Tim1 and Tim3 are not essential for experimental allergic asthma.

0.89 MB·English

by  Barlow, J L

doi:10.1111/j.1365-2222.2011.03728.x Clinical&ExperimentalAllergy,41,1012–1021
ORIGINAL ARTICLE ExperimentalModelsofAllergicDisease (cid:2)c2011BlackwellPublishingLtd
Tim1 and Tim3 are not essential for experimental allergic asthma
J.L.Barlow1,S.H.Wong2,S.J.Ballantyne3,H.E.Jolin1andA.N.J.McKenzie1
1MRCLaboratoryofMolecularBiology,Cambridge,UK,2MedImmune,GrantaPark,Cambridge,UKand3DepartmentofBiologyandHullYorkMedicalSchool,Centre
forImmunologyandInfection,UniversityofYork,York,UK
Clinical & Summary
BackgroundInitialstudiessuggestedthatpolymorphismsinTim1andTim3contributetothe
Experimental
developmentofairwayhyperreactivity(AHR)inanacutemousemodelofasthma.Thiswas
Allergy alsomirroredinhumangeneticstudieswherepolymorphismsinTim1andTim3havebeen
associatedwithatopicpopulations.
Objective Furtherstudies using anti-Tim1or -Tim3antibodies, or Timfusion proteins, have
alsosuggestedthatthesemoleculesmayfunctionasregulatorsoftype-1andtype-2
immunity.However,theirroleinthedevelopmentofAHRandairwayinflammationremains
unclear.GiventheproposedrolesforTim1andTim3intype-1andtype-2responses,we
soughttodeterminewhetherthesemoleculeswereimportantinregulatingantigen-driven
lungallergyandinflammation.
Method We used Tim1- and Tim3-deficient mice and determined how the development of
allergiclunginflammationwasaffected.
Results AHR was induced normally in the absence of both Tim1 and Tim3, although
Tim1-deficientmicedidshowasmallbutsignificantdecreaseincellinfiltrationinthelung
Correspondence:
DrJillianL.Barlow,MRCLaboratoryof andbloodeosinophilia.AlthoughTim3wasexpressedonCD41Tcellsintheallergiclung,
MolecularBiology,HillsRoad, Tim1expressionwasrestrictedtoCD861Bcells.
Cambridge,CB20QH,UK.E-mail: ConclusionsandclinicalrelevanceThus,Tim1andTim3arenotessentialfortheinductionof
[email protected] thetype-2responseinlungallergy.Thisiscontrarytowhatwasproposedinanumberof
Citethisas:J.L.Barlow,S.H.Wong,
otherstudiesusingneutralizingandactivatingantibodiesandquestionstheclinicalrelevance
S.J.Ballantyne,H.E.Jolinand
ofTim1andTim3fornovelallergytherapies.
A.N.J.McKenzie,Clinical&
ExperimentalAllergy,2011(41) Keywordsairwayhyperreactivity,asthma,eosinophils,Tim1,Tim3,type-2
1012–1021.
Submitted26November2010;revised7January2011;accepted28January2011.
which conferred susceptibility to ovalbumin (OVA)-in-
Introduction
duced AHR in an experimental mouse model of asthma
Asthmais acomplexchronic, inflammatory lungdisease [7].PolymorphismsinTim(Tcellimmunoglobulinmucin)
and is the most common chronic childhood disease 1(Havcr1)andTim3(Havcr2),twomembersofanewgene
affectingdevelopednations.TheTh2cytokinegeneclus- family discovered within the Tapr locus, were found to
ter,locatedonhumanchromosome5q31-35,isclearlyan correlate with AHR and Th2 cytokine production,
important genetic determinant in asthma susceptibility. although the relative importance of Tim1 and Tim3
Interleukin 4 (IL-4), IL-5, IL-9, and IL-13 have all been to this phenotype was unclear. McIntire et al. [8] have
shown to be essential components of the asthmatic since reported that atopic resistance can be correlated
response, includingairwayhyperreactivity(AHR),mucus with hepatitis A virus seropositivity and an insertion
hypersecretion,andBcellisotype-switchingtoIgE[1–6]. mutation in human TIM1, previously identified as the
However, mapping studies have also identified the Tapr hepatitisAvirusreceptor(HAVCR1),suggestingthatTIM1
(T cell and airway phenotype regulator) locus on mouse may provide a biological basis for the link between
chromosome 11, linked to the Th2 cytokine gene cluster, decreasingexposuretoHAVandtheincreasingincidence
of atopy in westernized society. Associations between a
Re-useofthisarticleispermittedinaccordancewiththeTerms number of mutations in both TIM1 and TIM3 and atopic
and Conditions set out at http://wileyonlinelibrabray.com/ disease have been found in other human asthma studies
onlineopen#OnlineOpen_Terms [9,10].
