductionofapoptosisinresponsetolive maturation.Thesedatasuggestacause-and-effectrelationshipbe-
bacteria were roughly similar to those observed using the direct tweenBMDCphenotypicmaturationandapoptosisinresponseto
inducers of apoptosis, actinomycin D or camptothecin. This, and bacterialactivation.
the ability of S. pneumoniae culture supernatants to induce apo- To further explore the potential relationship between BMDC
ptosis, suggested the possibility that live, but not heat-killed, S. maturationandapoptosis,wedeterminedthepotentialroleofGM-
pneumoniaeproduceatoxinthatdirectlyinducesBMDCapopto- CSF in comodulating these processes. GM-CSF is released early
sis.Inthisregard,thehemolysinactivityofGroupBStreptococcus afterbacterialinfectionandhasbeenshowntoinhibitapoptosisof
hasbeenshowntoinduceapoptosisinhumanmonocytes(7),and neutrophils (34), in addition to stimulating the generation of DC
the pneumolysin of S. pneumoniae induces neuronal and micro- precursors from the bone marrow. Thus, we wished to determine
glialapoptosis(16).Thus,wecomparedtheabilityofliveS.pneu- whether GM-CSF could likewise modulate BMDC apoptosis in
moniaecapsulartype2(strainD39)andpneumolysin-deficientmu- responsetoheat-killedbacteriaandwhetherthiscorrelatedwitha
tant(PLN)toinduceapoptosisinBMDCs.AsshowninFig.2B,at change in the level of phenotypic maturation. Addition of GM-
24h(cid:3)80%ofBMDCstreatedwithliveD39exhibitedhypodiploid CSFtoBMDCculturesatthetimeofadditionofbacteriasignif-
nucleivs(cid:4)20%forBMDCstreatedwithPLN,demonstratingacrit- icantly reduced the percentage of hypodiploid nuclei observed at
icalroleforpneumolysinintheinductionofrapidonsetapoptosisby 48 h (Fig. 4A). The difference observed in apoptosis induction at
livebacteria.Incontrast,bothD39andPLN,whenheat-killed,and 72 h between GM-CSF-treated and untreated groups was much
livePLNwereequallyefficientatinducingthedelayedonsetofap- less, suggesting that GM-CSF delays, but does not prevent, the
optosisseeninFig.2,demonstratingtheexistenceofasecondmech- onsetofapoptosis.TobeeffectiveinreducingBMDCapoptosis,
anismofapoptosisinductionthatwasunrelatedtobacterialviability GM-CSFwasrequiredduringthefirst24hafterbacterialexposure
orpneumolysinactivity.Pneumolysinisheat-sensitive,veryunstable (Fig. 4A) and needed to be present at high concentrations
in culture, and requires metabolic activity for its continued release ((cid:4)10pg/mlofGM-CSFwasineffective).Furthermore,whenvary-
(33).ThesedataindicatethatS.pneumoniaecaninduceDCapoptosis ing doses of GM-CSF were tested, a direct relationship was ob-
throughtwodifferentmechanisms.LivebacteriatriggerrapidBMDC servedbetweentheabilityofGM-CSFtoreduceapoptosisandan
TheJournalofImmunology 2359
FIGURE 4. InhibitionofbacteriainducedBMDCapoptosisbyGM-CSF.A,BMDCswereculturedintheabsence(medium)orpresenceofheat-killed
S.pneumoniaeinmediumcontaining10ng/mlGM-CSF((cid:7)GM-CSF)orinmediumwithoutGM-CSF((cid:1)GM-CSF)duringtheentireperiodofpulsewith
bacteria or only during the first 24 h of pulse. After 48 or 72 h, the percentages of hypodiploid nuclei were determined. Values are expressed as the
arithmetic mean of triplicate cultures (cid:5) SEM. B, Titration of the GM-CSF effect. BMDCs were pulsed with bacteria during 48 h in the presence of
increasingconcentrationsofGM-CSF,andthepercentagesofhypodiploidnucleiweredetermined.C,ExpressionofCD86andMHCclassIIbyflow
cytometryafter24hofpulsewithbacteriainthesameGM-CSFtitrationexperimentdepictedinB.
