OuterMembraneProteinAfromKlebsiella

pneumoniaeActivatesBronchialEpithelial
Cells:ImplicationinNeutrophilRecruitment
Thisinformationiscurrent
asofNovember11,2014.

MurielPichavant,YvesDelneste,PascaleJeannin,Catherine
Fourneau,AnneBrichet,AndrBernardTonnelandPhilippe
Gosset
JImmunol2003;171:66976705;;
doi:10.4049/jimmunol.171.12.6697
http://www.jimmunol.org/content/171/12/6697

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from http://www.jimmunol.org/byguestonNovember11,2014

TheJournalofImmunologyis
publishedtwiceeachmonthbyThe
AmericanAssociationof
TheJournalof
Immunology

OuterMembraneProteinAfromKlebsiellapneumoniae
ActivatesBronchialEpithelialCells:Implicationin
1
NeutrophilRecruitment
MurielPichavant,*YvesDelneste,PascaleJeannin,CatherineFourneau,*Anne
Brichet,AndreBernardTonnel,*andPhilippeGosset2*
Asidefromitsmechanicalbarrierfunction,bronchialepitheliumplaysanimportantrolebothinthehostdefenseandinthe
pathogenesisofinflammatoryairwaydisorders.Toinvestigateitsroleinlungdefense,theeffectofabacterialcellwall
protein,theoutermembraneproteinAfrom Klebsiellapneumoniae (kpOmpA)onbronchialepithelialcells(BEC)was
evaluatedonadhesionmoleculeexpressionandcytokineproduction.Moreover,thepotentialimplicationofthismechanism
inkpOmpAinducedlunginflammationwasalsodetermined.OurinvitrostudiesdemonstratedthatkpOmpAstrongly
boundtoBEAS2Bcells,ahumanBECline,andtoBECprimarycultures,resultinginNFBsignalingpathwayactivation.
Exposure to kpOmpA increased ICAM1 mRNA and cell surface expression, as well as the secretion of IL6, CXC
chemokineligand(CXCL)1,CXCL8,CCchemokineligand2,CXCL10byBEAS2Bcells,andBECprimarycultures(p<
0.005).WeanalyzedinvivotheconsequencesofintratrachealinjectionofkpOmpAtoBALB/cmice.InkpOmpAtreated
mice,atransientneutrophilia(withamaximumat24h)wasobservedinbronchoalveolarlavageandlungsections.Invivo
kpOmpApriminginducedbronchialepitheliumactivationasevaluatedbyICAM1andCXCL1expression,associatedwith
thesecretionofCXCL1andCXCL5inbronchoalveolarlavagefluids.Inthelung,anincreasedleveloftheIL6,CXCL1,
CXCL5,CXCL10mRNAwasobservedwithamaximumat6h.ThesedatashowedthatkpOmpAisinvolvedinhostdefense
mechanism by its ability to activate not only APC but also BEC, resulting in a lung neutrophilia. The Journal of
Immunology,2003,171:66976705.

Downloadedfrom http://www.

Epitheliumrepresentstheprimaryinterfacebetweentheexternaland
internal surroundings of the airways. Aside from its
mechanicalbarrierfunction,bronchialepithelium

isinvolvedbothinnormalhostdefenseandinthepathogenesisof
inflammatoryairwaydisordersbyitsimplicationininflammatory
cellrecruitment(1,2).Forthispurpose,bronchialepithelialcells

(BEC)3 expressvarioustypesofcelladhesionmolecules,suchas
ICAM1,conjugatedwithcytokineandchemokinesecretion,which
both orchestrate inflammatory cell migration toward the airway
wall. BEC are able to produce cytokines and growth factors,
particularly IL6, CXC chemokine ligand (CXCL)1 (macrophage
inflammatoryprotein2),CXCL8(IL8),CXCL10(IFNinduced
protein10),CCchemokineligand(CCL)2(monocytechemoat

*InstitutNationaldelaSanteetdelaRechercheMedicale,Unite416,Institut
Pasteur, Lille, France; Centre dImmunologie Pierre Fabre, St. Julien en
Gene`vois,France;and CliniquedesMaladiesRespiratoires,CentreHospitalier
RegionalUniversitaire,Lille,France
ReceivedforpublicationFebruary24,2003.AcceptedforpublicationOctober8,
2003.
Thecostsofpublicationofthisarticleweredefrayedinpartbythepaymentof
page charges. This article must therefore be hereby marked advertisement in
accordancewith18U.S.C.Section1734solelytoindicatethisfact.
1
ThisworkwassupportedbytheInstitutNationaldelaSanteetdelaRecherche
Medicale and the Pasteur Institute of Lille. M.P. is the recipient of doctoral
fellowshipsfromtheMiniste`redelEducationNationale.P.G.isamemberofthe
InstitutNationaldelaSanteetdelaRechercheMedicale.
2
AddresscorrespondenceandreprintrequeststoDr.PhilippeGosset,InstitutNa
tionaldelaSanteetdelaRechercheMedicale,Unite416,InstitutPasteurde
Lille,1,rueduProfesseurCalmette,BP245,59019Lillecedex,France.Email
address:Philippe.Gosset@pasteurlille.fr

Abbreviationsusedinthispaper:BEC,bronchialepithelialcell;CXCL,
CXCchemokineligand;CCL,CCchemokineligand;Omp,outermembraneprotein;
kpOmpA,OmpAofKlebsiellapneumoniae;DC,dendriticcell;TLR,Tolllikereceptor;
MFI,meanfluorescenceintensity;BAL,bronchoalveolarlavage;MAPK,mitogenacti
vatedproteinkinase;PKC,proteinkinaseC;PI3K,phosphatidylinositol3kinase.