Tim1andTim3inlungallergy 1013
Administration of a Tim1-human immunoglobulin CCCTTTCCTACCTAGC-30 and50-GGACAGGATCCCTTGCC
(Tim1HuIg) fusion protein, or ligation of Tim1, either by CCAGCACATAGGCACAAGTG-30.The2.0kb30homology
a proposed counter-ligand in the form of a Tim4HuIg arm was generated using RPCI21 PAC488-E20 as a tem-
fusionprotein[11],oranagonisticanti-Tim1(clone3B3) plate with PCR primers 50-ATTAGAACTAGTATT AGA-
monoclonalantibody[12],resultedinthecostimulationof CATCAAAGCAGGTGAGTAG-30 and 50-AATCCTACTAGT
Tcell activation and the enhancement of IL-4 and IFN-g GGCCTATATGGTAGATAACTATATG-30. The Havcr2 tar-
productionbyCD31Tcells.Invivoadministrationofthe getingvectorwaslinearizedandelectroporatedintoE14.1
anti-Tim1 antibody (clone 3B3) prevented the develop- 129J EScellsand used togenerate micehomozygous for
ment of respiratory tolerance and increased pulmonary the gene by standard methods.Tim3(cid:3)/(cid:3)mice, and corre-
inflammation [13], suggesting that blocking Tim1 func- spondingwild-typeanimals,werederivedfromtheorigi-
tion may suppress type-2 responses such as those ob- nal chimeras. Sequencing of Tim1 and Tim3 transcripts
served in allergic inflammation. Indeed, Encinas and from BALB/c and 129 sources identified no polymorph-
colleagues showed a down-regulation of OVA-induced isms between these strains; thus, mice were backcrossed
allergic inflammation following anti-Tim1 antibody forsixgenerationstotheBALB/cstrainbeforeintercross-
(clone 222414) treatment. However, recent reports have ing to generate homozygous mice. Genotypes were
demonstratedthattheinteractionsofantibodiesrecogniz- screened by southern analysis or PCR using primers,
ing different epitopes on Tim1 can mediate diametrically 50-TGGTAAGAATGCCTATCTGCC-30and50-TCTGTAGAC-
opposite effects. Anti-Tim1 antibodies, distinguishing CATATCCTGGAGTTC-30. The oligonucleotide specific to
specific epitopes in both the immunoglobulin (Ig) region the inserted neomycin cassette was 50-CTATCAGGACA-
and the mucin domain, were shown to reduce lung TAGCGTTGGCTACC-30. All mice were maintained in a
eosinophiliaandlunginflammation,whereasanantibody specific pathogen-free environment in SABU/CBS facil-
directed at a different epitope in the mucin domain itiesaccordingtoUKHomeOfficeregulations.