inhibitory effect on phenotypic maturation (Fig. 4, B and C), re- Although addition of GM-CSF allowed for the generation of
inforcingtheviewthatdelayedapoptosisinresponsetobacterial immatureDCsfrombonemarrowprecursors,allexperimentswere
stimulationisareflectionofterminalmaturation. conducted in the absence of GM-CSF. As illustrated in Fig. 4, A
FIGURE 5. DNAfragmentationinducedbyS.pneumoniaedoesnotrequirebacterialinternalization.A,FlowcytometricanalysesofPI-stainedBMDC
nucleitodeterminepercentagesofhypodiploidnucleiafter24-hpulsewithlive(left)or72-hpulsewithheat-killed(right)S.pneumoniaecapsulartype
14withorwithoutpretreatmentwithCtD.B,PercentagesofhypodiploidnucleiinadifferentsetofsimilarexperimentsinwhichBMDCswerepretreated
witheitherCtAorCtD.C,MFIratio(%)ofcellsurfacemarkerexpressionat24honBMDCspulsedwithheat-killedS.pneumoniaetreatedoruntreated
withCtD.D,Cytokinessecretedasratio(%)oftreatedoruntreatedwithCtDasinC.Untreatedcellswereincubatedwiththesamevolumeofthediluent
DMSO.Oneexperimentrepresentativeoffiveisshown.
2360 BACTERIALINDUCEDDCAPOPTOSIS
FIGURE 6. HeavilyencapsulatedS.pneumoniaearenoteffectiveinducersofBMDCmaturationorapoptosis.A,KineticsofBMDCinternalizationof
heat-killedS.pneumoniaeofaheavilyencapsulatedtype3strain(WU-2),itsnonencapsulateddefectivemutant(JD6/11),orthemoderatelyencapsulated
type14strain(Pn14)labeledwiththefluorescenttracerCM-DiIwhenusedatbacteria:BMDC200:1ratio.B,Concentrationsofcytokinessecretedinto
themediumafter6h(upperpanel)and24h(lowerpanel)ofpulsewiththesame200:1bacteriatoBMDCratio.Statisticallysignificantdifferencesbetween
theconcentrationsofcytokinesinthesupernatantsofBMDCspulsedwithWU-2andJD6/11areindicatedbydots((cid:1))andforthecomparisonbetweenWU-2
andPn14byasterisks((cid:1)).C,PercentagesofincreaseintheexpressionofseveralphenotypicmarkersofBMDCmaturationandCD11c,after24hofpulse
ata500:1bacteriatoBMDCratio.D,Percentageofhypodiploidnucleiobtainedafter72hofpulsewithdifferentstrainsofS.pneumoniaeatdifferentratios.
E,TimecourseofDNAfragmentationatan800:1bacteriatoBMDCratio.
andB,GM-CSFwasnotrequiredforthesurvivalofBMDCsover BMDCsandindeedeffectedamodestenhancement(Fig.5C).In
a72hcultureperiod,followingtheirgenerationafter6–8daysof contrast,CtDstronglysuppressedthesecretionofbothIL-12and
invitrocultureinthepresenceofGM-CSF. TNF-(cid:1)andpartiallyinhibitedIL-10release,buthadnoeffecton
IL-6(Fig.5D).Thesedatastronglysuggestthatinteractionofbac-
InductionofBMDCapoptosisdoesnotrequirethe
teriawithstructuresexpressedontheBMDCsurfaceissufficient
internalizationofbacteria
tomediatethecellsignalingrequiredforbothmaturationandin-
Macrophageshavebeenshowntoundergoapoptosisasaresultof duction of apoptosis. Nevertheless, the modest enhancement of
mediator release consequent to intracellular killing of bacteria phenotypicmaturationseeninthepresenceofCtmightreflectthe
(11).Byanalogy,thesedataraisethepossibilitythatbacterianeed decrease in release of IL-10.4 The data also suggest that neither
tobeinternalizedtodelivertherequisitesignalingforBMDCap- IL-12norTNF-(cid:1)iscriticalforapoptosisinduction.