ries (3, 4). In addition to their

chemotactic activity for
neutrophils, CXCL8 and other
CXCchemokineswithanELR
aminoacidmotifactivatethese
cells.CXCL10isimplicatedin
Th1 lymphocyte recruitment
(5). Conversely, CCL2 is a
potent chemoattractant and
activator of monocytes,
macrophages, and T
lymphocytes. So, BEC
activationmaybeimplicatedin
thelungdefensemechanismby
itsabilitytorecruitgranulocytes
andmononuclearcells.
Among the agents
responsible for resident airway
cell activation, many different
stimuli such as fungi, viruses,
pollutants, and bacteria are
present in inhaled air.
Klebsiella pneumoniae is a
Gramnegative

aerobic
organism, which is a major
pathogen for nosocomial
pneumonia (6). The clearance
of this bacteriafrom the lungs
requires effective host defense
mechanisms, to which the
bacterial surface takes part.
Threecomponentsof theouter
wallofGramnegativebacteria
aresuspectedtobeinvolvedin
development of immunity:
LPS, membrane proteoglycans,
and outer membrane proteins
(Omp)(7,8).OmpAisoneof
themajorproteinsoftheouter
membrane of Gramnegative
bacteria. Thisprotein is highly
conserved among the
enterobacteriacaefamilyandis
thought to consist of two
domains.Whereastheeffectsof
LPS and membrane
proteoglycansonimmunecells
have been largely described
(reviewed in Refs. 7 and 9),

only few studies evaluated

Omp properties, mainly
focused

on

its
immunomodulatory function
(8, 10 12). It has been
reportedthattherecombinant
OmpA of
Klebsiella
pneumoniae (kpOmpA) is a
potentcarrierprotein(1315)
that binds to, is internalized,
and activates macrophages
(16) and professional APCs
such as dendritic cells (DC)
(14).kpOmpAalsofavorsthe
crosspresentation

of
exogenous Ags and the
induction of cytotoxic
responses (14). These
interactions

between
kpOmpA and immunological
cells may represent an initi
ating event in acquired host
defensemechanisms.

unol.org/byguestonNovember11,2014
Copyright2003by

TheAmerican

6698
Incontrast,OmpAinvolvementinlunginflammation,and
particularly its action on BEC, is not documented. The
purpose of this work was to identify potential interactions
between kpOmpA and BEC in vitro and in vivo and
secondarilythedevelopmentoflunginflammation.Ourresults
demonstratedthatkpOmpAbindstoBECandinducesadhesion
molecule and cytokine expression by these cells. In vivo,
kpOmpAtriggersBECactivationandaneutrophilinflux.

MaterialsandMethods
Cytokines,Abs,andotherreagents
kpOmpAwasexpressedin Escherichiacoliandpurifiedasdescribed
asanendotoxinfreepreparation(14).Thefollowingmaterialswere
purchased:DMEM/F12medium(Invitrogen,CergyPontoise,France),
airway epithelial cell growth and basal medium (Promocell,
Heidelberg,Germany),antibiotics(penicillinGsodium,10,000U/ml;
streptomycinsulfate,10,000mg/ml;andamphotericinB,25g/ml);and
2mMLglutamine(Invitrogen),collagenG(3mg/mlin12mMHCl;
Biochrom,Berlin,Germany),FCS,UltroserG,trypsin(containing1
mMEDTA);agarose,PBS,TRIzolreagent(LifeTechnologies,Grand
Island, NY), chloroform (Merck, Fontenay sous Bois, France) and
isopropanol (Carlo Erba, Milan, Italy), gelstar (FMC bioproducts,
Rockland,ME);andrecombinanthumanTNF,IFN(R&DSystems,
Abingdon, U.K.); SB203580, RO318220, PD98059, genistein
(Calbiochem,SanDiego,CA),LY294002(SigmaAldrich,St.Louis,
MO).ThefollowingmousemAbwereused:anticytokeratin5/6/18Ab
(NeoMarkers,Fremont,CA);antiICAM1mAb(IgG1),cloneB159
and the isotype control (IgG1), clone MOPC 21 (BD Biosciences,
Erembodegem,Belgium),aswellasthesecondaryAbsFITCorPE
labeled streptavidin (SigmaAldrich). Neutralizing antihuman Toll
likereceptor(TLR)2andTLR4mousemAbwerepurchasedbyeBio
science(SanDiego,CA).

Cellculture
Human bronchial epithelial biopsies were obtained by fiberoptic
bronchoscopy from patients who were being investigated for
bronchopulmonary carcinoma. Biopsies were taken largely at a
distancefromthetumor.Histologicfeaturesofthebronchialmucosa
were normal in all specimens. All procedures were reviewed and
approvedbyHospitalInstitutionalReview

kpOmpAACTIVATES
BECs
Boardandwritteninformedconsentwasobtainedfromallsubjectsin
cludedinthestudy.
BEC were cultured as previously described (17). Briefly, one
explant(0.50.5mminsize)wasplacedonsterileplasticdishescoated
withcollagenGmatrix(typeIandIIIcollagen).Afteranadherence
phase,explantswereculturedinDMEM/F12mediumcontaining2%
UltroserG,1%antibiotics;and2mM Lglutamine.Culturemedium
waschangedevery34days.ExplantswerecultureduntilBECwere
confluent.Then,explantsweretransferredthreetimestonewdishesto
initiatenewBECprimarycultures.Aftertransfer,confluentepithelial
cellswereincubatedfor24hwithmediumalone(negativecontrol)and
thenincubatedwithkpOmpAinfreshmedium.Epithelialphenotype
( 96%) was confirmed by staining with anticytokeratin 5/6/18 Ab
(datanotshown).
BEAS2B cells were obtained from the American Type Culture
Collection(Manassas,VA).Thiscelllinewasderivedfromhuman
bronchialepitheliumtransformedbyanadenovirusSV40hybridvirus.
BEAS2Bcellswereculturedin75cm2 cultureflasks,untilpassage
20,andmaintainedinBECgrowthmedium,containing1%antibiotic
solution. After seeding, 5 105 cells were plated on sixwell cluster
plates(Costar,Cambridge,MA)withcollagenGcoating.Confluent
cellswerethenculturedinBECbasalmediumfor24h.
Cell activation was achieved by addition of endotoxinfree
recombinantkpOmpA(240g/ml)oracontrolglycoproteinsuchas
BSA.Insomeexperiments,TNF(200U/ml) withor withoutIFN
(100U/ml)wereaddedinthepresenceornotofkpOmpA.Activation
byLPS(serotype055B5;100ng/ml)andkpOmpAinthepresenceor
notofpolymyxinB(50U/ml)(SigmaAldrich)wasalsoperformedas
acontrol.Supernatantswerecollectedafter6or24hincubationand
cellswerelysedinTRIzolreagenttoprepareRNA.