actually exacerbated the allergic response [14]. Further-
more, even anti-Tim1 antibodies recognizing the same
RNApreparation
epitope (clones 3B3 and RMT1-10), but with different
avidities for Tim1, have been reported to elicit opposite RNA was prepared from na¨ıve tissues and from in vitro
functional effects [15]. Administration of a neutralizing differentiated T helper cells [17] using RNAzolB, accord-
anti-Tim3 antibody (clone 8H7), although ineffective ing to the manufacturer’s instructions (AMS Biotechnol-
when administered during an OVA sensitization and ogy, Oxford, UK). Tim3 RT-PCR primers were as follows:
challenge model of asthma, was able to induce a partial 50-AGGTCATTGGAAAATGCTTATGTGTTTGAG-30 and
decrease in AHR and Th2 cytokine production following 50-CAGTAGGTCCCATGGTCATCC-30, 50-CTGCTGCAGGA
the transfer of OVA-specific in vitro differentiated Th2 TACAGTTCCC-30 and 50-GAAATTAAGGACTCTGGAGA
cells[16]. AACGATCAGAAC-30, 50-GAAATTAAGGACTCTGGAGAA
Thus, studies to date leave an unclear picture as to the ACGATC-30 and 50-GCTACGTCAACAGAAAGCAGCCAT
importance of Tim1 and Tim3 in allergic asthma re- CC-30. HPRT primers and conditions were as described
sponses. To clarify the functional requirement for Tim1 previously[18].
and Tim3, we have analysed Tim1- and Tim3-deficient
mouse lines and assessed the potential roles of these
Flowcytometry
molecules in an in vivo model of antigen-driven acute
lungallergy. Lung cell suspensions, treated with 720mg/mL collage-
nase D (Amersham, Bucks, UK), were analysed for the
expressionofTim1andTim3byflowcytometryusingthe
Materialsandmethods
FACScalibur (BD, Biosciences, Oxford, UK) and FlowJo
analysis software. Biotin-conjugated anti-Tim1 (clone
TargeteddisruptionofthemouseTim1andTim3genesin
RMT1-4, eBioscience SanDiego, CA, USA), biotin-conju-
embryonicstemcells
gated anti-Tim3 (clone 8B.2C12, eBioscience), and PE-
The Havcr1 (Tim1(cid:3)/(cid:3)) mice were generated as described conjugatedstreptavidinwereused.
previously[17].TheHavcr2(Tim3(cid:3)/(cid:3))replacementvector
was constructed to insert the neomycin resistance gene
Sensitizationandallergenexposure
into exon 2 of the Havcr2 gene. This deleted the nucleo-
tidesencoding71aminoacids(Phe57–Lys127)ofthe281 BALB/cmice(6–12weeks)weresensitizedbyanintraper-
aminoacidcodingsequenceoftheTim3gene.The4.1kb itonealadministrationofOVA(20mg/injection,endotoxin
50 arm of homology was generated using E14.1 129J low – removed using Affiniti Pak Endotoxin Removing
embryonic stem (ES) cell DNA as a template with PCR Gel Columns (Pierce, Rockford, IL, USA) according to
primers 50-CAGCAATGCGGCCGCGTAAGCCAAATAAA the manufacturer’s instructions) in PBS (endotoxin free
(cid:2)c2011BlackwellPublishingLtd,Clinical&ExperimentalAllergy,41:1012–1021
1014 J.L.Barlowetal
phosphate buffered saline, Sigma-Aldrich Dorset, UK), QuantikineELISAkit(R&DSystems,Abingdon,UK).OVA-
complexed in a 1:1 ratio with alum (Imjects, Thermo specificserumIgELISAswereundertakenusingimmuno-
Scientific, Loughborough, UK), at days 0 and 12. Control sorbent plates coated with 5mg/mL OVA, overnight, and
micereceivedPBSonlywithalum.Aerosoladministration thenasdescribedpreviously[17].
of 1% OVA was undertaken on days 19, 20, and 21 for
20min/day.ControlanimalsreceivedPBS.
Statisticalanalysis
The significance of the differences between experimental
Measurementofairwayresistance
groupswasanalysedusingatwo-wayANOVAwithBonfer-
On day 22, animals were analysed using invasive lung roni post-test (for dose response to methacholine) or the
function measurements (EMMS, Bordon, UK) to assess Studentunpairedt-test.
AHR. Animals were anaesthetized, tracheostomized, and
placedonaventilator(MiniVent845ventilator,EMMS)at
Results
arateof150breaths/min,withatidalvolumeof0.15mL.