optosis. Thus, we used either CtA or CtD to block BMDC inter-
nalization of bacteria and measured the subsequent effects on re- Heavyencapsulationcaninterferewithbothphenotypic
lease of cytokines, induction of phenotypic maturation, and maturationandapoptosisinducedbyheat-killedS.pneumoniae
development of hypodiploid nuclei (i.e., apoptosis) (Fig. 5). Ad-
Our observation (Fig. 5) that heat-killed S. pneumoniae induces
ditionofCttoBMDCcultures,intheabsenceofbacteria,induced
phenotypicmaturationandapoptosisintheabsenceofsignificant
minimalapoptosis((cid:4)12%)relativetoBMDCsculturedinDMSO
internalization strongly suggests that these processes were medi-
diluent or medium alone ((cid:4)5%) after 24 h of culture. Treatment
ated by an interaction of the bacteria with cell surface BMDC
withCteffectivelyblockedtheinternalizationofCM-DiI-labeled
bacteria(MFIofCtA-treatedBMDCswas2.5–3.8%oftheMFIof
untreated BMDCs; data not shown). Neither CtA nor CtD pre-
4J.ColinoandC.M.Snapper.OpposingsignalsfromPAMPSandIL-10arecritical
ventedapoptosisinducedbyeitherliveorheat-killedbacteria(Fig.
foroptimaldendriticcellinductionofinvivohumoralimmunitytoStreptococcus
5,AandB).Likewise,Ctdidnotinhibitphenotypicmaturationof pneumoniae.Submittedforpublication.
TheJournalofImmunology 2361
receptors. In the next series of experiments, we wished to deter- BMDCs stimulated with WU-2 (Fig. 6C). Furthermore, at 72 h,
minewhetherthebacterialcomponentsinvolvedinthisinteraction the percentage of hypodiploid nuclei was markedly reduced in
were present within the bacterial capsular and/or subcapsular do- BMDCs stimulated with WU-2 at any ratio between 50 and 800
main. In light of previous studies demonstrating that polysaccha- bacteriaperBMDC(Fig.6D).Kineticstudiesindicatethatheavy
rideencapsulationcaninfactmaskunderlyingimmunostimulatory encapsulationsignificantlydelayed,butdidnoteliminate,theon-
bacterialsubcapsularstructures,wecomparedphenotypicmatura- setofBMDCapoptosis(Fig.6E).Thesedatafurtherreinforcethe
tion and apoptosis induction of BMDCs in response to either a notion that delayed BMDC apoptosis is a reflection of terminal
heavily encapsulated WU-2 strain of S. pneumoniae type 3 or its maturation, both being related to signaling mediated by interac-
nonencapsulated isogenic mutant, JD6/11 (28). As a further con- tionsbetweenBMDCcellmembranereceptorsandbacterialsub-
trol,weusedalow-encapsulatednonvirulentS.pneumoniaetype capsular structures, without a requirement for internalization or
14strain(Pn14).BothmorphologyandsizeofPn14colonies(sim- bacterialprocessing.
ilar to JD6/11 colonies) confirmed a much lower level of encap-
PurifiedPGorintactS.pneumoniaeinduceBMDCapoptosisin
sulationthanforWU-2.