QuantificationofmRNAexpression
TotalRNAwerepurifiedandreversetranscriptedusingoligodTprimers
and Superscript reverse transcriptase (Invitrogen). In BEAS2B cells,
mRNAexpressionforhumanIL6,IL18,CCL2,CCL5,CXCL8,CXCL10,
andICAM1wasevaluatedbyatwostepsemiquantitativeRTPCRusing
GAPDH mRNA as a reference. In mouse lungs, murine IL6, CCL2,
CXCL1, CXCL5, CXCL10, and ICAM1 mRNA expression was
determinedbythesamemethodusingactinmRNAasareference.The
primersequencesandthesizeoftheproductarereportedinTableI.After
gelelectrophoresisandstainingwithgelstar,theintensityofeachbandwas
measuredwithGelAnalystsystem(Claravision,Orsay,France).Results

Downloadedfrom http://www.jimmunol.org/byguest
TableI.PrimersforRTPCR
analysisofcytokine,chemokine,and
adhesionmoleculeexpressiona
Target

Product(bp)

hGAPDH

206

mactin

353

hIL6

260

hIL18
512
5 -GTC TTC ACC ACC ATG GAG

5
5
5
5
5
5
5

-CCA
-GTC
-CTC
-TCA
-GAT
-GGC
-GCC

AAG TTG TCA

mIL6
GGG
GCG CCC
CTT AAT GTC
mCCL2
ATG
AGG AGA
GAG TTG TCA
ACA
mCXCL1
GGT CAT
GGA TCC CGC

TGG ATG
352GCA
CAG
ACG CAC
273GCC
CTT
TGT CCT
GAC
530AGA
GCC CGC

5
5
5
5
5
5
5

-CCA
-GTG
-GGT
-TCA
-AGG
-AAC
-GAC

ACG
ACA
AGC
GCC
TGC
GGA
GAG

TCT
ACC
TAT
AGA
TGA
GAA
ACC

CGT
ACG
GGT
TGC
AGA
AGA
AGG

CCT
GCC
ACT
AGT
CCT
AGA
AGA

TCT TC-3
TTC CCT A
CCA-3
TAA CG-3
TAG GG-3
CAG ACT G
AAC AG-3

onNovember11,2014
a

h,human;m,mouse.

TheJournalofImmunology
wereexpressedasaratio:geneofinterest/GAPDHoractinmRNAfor
thehumanorthemousegenes,respectively.

Flowcytometricanalysis
BECwerebrieflytreatedwithtrypsinsolutionand,afterneutralization
ofproteinaseactivity,removedfromplatesbyrepeatedpipetting.The
bindingofkpOmpAonBECwasevaluatedbytheirincubationfor30
minat37CwithbiotinylatedkpOmpA(20g/ml)ortetanustoxinC
(20g/ml)asacontrol.Afterwashings,cellswereincubatedwithPE
labeled streptavidin for another 30 min and then washed twice. To
blockthebindingofkpOmpA,neutralizingantiTLR2andantiTLR4
mAbs(20g/ml) werepreincubatedwithBEAS2Bcellsfor30min
beforetheadditionofbiotinylatedkpOmpA.
ToassessthemodulationofICAM1expressiononBEAS2Bcells,
cellswerestimulatedinpresenceornotofkpOmpA(8,20,or40g/ml)
for24h.FITClabeledantiICAM1mAbandtheisotypecontrolwere
added for 30 min to cells, and then washed twice. Cells were
resuspended and fixed in PBS with 0.25% paraformaldehyde.
Fluorescencewasanalyzedon10,000eventsusingaflowcytometer
(FACSCalibur;BD Biosciences).Previousexperimentsshowedthat
BEC treatment with trypsin did not affect ICAM1 expression
comparedwithcellsdetachedwithPBSplusEDTA2mM(datanot
shown).
Results were expressed as difference between mean fluorescence
intensity (MFI) with specific Ab minus the isotype control MFI
(MFI).

Cytokinemeasurement
ConcentrationsofhumanIL6,CCL2,CCL5,CXCL1,CXCL8,and
CXCL10inBECculturesupernatantsweredeterminedbysandwich
enzymeimmunoassay(R&DSystems).LevelsofmurineIL10,IL12,
IFN,TNF,CXCL1,andCXCL5(R&DSystems)weredetermined
byELISAinbronchoalveolarlavage(BAL)fluids.