After recording stable baseline pulmonary resistance for
Allergiclunginflammationcanbeinducedintheabsence
3min, increasing concentrations of acetyl-b-methylcho-
ofTim1
line chloride (methacholine) (Sigma-Aldrich) were admi-
nisteredbyaerosolfor10sateachconcentrationusingan Airway obstruction is the most debilitating symptom of
ultrasonic nebulizer, and pulmonary resistance was re- asthmaandisaresultofexcessivecontractionofsmooth
corded for a 3-min period. eDaq software was used to muscle. Bronchoconstriction can be determined during
analyseairwayresistance. allergiclunginflammationusinginvasiveplethysmogra-
phy to directly measure airway resistance in response to
lungagonists,e.g.methacholine.Toinvestigatearolefor
Tissuecollection
Tim1inallergicdisease,wildtypeandTim1(cid:3)/(cid:3)micewere
Lungswerefixedinformalin(10%formaldehydeina0.9% first sensitized intraperitoneally and then challenged in
salinesolution)andstainedwithGiemsa,forinflammatory the lung with aerosolized OVA to induce a type-2
infiltrate,andperiodicacid-Schiff(PAS)formucus-produ- mediatedinflammatoryresponse.Asexpected,theairway
cinggobletcells.Airwaymucusproductionandnumbersof resistance in OVA-treated wild-type mice increased com-
infiltratingcellsaroundbloodvesselsweremeasuredblind pared with PBS-treated control mice (Fig. 1a). Tim1(cid:3)/(cid:3)
using numerical scoring systems. Mediastinal lymph node micedisplayedaminor,althoughstatisticallysignificant,
cells were re-stimulated with 100mg/mL OVA or 1mg/mL reductioninairwayresistanceatthe20mg/mLmethacho-
purifiedanti-CD3andcellsupernatantsweretakenata72h line dose, but overcame this deficit at higher doses
time-point.BALfluidwascollected,accordingtopreviously (40–80mg/mL).
describedprotocols[19]. We also investigated the requirement for Tim1 during
the allergic lung inflammatory response. The total cell
counts from the bronchoalveolar lavage (BAL) showed
Differentialwhitebloodcellcountsand3[H]-thymidine
that the numbers of lymphocytes, macrophages, eosino-
cellproliferationassay
phils, and polymorphonuclear leucocytes (PMNLs) were
Blood smears were stained with Giemsa (Sigma-Aldrich), equivalent in the PBS-treated Tim1(cid:3)/(cid:3) and wild-type
following the manufacturer’s instructions. Differential mice(Fig.1b).OVAtreatmentledtoaminor,butstatisti-
cellcounts weremadeblind on100whitebloodcellsper cally significant, decline in lymphocyte and eosinophil
slide under oil and at a (cid:4)63 magnification. Blood was cell numbers in the BAL of Tim1(cid:3)/(cid:3) mice, as compared
incubated at 41C overnight and serum was collected by with wild-type mice (Fig. 1b). However, eosinophils, the
centrifugation. Measurement of cell proliferation was maininfiltratingcell,stillincreasedsignificantlyinOVA-
undertaken as described previously [17] with or without treatedTim1(cid:3)/(cid:3)micecomparedwithPBS-treatedcontrol
30mg/mL IgM (AffiniPure F(ab0) , m chain specific, Jack- mice(Fig.1b).
2
sonLaboratories,BarHarbor,ME,USA). To explore whether the reduction in infiltrating cells
resulted from an inability to migrate or home efficiently,
or from a deficit in circulating eosinophils, we examined
Cytokineandserumimmunoglobulinenzymelinked
blood cell composition. Differential blood cell counts of
immunosorbentassays
lymphocytes, monocytes, eosinophils, and PMNLs were
CytokineELISAutilizedthesandwichformatwithcapture performedonsamplesfromthePBS-andOVA-challenged
and detection antibodies purchased from Becton Dickin- mice.Bloodeosinophilsconstituted(cid:5)2%ofleucocytesin
son. ELISAs were performed according to the Becton wild-type and Tim1(cid:3)/(cid:3) PBS control mice. In wild-type
Dickinson ELISAprotocol. IL-13was measured using the OVA-treated mice, this was increased to around 12%.