aMyD88-dependentmanner
After 1 h ofbacteria-BMDC coculture, encapsulation had no
significant effect on the rate of internalization of nonopsonized Wenextwishedtodirectlydeterminewhethersubcapsularbacte-
bacteriabyBMDCsinvitro,assuggestedbythesimilarslopesof rial structures were capable of mediating BMDC maturation and
thekineticcurvesofinternalizationofthethreestrains(Fig.6A). apoptosis and whether this involved a MyD88 (TLR)-dependent
ThedelayedkineticsobservedfortheheavilyencapsulatedWU-2 process. To accomplish this, we used an LPS-free preparation of
strain may be explained by the greater flotation coefficient and purified PG from Staphylococcus aureus, which has previously
electrostatic repulsion mediated by the thick layer of capsular beenshowntobeaTLR-2ligand.PGinducedBMDCstosecrete
polysaccharide, which delayed optimal BMDC-bacterial contact cytokines (IL-6, IL-12, TNF-(cid:1), and IL-10) in a dose-dependent
afterinitiationofculture. manner(Fig.7A)andtoup-regulatecellsurfacemarkersofmat-
Encapsulation also had no significant effect on induction by uration (MHC class I, MHC class II, CD25, CD40, and CD86)
BMDCs of the cytokines TNF-(cid:1)and IL-12 (Fig. 6B), whose se- (Fig.7B).Inductionofcytokinesecretionwascompletelydepen-
cretionwasinhibitedbyCtD(Fig.5).Incontrast,IL-10secretion dent, whereas phenotypic maturation was partly dependent, on
and, to a lesser extent, IL-6 secretion were significantly lower in MyD88 signaling, in response to either PG (Fig. 7, A and B) or
response to WU-2. Thus, these observations are consistent with intactS.pneumoniae(Fig.7Banddatanotshown).
thoseusingCt(Fig.5)andstronglysuggestthatsignalsoriginating PGalsoinduceddelayedBMDCapoptosisinadose-dependent
fromBMDCcellsurfacestructuresaresufficientforinductionof manner(Fig.7C)withkineticssimilartothoseobservedforintact
IL-6 and in part IL-10 secretion and that heavy polysaccharide bacteria.Asobservedforphenotypicmaturation,apoptosisinduc-
encapsulationcanmasktherelevantbacterialsubcapsularstimuli. tioninresponsetoeitherPGorintactbacteriawaspartlyMyD88
In agreement with the hypothesis that heavy encapsulation in- dependent.ThesedatasuggestthatintactS.pneumoniaecanstim-
terfered with surface signaling, the up-regulation of phenotypic ulate BMDC maturation and apoptosis, at least in part, through
markersofmaturationinresponsetobacteriaat24h,whichdoes MyD88-dependentsignalingviaTLR-2interactionwithPGinthe
notrequireinternalization(Fig.5C),wasdramaticallyimpairedin bacterialcellwall.
FIGURE 7. InductionofBMDCapoptosisinboth
wild-type and MyD88(cid:1)/(cid:1) mice in response to PG.
BMDCs obtained from wild-type (MyD88(cid:7)/(cid:7)) or
MyD88(cid:1)/(cid:1)werestimulatedwithvariableconcentra-
tionsofPG(A)and,after20hofculture,theconcen-
trationofcytokinessecretedintothemediumwasde-
termined by ELISA. Concentrations secreted by
MyD88(cid:7)/(cid:7) are shown with a continuous line, and
those secreted by MyD88(cid:1)/(cid:1) are shown as broken
lines.B,BMDCswerestimulatedfor20hwith100
(cid:2)g/ml PG or with 800 bacteria per BMDC, and the
expressionofdifferentmarkersofphenotypicmatura-
tionweredeterminedandexpressedaspercentagesof
the levels expressed in nonstimulated BMDCs. C,
Time course of DNA fragmentation induced during
thefirst72hofstimulationwithdifferentPGconcen-
trationsinwild-typeMyD88(cid:7)/(cid:7)BMDCs.D,Theper-
centagesofhypodiploidnucleiat72hinMyD88(cid:7)/(cid:7)
andMyD88(cid:1)/(cid:1)BMDCsstimulatedwithdifferentcon-
centrationsofPGorR36A(bacteria).