EMSA
BEAS2Bcellswereculturedinmediumalone,orinpresenceofTNF
(200U/ml)(positivecontrol)orkpOmpA(5,20,and40g/ml)for2h.
Afterwashing,nuclearproteinswereextractedbystandardprocedures
(18). The probes used for the gel retardation assay contained the
consensusB(5CAGCGGCAGGGGAATTCCCCTCTCCTT
AGG TT3 ) binding site. The 5 end 32Plabeling of the double
stranded oligonucleotide and EMSA were performed by standard
procedures. Membranes were exposed to a PhosphoImager screen
(MolecularDynamics,Sunnyvale,CA)andtheintensityofthebands
werequantifiedbyusingacomputerimageanalysissystem.

Transienttransfectionassays
BEAS2B cells, grown to 70 80% confluence in BEC growth
medium and starved during 24 h in BEC basal medium, were
transiently transfected by lipofection using lipofectamin (Invitrogen)
techniquewithreporterandexpressionGL3plasmidscontainingor
not a tandem repeat of an NF B site and luciferase promoter.
Luciferaseassayswereperformed24haftertransfectionaspreviously
described(19).

kpOmpAintratrachealinjectioninmice
Sixto10wkfemaleBALB/cmicewereanesthetizedbyi.p.injection
ofavertin(SigmaAldrich)(2.5%v/vinPBS).FiftymicrolitersofBSA
(100 g) or kpOmpA solution (20 or 100 g) were administrated

intratracheally under direct vision through the opening vocal cords

usinga23Gmetalcatheterconnectedtotheoutletofamicropipette.
Miceweresacrificedafter6h,1,2,3,and7days,andBALfluidswere
performedbyPBSinstillation(1ml).

ImmunohistochemistryforICAM1andCXCL1expression
inmicelungtissue
Lung specimens were fixed with Immunohistofix and embedded in
Immunohistowax (Aphase, Mormont, Belgium). After
permeabilization, sections were overlaid overnight with rabbit anti
mouseICAM1(BDBiosciences)orCXCL1Abs(R&DSystems).Ab
binding was detected after a 2h incubation with biotinconjugated
goatantirabbitIgGAb(dilution,1/400)atroomtemperature,followed
byextravidinalkalinephosphataseincubation(dilution,1/200;Sigma
Aldrich)for30min.ColordevelopmentwasobtainedwithaFastRed
solution (SigmaAldrich). Slides were washed three times between
each step. Gills hematoxylin was used to counterstain. Leukocyte
infiltrate was shown by MayGrunwald Giemsa staining (Sigma
Aldrich)onlungsections.

669
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at
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eofnonparametrictests:theWilcoxontestforpaireddataortheMann
WhitneyUtestforunpaireddata.Avalueofp0.05wasconsideredas
significant.

Results
kpOmpAbindstoBEC
Binding of biotinylatedkpOmpA was analyzed by flow
cytometrytothehumanBEClineBEAS2BandBECprimary
cultures.Fig.1showsthatkpOmpA(20g/ml)stronglybound
toBEAS2Bcells(Fig.1A)andBECprimarycultures(Fig.
1B). In contrast, no binding of biotinylatedtetanic toxin, a
bacterial protein with carrier properties, was detected.
BiotinylatedkpOmpAbindingwasinhibitedbyadditionofa
10fold excess of unconjugated kpOmpA (data not shown).
PreviousstudiesreportedthatTLR2isinvolvedinkpOmpA
signalinginmacrophageandDC(14,20).Therefore,wetested
the effects of antiTLR2 and TLR4 blocking Abs on
kpOmpAbindingtoBEAS2Bcells.Nomodificationwasob
served, suggesting, in agreement with others (14, 20), that
TLR2 and TLR4 are not involved in kpOmpA binding to
BECbutratherthanincellactivation(datanotshown).
kpOmpAinducesICAM1mRNAandcellsurface
proteinexpressioninBEAS2Bcells
Studies in BEAS2B cells were conducted to determine
whether kpOmpA may affect ICAM1 expression. ICAM1
mRNAexpressioninresponsetokpOmpA(8or20g/ml)was
studiedbyRTPCRat6and24hstimulation.BEAS2Bcells
incubatedwithmediumaloneexpressedlowlevelsofICAM1
mRNA(Fig.2A).ICAM1mRNAwasstronglyincreasedwith
8and20g/mlkpOmpA,at6h,whereasthislevelwascloseto
baselinelevelsat24h.
kpOmpAeffectsonICAM1cellsurfaceproteinexpression
weretestedaftera24hincubation.Fig.2BshowsthatBEAS
2BcellsconstitutivelyexpressICAM1,aspreviouslyreported
(17).kpOmpAincreaseddosedependentlyICAM1expression
( p 0.01comparedwithunstimulatedcells).MFIwas3,4,
and5fold

FIGURE1.kpOmpAbindstoBECs.BEAS2Bcells(A)andBECpri

marycultures(B)wereincubated,for30min,at37C,withmedium
(thin), biotinylatedtetanic toxin (20 g/ml) (bold), or biotinylated
kpOmpA (20 g/ml) (shaded), washed, and analyzed by FACS. The
binding was revealed by PElabeled streptavidin. Results are
representativeofoneofsixexperiments.

Downloadedfrom http://www.jimmunol.org/byguestonNovember11,2014

6700

kpOmpAACTIVATESBECs

FIGURE2.kpOmpAupregulatesICAM1mRNA
expressionandcellsurfaceexpressiononBEAS2B
cells. A, ICAM1and GAPDH mRNA expression
wereanalyzedbyRTPCRonBEAS2Bepithelial
cells cultured for 6 or 24 h, with medium alone
(control) or 8 and 20 g/ml kpOmpA. One
representative of three separate experiments is
shown. B, Membrane ICAM1 expression was
measuredaftera24hincubationbyflowcytometry
onBEAS2Bepithelialcells,culturedwithmedium
alone()oractivatedwith840g/mlkpOmpA(u).
ResultsareexpressedasMFI.DataaremeanSEM
(n6).,p0.01comparedwithunstimulatedcells.