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Tim1andTim3inlungallergy 1015
Fig.1.(a)AirwayresistanceinwildtypeandTim1(cid:3)/(cid:3)micemeasuredusinginvasiveplethysmography.Resultsareshownforlungresistanceabovethe
baselinereading.(cid:2)(cid:2)(cid:2)P=o0.001betweenwild-typePBSandovalbumin(OVA)-treatedmice.‰P=o0.05betweenwild-typeandTim1(cid:3)/(cid:3)OVA-treated
mice.(b)DifferentialcellcountswereperformedontheBALfluidin250mLfromwild-typeandTim1(cid:3)/(cid:3)mice.(c)Thepercentagesofeosinophilsin
peripheralbloodwereassessedbydifferentialcellcounts.(d)Cellularinfiltrationand(e)mucusproductiondeterminedbyanalysisoflungsections
stainedwithGiemsaorperiodicacid-Schiff,respectively.(cid:4)20magnification.Dataarerepresentativeoftwotothreerepeatexperiments(n=8–10).
However, OVA-treated Tim1(cid:3)/(cid:3) mice failed to develop AsTim1isreportedlyexpressedonCD41Th2cells,we
equivalentlysevereeosinophilia(Fig.1c).Nootherblood investigated the proliferation and cytokine responses in
cell types were altered by the Tim1 deficiency (data not OVA-restimulated cells cultured from the draining med-
shown). The coincident decreasein the levelsof circulat- iastinal lymph nodes. Cells from OVA-challenged mice
ing eosinophils and lung infiltrating eosinophils implies produced very little antigen-specific IL-2 and no IFN-g
thatthedeficitincellularinfiltratearisesfromthereduc- (datanotshown),butelevatedlevelsoftheTh2-cytokines
tionincirculatingcellsratherthananinabilitytomigrate IL-4 and IL-5 (Fig. 2a). The levels of IL-5 were slightly
tothelungs. reduced in the Tim1(cid:3)/(cid:3) samples, but this did not attain
The deficit in cellular infiltrate observed in the BAL statistical significance. Although IL-4 could not be de-
of Tim1(cid:3)/(cid:3) mice was also mirrored in the lung tissue. tectedintheBALfluid,OVA-inducedIL-5andIL-13levels
Here, the perivascular cell infiltrate score was again were found to be normal in Tim1(cid:3)/(cid:3) mice (Fig. 2b). The
significantly lower, but not completely abrogated, in proliferation of mediastinal lymph node cells from wild-
the Tim1(cid:3)/(cid:3) lungs (Fig. 1d). It is not clear whether this typeandTim1(cid:3)/(cid:3)mice,inresponsetoOVA,alsodemon-
relates to a decrease specifically in lung eosinophils. stratednoappreciabledifferencesbetweentheOVA-chal-
Mucus production, identified by PAS staining of airway lenged groups (data not shown). Collectively, these data
epithelium, was similar in the OVA-treated wild type and show that OVA-induced Tcell proliferation and cytokine
Tim1(cid:3)/(cid:3)groups(Fig.1e). responses do not appear to be significantly affected by
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1016 J.L.Barlowetal
Fig.2.Ovalbumin(OVA)-specificandpolyclonalcytokineproductionwasdeterminedfrom(a)restimulatedmesentericlymphnodecellculturesand(b)
BALfluidsamplesbysandwichELISA.Dataarerepresentativeoftworepeatexperiments(n=8–10).