2362 BACTERIALINDUCEDDCAPOPTOSIS
FIGURE 8. Heat-killed S. pneumoniae-induced
BMDCapoptosisisdependentoncaspaseactivity.Per-
centagesofhypodiploidnucleiinBMDCstreatedwith
50(cid:2)Mofthespecificinhibitorsofthecaspaseactivities
indicatedintheabsence(inhibitor)orinthepresence
(inhibitor(cid:7)bacteria)ofheat-killedS.pneumoniaefor
72h(left)orliveS.pneumoniaefor5h(right).BMDCs
pulsedwithlivebacteriawereculturedforanadditional
20-hperiodinthecontinuedpresenceofcaspaseinhib-
itorbeforeanalysisofDNAfragmentation.
Inhibitionofselectedcaspasesblockdelayed-onset,butnot volved in cytokine processing and inflammation, had no effect.
pneumolysin-dependent,BMDCapoptosis However,inhibitionofcaspase-13activity,anothermemberofthe
caspase-1subfamily,deeplyimpairedapoptosis(Fig.8).
We detected the induction of caspase-8 activity in BMDCs des-
Instrikingcontrast,apoptosisinducedbylivebacteriawasnot
tined to undergo apoptosis in response to heat-killed S. pneu-
affected by the inhibition of any of the caspase activities tested,
moniae(Fig.1).Totestwhethertheactivityofcaspase-8,aswell
indicating that caspase activation is not a major requirement for
asthatofothercaspases,wascriticalfortheonsetofapoptosis,we
inductionofpneumolysin-dependentBMDCapoptosis.
irreversiblyblockedtheactivityofspecificcaspasesbytreatment
withcell-permeablepeptidederivatives.AsshowninFig.8,ofthe
IntactS.pneumoniaeinducesBMDCapoptosisinaCD95/
two initiator caspases (caspase-8 and caspase-10) recruited into
CD95L-independentmanner
apoptosissignalingcomplexesassociatedwithdeathreceptors(35,
36),theinhibitionofcaspase-10activitycompletelyabrogatedthe We demonstrated that caspase-8 activity is induced in BMDCs
abilityofheat-killedbacteriatoinduceBMDCapoptosis,whereas destinedtoundergoapoptosisinresponsetointactS.pneumoniae
inhibitionofcaspase-8activityhadonlyapartial,thoughsignifi- (Fig.1)andthatcaspase10and,toamuchlesserextent,caspase
cant,effect.Theseresultssuggestthatheat-killedbacteriainduced 8 are critical for induction of apoptosis. Because both caspase 8
BMDC apoptosis through death cell receptor signaling, where andcaspase10havebeenimplicatedinCD95-dependentinduction
caspase-10playedacriticalroleasinitiator. of apoptosis (36, 37), we wished to determine whether CD95/
The inhibition of caspase activities located downstream of the CD95L interactions were involved in S. pneumoniae-induced
apoptosis signaling cascade, such as caspase-3 or caspase-9, had BMDCapoptosis.AsshowninFig.9A,BMDCsfromLPS-defec-
only partial inhibitory effects (Fig. 8), suggesting multiple path- tive C3H/HeJ mice constitutively expressed cell surface CD95,
waysofamplification,andasexpected,theinhibitionofmembers with progressive up-regulation in response to bacteria, but not in
ofthecaspase-1subfamily,caspase-1andcaspase-4,primarilyin- the presence of medium alone over a 72 h period. However, as
FIGURE 9. S.pneumoniae-inducedBMDCapoptosisisindependentofCD95orCD95Lexpression.A,TimecourseofcellsurfaceexpressionofCD95
(MFI)duringthepulsewithR36AstrainofS.pneumoniae.FilledhistogramrepresentstheexpressionofCD95inBMDCsderivedfromC3H/HeJmice
culturedduring72hinmediumalone.B,PercentageofhypodiploidnucleiinBMDCsderivedfromMRL/lpr,C3H/gld,andcontrolC3H/HeJmice,cultured
bytriplicateinthepresenceorabsenceof200:1bacteriatoBMDCduringthepulsetimeindicated.