0.01) compared with

unstimulated cells after 24 h
higherthaninmediumalone,inthepresenceof8,20,and40g/ml(Fig.3C).Takentogether,these
kpOmpA,respectively.
datashowthatkpOmpAtriggers
CCL2, CXCL8, CXCL10, and
kpOmpAinducescytokinemRNAexpressionandsecretionin
IL6 mRNA expression and
BECs
secretioninBEC.

EffectofkpOmpAoncytokineproductionbyBEAS2Bcellsand
BECprimarycultureswasanalyzed.kpOmpAeffectsonmRNA
expressionforCCL2, CXCL8, CXCL10, IL6, andIL18were kpOmpAactivates
quantifiedinBEAS2BcellsbyRTPCR.CytokinemRNAtran intracellularsignaling
scriptswereundetectableinrestingBEAS2Bcells,exceptforILpathwayinBEAS2Bcells

18,asshowninFig.3A.However,CCL2,CXCL8,CXCL10,andTo investigate transduction

IL6 mRNA levels increased in a dosedependent manner afterpathwayinvolvedinkpOmpA
treatmentwith8and20g/mlkpOmpA.CytokinemRNAlevels activation,differentinhibitors
reachedamaximumat6hpostexposure,andpersistedat24h. ofproteinkinasesweretested
SuchmodulationwasnotobservedforIL18(Fig.3A).
on chemokine production
kpOmpAinducedmRNAexpressionwasassociatedwiththedose (Table II). After kpOmpA
dependentproductionofCCL2,CXCL1,CXCL8,CXCL10,andIL6 stimulation, P38 mitogen
release,inBEAS2Bcellsupernatantsafter24hofstimulation( pactivated protein kinase
0.01forthesecytokines)(Fig.3B).CXCL1andCXCL8production(MAPK)

inhibitor
was strongly induced by 480 and 110fold, respectively, in the (SB203580) reduced CCL2,
presence of 20 g/ml kpOmpA compared with medium alone. CXCL8, and CXCL10
Moreover, treatment with polymixin B (an inhibitor of endotoxin secretionby39,40,and34%,
activation)didnotaffectCCL2andCXCL8production,whereasit respectively, whereas the
neutralized the effect of 10 ng/ml LPS (data not shown). Similar extracellular signalregulated
resultswereobtainedwithBECprimarycultures:kpOmpA(20g/ml) kinase 1/2 MAPK inhibitor
increased significantly the production of CCL2, CXCL1, CXCL10, PD98059 had no effect. The
inhibitor

of
andIL6(p0.05),andCXCL8(p
phosphatidylinositol 3kinase
(PI3K) LY294002 inhibited
in a higher manner CCL2,
CXCL8, and CXCL10
secretion (62, 49, and 60%,
respectively).ProteinkinaseC
(PKC)inhibitor,referredtoas
RO318220, also decreased
CCL2 (45%) and CXCL10
(70%) production, but not
CXCL8(20%).
NFBnucleartranslocation
was also investigated in
BEAS2BcellsbyEMSA.In
theseexperiments,NFBwas
activatedinadosedependent
manner with kpOmpA (Fig.

4A). TNF induced

translocation was used as a
positivecontrol.
To evaluate NF B
promoter activity, the
luciferase activity was
measuredinlysatesofBEAS
2B cells transfected with the
reporter plasmid pGL3,
containing or not a tandem
repeatofanNFBsite(Fig.
4B). The data showed that
kpOmpA(20g/ml)orTNF

FIGURE 3.
kpOmpA
induces
cytokine
mRNA ex
pression and
production
byBEAS2B
cells and
BECprimary
cultures. A,
Cytokine
mRNA
expression
was
quantified in
BEAS2B
cells,
cultured for
6 and 24 h
withmedium
alone, or
kpOmpA (8
and20g/ml).
CCL2,
CXCL8,
CXCL10,
IL6, IL18,
and GAPDH
mRNA
expression
were
evaluated by
RTPCR.
One
representativ
e of six
experiments
is shown.
CCL2,
CXCL1,
CXCL8,
CXCL10,
and IL6
cytokine
production
was
measured
after a 24h

incubation
ELISA

supernatants
BEAS2B
(B) cultured
mediumalon
8(u),and20
kpOmpA (f)
BEC pr
culture

exposed to
g/ml kpO
(right colum
not (left col
B and C, R
wereexpress
themeanSE
6

experiments.
0.05 and , p
compared
unstimulated

Downloadedfrom http://www.jimmunol.org/byguestonNovember11,2014

TheJournalofImmunology

6701

TableII.ModulationofkpOmpAinducedchemokineproduction
CCL2

Medium
kpOmpADMSO
SB203580
LY294002
RO318220
PD98059

142.5 52.3
1186.5 274.5
758 129.5
469 44.4
677 64.8
1337.5 357.7

CXCL8

103 75.4
755.5 176.1
487.25 104.3
414 75.2
614.5 126
941.25 313.8

CXCL10

30.25 13.3
78.25 27.9
55.25 12.4
47.5 16
39 10.8
70.25 21.5

(control),kpOmpA(20g/ml),or
TNF (200 UI/ml) for 24 h.
Epithelial cells were harvested,
andluciferaseactivityinprotein
equivalent cell lysates was
assessed as described in
MaterialsandMethods.Results
are representative of one of
threeexperiments.

a
Modulation of kpOmpAinduced chemokine production by inhibitors of the
MAPKP38(SB203580),extracellularsignalregulatedkinase1/2(LY294002),PKC
(RO318220),PI3K(PD98059),orthevehicle(DMSO).ChemokinelevelsinBEAS
2Bcellsupernatants(24h)wereexpressedinpicogramspermilliliterasthemean
SEMofthreeseparateexperiments.