Tim1 deficiency during the type-2-driven allergic lung the Tim1 expression during lung allergy coincides with
response. CD86 expression on the B cells, suggesting that it is
expressed on activated B cells capable of co-stimulation
of T cell activation (Fig. 3c). Although B cells have not
Tim1ispredominantlyexpressedonactivatedBcells,not beenshowntobeessentialforallergiclunginflammation,
Tcellsduringallergiclunginflammation the expression of Tim1 on CD191CD861 cells led us to
investigate whether Tim1(cid:3)/(cid:3) mice had defects in B cell
The lack of a demonstrable effect on type-2 cytokine function following the induction of lung allergy. To test
production is at odds with the existing dogma regarding this, the production of OVA-specific serum Igs was ana-
Tim1bioactivityanditsreportedexpressiononTh2cells. lysed. In wild-type mice, OVA treatment induced a large
Thus, we undertook extensive studies to demonstrate increase in OVA-specific IgG and IgE antibodies, and
1
convincing staining for Tim1 on the surface of na¨ıve T some IgA and IgG antibodies, as well as an increase in
2b
cellsordifferentiatedTh1orTh2cells.Duringouranalysis totalIgEproduction(Fig.3dande).Nodefectswerefound
of the cell surface expression of Tim1, we used the anti- in serum antibody production during allergic lung in-
Tim1 antibody RMT1-4 (16) due to it consistently recog- flammation in Tim1(cid:3)/(cid:3) mice. Similarly, the proliferative
nizing Tim1 as assessed by its binding to native Tim1, response of B cells from Tim1(cid:3)/(cid:3) mice following IgM
transgenicallyexpressedTim1ofeithertheBALB/corthe stimulationinvitrowasalsofoundtobenormal(Fig.3f).
C57BL/6allotype(datanotshown),anditsfailuretostain Therefore,Bcellfunctionwasnotfoundtobeaffectedby
cellularproductsintheTim1-deficientmice(Fig.3a). deficiencyinTim1.
Significantly, we were unable to find any detectable
Tim1 expression by flow cytometric analysis on
Tim3isexpressedonCD11c1myeloidcellsand
CD31CD41 or CD31CD81 T cell populations in either
CD41CD251Tcellsduringallergiclunginflammation
na¨ıve or allergic lung tissue (data not shown). However,
further investigation of the expression pattern of Tim1 Tim3,likeTim1,hasalsobeenassociatedwithanumberof
showed that it was detectable on CD4(cid:3)CD8(cid:3) cells, and humandiseasesincludingmultiplesclerosisandrheuma-
restricted within that population to CD191 B cells from toid arthritis, and has been shown to be expressed on
na¨ıvelungtissue(Fig.3aandb).Wefoundnoevidencefor CD41 Th1 cells in mice. Components of the type-1
Tim1expressiononanyothercellpopulationatthistime- immune response have previously been shown to affect
point. Furthermore, Tim1 was up-regulated on B cells theallergiclungresponse.Toaddressthepotentialroleof
after induction of allergic lung inflammation (Fig. 3b). Tim3inexperimentalasthma,wedevelopedamouseline
Additionally, further phenotypic analysis indicated that deficient for Tim3 (Havcr2) by gene targeting in ES cells
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Tim1andTim3inlungallergy 1017
Fig.3.(a)MeanfluorescenceintensityofTim1expressiononCD4(cid:3)CD8(cid:3)inthelungasassessedbyflowcytometry.(b)Leftpanel,histogramofCD19(cid:3)
andCD191gatedpopulationsinthelung.Middleandrightpanel,Tim1expressionwithintheCD19(cid:3)gateandCD191gate,respectively,inthelung.
Greyline,PBS-treatedmice;blackline,ovalbumin(OVA)mice;filledgrey,isotypecontrol.(c)OVA-specificIgA,IgE,IgG ,andIgG ,and(d)totalIgE
1 2b
productioninserum.(e)MediastinallymphnodecellproliferationinresponsetomediaonlyorIgM,measuredbytheincorporationof3[H]-thymidine.
Dataarerepresentativeoftworepeatexperiments(n=8–10).