inducedNFBactivation,asevidencedbytheincreaseinlucif
erase activity (10 and 13fold, respectively). No luciferase
activitywasobservedwhencellsweretransfectedwithplasmid
FIGURE 4. kpOmpA activates
withoutNFBsite.

the NF B signaling pathway in

kpOmpApotentiatesTNFandTNFIFNeffectsonCXCL8
BEAS2Bcells.A,BEAS2Bcells
andCCL2productionbyBEAS2Bcells
were incubated with medium
alone (negative control), TNF
Toinvestigatethepotentialsynergybetweeninflammatorycyto
(positivecontrol),orkpOmpA(5,
kinesandkpOmpA,BEAS2BcellswereactivatedbyTNFand 20, and 40 g/ml). After 2 h of
TNFIFN.CXCL8andCCL2productionwasupregulatedin incubation, nuclear protein
thepresenceofthesecytokinesandkpOmpAalone(Fig.5).BEC extractswerepreparedandEMSA
costimulation with kpOmpA and TNF , or TNF IFNhad afor NF B were performed as
synergistic effect on CXCL8 and CCL2 production. The dosedescribed in Materials and
dependenteffectofkpOmpAwasalsoobservedinthepresenceMethods. Results are
ofTNFandTNFIFN.ThesedatademonstratedthatkpOmpArepresentative of one of three
potentiatesTNFandTNFIFNeffectsonCXCL8andCCL2 experiments. B, BEAS2B cells
were transiently transfected with
productionbyBEAS2Bcells.
GL3reporterplasmidscontaining
(f),ornot(u),atandemrepeatof
an NF B site. Cells were
incubated with medium alone

F
I

I
n
T
o
I
n

FIGURE6. kpOmpAintratrachealinjectioninmiceinducesneutrophil
influxinBALfluids.MicereceivedanintratrachealinjectionofBSA(100
g)orkpOmpA(20or100g).BALcellswerespunonslidesandstained
withMayGrunwaldGiemsa.Thesedatashowedpercentagesofmacro
phagesandneutrophilsinBALfluidscollectedat6h,1,2,3,and7days
afterinstillation.ResultsareexpressedasmeanSEMforthreemiceand
arerepresentativeofthreeexperiments.

Downloadedfrom http://www.jimmunol.org/byguestonNovember11,2014

6702
103 cells and 36 103 cells, respectively) compared with the
number obtained after BSA instillation (42 10 3 cells). In con
trast, the absolute number of neutrophils was significantly in
creased6and24hafterinstillationof100gofkpOmpA(3410 3
cells and 42 103 cells, respectively) in comparison with naive
mice(6103cells)(p0.05).Thiswasconfirmedbytheanalysis
ofBALcelldifferentialcountinmicereceivingkpOmpA(Fig.
6). Whereas BAL cells from control mice (receiving BSA)
consistedpredominantlyofmacrophages,apronouncedinfluxof
neutrophils was found in BAL of mice primed with 100 g of
kpOmpA,withamaximumatday1(85%totalcells).kpOmpA
exhibited an in vivo dosedependent effect as intratracheal
injectionof20ginducedalowerneutrophilinfluxonday1(55%
total cells). Neutrophil percentage progressively returned to
baselinelevelatday7.Lymphocyte,eosinophil,andepithelial
cellcountswerenotmarkedlymodifiedafterkpOmpAinjection
(datanotshown).
Neutrophilinfluxwasalsoobservedonlungsectionscollected
6or24hafterkpOmpAinjection.Neutrophilsarepresentinboth
alveolarspaces,bronchialwalls,andairwaylumen(Fig.7,Aand
B).Thecellularinfiltratereacheditsmaximumat24h,andre
solvedafter7days(datanotshown).Incontrast,fewinfiltrating
leukocyteswereobservedinlungsectionsofmiceprimedwith
BSA(datanotshown).TodemonstrateinvivoBECactivation
afterintratrachealinjectionofkpOmpA,ICAM1expressionand
CXCL1productionwasmeasuredbyimmunohistochemistryon
lungsections.kpOmpAupregulatedICAM1expressionwithin
BECbutalsowithinpneumocytesandendothelialcells(Fig.7,E
and F). ICAM1 expression was similar at 6 and 24 h
postexposure

kpOmpAACTIVATES
BECs
to kpOmpA. In contrast, a faint staining for ICAM1 was
detectedwithinBECafterBSAexposure(Fig.7D).Concerning
CXCL1synthesis,BECandalveolarmacrophageswerestrongly
positiveforthischemokineafterkpOmpAtreatment(Fig.7, I
and J)incontrastwithcontrolmicereceivingBSA(Fig.7H).
CXCL1 staining was mainly detected at 6 h (Fig. 7I) but
decreasedat24h(Fig.7J).

In addition, lung inflammation was evaluated by the

measurementofcytokineandchemokineproduction.Inlung
homogenates, mRNA expression for CXCL1, CXCL5,
CXCL10, and IL6 was transiently increased at 6 h after
kpOmpAinjection(Fig.8),butCCL2mRNAlevelswerenot
affected(Fig.8).ICAM1mRNAexpressionwassimilarly
upregulatedafterkpOmpAexposureat6h.Expressionof
CXCL1mRNAwasassociatedwiththeconcomitantrelease
ofthischemokineinBALfluidsat6h;CXCL5production
was also progressively induced (Fig. 9). In addition to
chemokine production, kpOmpA instillation also triggered
the secretion of inflammatory and immunomodulatory
cytokines. kpOmpA intratracheal injection transiently up
regulatedIL10,IFN,andTNF,(at6h)inBALfluids
whereasitdidnotincreaseIL12levels(Fig.9).

Discussion
Effectivelungdefenseagainstbacterialinfectionisprimarily
dependentontherapidclearanceofmicroorganismsviathe
participation of two phagocytic cells: neutrophils and
macrophages.Inflammatorycellrecruitmentandactivation
require complex interactions involving the secretion of
activatingandchemotactic

Downloadedfrom http://www.jimmunol.org/byguest
FIGURE 7. Histologic evaluation
of lung inflammation in mice
primed with BSA or kpOmpA.
Lung sections from mice
sacrificed 24 h after intratracheal
injectionofkpOmpA(100g)were
stainedbyeosinhematoxylinand
observedatamagnificationof500
(A) or 1250 (B). Neutrophils
(arrows)werepresentinbronchial
lumen(BL),bronchialepithelium,
and alveolar spaces (AS). BAL
immunohistochemistry

was
performed for ICAM1 on lung
sectionsfrommicetreatedfor6h
with BSA (D) or exposed to
kpOmpAfor6h(E)or24h(F),in
comparisonwithanisotypecontrol
(C).Sameexperimentsweredone
for CXCL1: mice treated for 6 h
with BSA (H) or exposed to
kpOmpA for 6 h (I) or 24 h (J),
compared with an isotype control
(G). BECs (arrow) and mac
rophages (arrow head) were
positive for both ICAM1 and
CXCL1. The magnifications were
500.

onNovember11,2014

TheJournalofImmunology
TLR2 is involved in the
recognition of diverse bacteria
and their products, including
Grampositive bacteria and
peptidoglycans,whereasTLR4
has been identified as the
receptorforLPS,anendotoxin
ofGramnegativebacteria(22).
Blocking antiTLR4 Abs did
notinhibitkpOmpAbindingto
BEC. kpOmpAmediated
activation is not due to LPS
because endotoxin is
undetectable in kpOmpA
preparation and polymyxin B
does not neutralize kpOmpA
effectsonbronchialepithelium.
Using TLR2transfected cells
and DC from TLR2deficient
mice, Jeannin et al. (14).
demonstrated that TLR2 is
FIGURE8.kpOmpAupregulatesCXCL1,CXCL5,CCL2,CXCL10,ILinvolved in kpOmpAmediated
6, and ICAM1 mRNA levels in the lung parenchyma after local ad signalingbutisnotrequiredin
ministrationof kpOmpA.Mice were primedwithasingleintratracheal kpOmpA binding to and
administrationofBSA(thin)orkpOmpA(bold)(100g).Therightlung
endocytosis
wasprocessedtoassesscytokinemRNAexpressionbysemiquantitative
RTPCR,at6,24,and48hafterinstillationincomparisonwithcontrol
mice(t0).mRNAexpressionforCXCL1,CXCL5,CCL2,CXCL10,IL6,
andICAM1andactinwasanalyzed.Dataareexpressedasthemeanof
theratiogeneofinterest/actinforthreemiceandarerepresentativeof
oneofthreeseparateexperiments.

factors as well as adhesion molecule expression. Although mac

rophages may initially encounter bacteria, the microorganisms are
alsoabletocolonizeairwayepithelium.Increasingevidenceshows
thatBECarealsoimplicatedinthecontrolofthelocalinflammatory
reaction (1, 2). In this way, among pathogenassociated molecular
patterns, different bacterial membrane components, including LPS
andglycoproteins,havebeenshowntoactivateBEC.Inthisstudy,we
demonstratedthatkpOmpAbindstoandactivatesBECparticularly
through the NF B pathway. This activation induces cytokine and
chemokine production and enhances ICAM1 expression. In vivo
intratracheal kpOmpA injection induces an inflammatory reaction
associated with BEC activation as shown by ICAM1 and CXCL1
expressiononepithelialcells.
First,wereportthatkpOmpAbindstoBECinasaturablefashion
to similar levels as observed with monocytederived DC (data not
shown),suggestingatleastthatoneofitsreceptorsisexpressedon
epithelial cells. The innate immune system recognizes diverse
pathogenassociated molecular patterns by members of the TLR
family,leadingtoaninflammatoryreaction(21).TLR2andTLR4
are expressed by numerous cell types such as monocytes,
macrophages, DCs, endothelial cells, and mucosal epithelial cells.

6703
n
e
M
o

FIGURE 9. Intratracheal
injection of kpOmpA in mice
increases IL10, IFN , TNF
CXCL1, and CXCL5 levels in
BAL fluids.Micewereprimed
by a single intratracheal
administrationofkpOmpA(100
g).IL12,IL10,IFN,TNF,
CXCL1, and CXCL5 levels in
BAL fluids were assessed by
ELISA,6h(u)or24h(f after
instillation, in comparison with
naivemice().Datashownare
representative of one of three
separate experiments, and are
expressedasthemeansSEMof
threemice.,p0.05and,p0.01
comparedwithBSA.

byDC(14,20).Wealsoshow
herethatblockingantiTLR2
Abs do not affect kpOmpA
binding to BEC. These
observations suggest that
endocyticreceptor(s)mightbe
responsibleforthecaptureof
kpOmpA and for the
subsequent mobilization of
TLR2 to activate the
signaling cascade (23).
Nevertheless, activation of
airway epithelial cells by
TLR2 ligands does not

Downloadedfrom http://www.jimmunol.org/byguestonNovember11,2014

6704
factorsforneutrophilsthroughtheinteractionwithCXCR1
and2
(5). CCL2 and CXCL10 are implicated in the recruitment of
mononuclear cells through the interaction with CCR2 and
CXCR3. Both chemokines are involved in the recruitment of
monocytes/macrophages and they are also chemotactic for
different T cell subsets including memory T and Th1 cells,
respectively(27).

Inourinvivomodel,intratrachealkpOmpAadministration
induced mRNA expression of several chemokines in the
lung, including CCL3 (macrophage inflammatory protein
1), CXCL1, CXCL5, andCXCL10.Inthemurinemodel,
kpOmpA instillation caused an important and transient
accumulationofneutrophilsinlungandBALfluids,probably
mediatedbytheproductionofchemoattractantfactorssuch
as CXCL1 and CXCL5. Interestingly, CXCL5 production
seems to be delayed (maximal at 24 h) compared with
CXCL1secretion(at6h),suggestingthatthesechemokines
act sequentially in this model. Both CXCL1 and CXCL5
potentlyactivateneutrophilsviathemurineCXCL8receptor
homologue,resultinginincreasedMac1expressionandin
respiratoryburstactivity(2830).Therecruitmentandthe
subsequentactivationofneutrophilsplayakeyroleinlung
bacterialclearanceandimprovedsurvival(31,32).CXCL5is
predominantly produced by fibroblasts and lung epithelial
cells. Macrophages are reported to be the main source of
CXCL1 (33, 34). However, immunochemistry analysis
showedthatbothmacrophagesandBECarestronglypositive
forCXCL1expressionafterexposuretokpOmpA,suggesting
that both cell types are involved in this process. The
adhesion molecule ICAM1 participates in leukocyte di
apedesis by mediating firm adhesion to endothelial or
epithelialcellsandbythiswayisinvolvedinhostdefense
(35). We also reported that ICAM1 expression is up
regulatedafterkpOmpAexposureinalveolarandbronchial
epithelium and in endothelium, witness of the local

inflammation.InhumanBEC,thereissomespecificityinthe
kpOmpAderived adhesion molecule expression, because
kpOmpAaloneorinpresenceofTNFdoesnotmodulate
VCAM1expression(datanotshown)incontrasttoICAM1.
Therefore,thesedatademonstratedthatchemokinesecretion
andadhesionmoleculeexpression,inresponsetokpOmpA,
specificallydirectneutrophilmigrationacrosstheepithelial
barrieratinfectedmucosalsites.
The in vivo production of inflammatory and
immunostimulatory cytokines, such as TNF and IL10, was
investigated in the BAL. In lung, TNF is predominantly
producedbymacrophages,butBECmayalsosecreteit(1).This
cytokine improves lung bacterial clearance through an up
regulation of adhesion molecule expression and secretion of
chemokines including CXCL1 (36). In our study, kpOmpA
inducesaweakproductionofTNFbyBEC,whereasIFNand
IL10 remain undetectable in BEC supernatants (data not
shown).Inaddition,kpOmpApotentiatestheeffectsofTNF,
and TNF IFN on CXCL8 and CCL2 production by BEC.
kpOmpAactivatesmacrophages,whichproducehighamountsof
TNF (16). In turn, these mediators may expand macrophage
activation. So, in vivo, this may result in a proinflammatory
amplificationloopbetweenmacrophagesandepithelialcells.In
contrast, IL10 is detrimental to innate and cellmediated im
munity in the lung, particularly by blocking the synthesis of
proinflammatorycytokines,includingIFN,IL1,TNF,IL12,
andchemokineproduction(37).Invivo,neutralizationbyanti
IL10Abresultsinanenhancedbacterialclearance(38)whereas
IL10secretionservestolimittheproductionofproinflammatory
cytokinesinthismodel(38,39).Duringinfection,IL10seems
tohaveadualeffect:detrimentalonthebacterialclearanceand
beneficialontheinflammationasshowninsepticperitonis(40).
Inourstudy,IL10productionreachesitsmaximumat6hand
persists at 24 h, whereas the production of chemokines and
proinflammatorycyto

kpOmpAAC
ki
n
es
re
tu
rn
e
d
to
b
as
el
in
e.
T
h
es
e
d
e
m
o
ns
tr
at
e
d
th
at
k
p
O
m
p
A
in

duced inflammatory burst was associated with the

productionofantiinflammatorymediators(IL6andIL10).
Thedifferentcellpopulationsinthemucosalbarrierare
equippedtosenseandrespondtothemolecularcontentsin
the lumen and to translate this molecular information into
signalsthatcanreachlocalordistantsiteswithinthebody.In
additiontoitseffectonBEC,kpOmpAinteractsandactivates
DC, inducing their maturation and delivering Ag into the
MHCclassIpresentationpathway(14).However,theIL12
productioninducedbykpOmpAinmacrophagesandDCis
modest compared with that obtained after LPS or CD40
dependentactivation(16).Inthiscontext,thelackofIL12
secretioninBALfluids,afterkpOmpAtreatmentmaybedue
tothecaptureofthiscytokinebyIL12Rpositivecells.In
vivo IL12 secretion by kpOmpAactivated APC may also
requiretheactionofacoactivator.Nevertheless,IFNlevels,
whose production can be induced by IL12, are increased
after kpOmpAinstillation. As IFNproduction isobserved
after6h,kpOmpApresumablydirectlyinducesitsproduction
by immunocompetent cell activation. Further studies are
required to elucidate this point. Because IFN plays an
essential role in the development of adaptative immunity
(41),kpOmpAmayorchestratethetransitionfrominnateto
adaptive immunity, and the polarization of this immune
responsetopromotecellmediatedimmunity.
Inconclusion,thepresentdataidentifyanewmechanism
by which the bronchial epithelium is involved in anti
infectious defense. BEC exposed to kpOmpA show an
increased expression of adhesion molecules and an up
regulationofchemokinesecretion, whichareimplicatedin
vivo in inflammatory cell recruitment toward the lung. In
addition to endotoxin and glycoprotein, OmpA is also an
importantcomponentofthebacterialwallimplicatedinthe
developmentofadefensemechanism.

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We greatly thank Philippe Marquillies for his helpful technical

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