(supporting information Fig. S1). Tim3(cid:3)/(cid:3) mice were in OVA-treated mice (Fig. 4b). Thus, although Tim3 has
healthyanddisplayednoovertphenotypicabnormalities. beenassociatedinpreviousreportswithtype-1immunity,
WefirstwantedtoascertainTim3expressionduringthe ourdatashowthatTim3isup-regulatedduringthetype-2
allergic response. Lung tissue was taken from wild-type responseinallergiclunginflammation.
and Tim3(cid:3)/(cid:3) mice, in which lung allergy had been
induced. Tim3 expression in these mice was compared
with that in PBS control mice. No differences in the Tim3isnotrequiredfortheinductionofallergiclung
percentage of CD41 or CD81 T cells, CD191 B cells, or inflammation
CD11c1 alveolar macrophages were observed between
wild-type and Tim3(cid:3)/(cid:3) mice (data not shown). Tim3 was As Tim3 was expressed in the lung during allergic
expressed in na¨ıve wild-type mice only on CD11c1 lung responses, we next wanted to test its functional signifi-
cells (Fig. 4a). The expression of Tim3 was increased on canceinthoseresponses.Wewereunabletomeasureany
CD11c1 cells in OVA-treated mice. Although CD11c is detectabledifferencesinairwayresistance,comparedwith
normally considered to be a surface marker for dendritic theOVA-treatedwild-typemice,causedbytheabsenceof
cells, in the lung, it is expressed mostly on alveolar Tim3(Fig.5a).
macrophages. Tim3 expression was also found on In agreement with the airway resistance data, the
CD41CD251cells.Notably,thesecellswereonlyobserved absence of Tim3 also exerted no detectable effect on the
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1018 J.L.Barlowetal
Fig.4.(a)HistogramsshowingTim3expressiononCD11c1gatedcellsand(b)TcellsinthelungsofmicetreatedwithPBSorovalbumin(OVA).Tcells
weregatedasbeingCD41CD251/(cid:3)byflowcytometry.Averagepercentagesforthreemicepergroupareshown.Regulartext,wildtype;italictext,
Tim3(cid:3)/(cid:3).Dataarerepresentativeoftworepeatexperiments.
lunginflammatoryresponse.Cellularinfiltrationintothe duction were also found to be normal in the Tim3-
lung, as assessed by BAL cell counts and lung histology, deficient mice, as were type-2 cytokine responses in the
was comparable to wild-type controls (Fig. 5b and c). BAL fluid, as measured by IL-5 production (Fig. 5d–f).
Furthermore, blood eosinophilia and airway mucus pro- Cytokineproductionfromdraininglymphnodecells,and
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Tim1andTim3inlungallergy 1019
Fig.5.(a)Airwayresistanceinwild-typeandTim3(cid:3)/(cid:3)measuredusinginvasiveplethysmography.Resultsareshownforlungresistanceabovethe
baselinereading.(b)DifferentialcellcountswereperformedontheBALfluidin250mLfromwild-typeandTim3(cid:3)/(cid:3)mice.(c)Cellularinfiltrationand(d)
mucus production determined by analysis of lung sections stained with Giemsa or PAS, respectively. (cid:4)20 magnification. (e) The percentages of
eosinophils in peripheral blood were assessed by differential cell counts. (f) IL-5 production in the BAL fluid as measured by ELISA. Data are
representativeoftwotothreerepeatexperiments(n=8–10).
serumIgproduction,includingOVA-specific IgEproduc- CSF, may be suppressed at other time-points in the
tion, were also unchanged by the absence of Tim3 (data response, thereby underlying this deficit in circulating
notshown). eosinophils.
Encinas et al. [13] previously demonstrated, using an
anti-Tim1 mAb (clone 222414), that pulmonary inflam-
Discussion mation, and IL-10 and IL-13 levels were reduced follow-
ing anti-Tim1 administration, suggesting that Tim1 was
Using Tim1(cid:3)/(cid:3) and Tim3(cid:3)/(cid:3) mice, we have investigated required to induce normal allergic lung inflammation.
the role of these molecules in the regulation of type-2 Although we observed minor reductions in eosinophil
responses during lung allergy.Although previous reports infiltration, we did not detect a significant reduction in
have implicated both Tim1 and Tim3 as being important type-2 cytokine production from cells isolated from the
components of the T cell cytokine response, we now draininglymphnodesandTim1wasnotessentialforAHR
demonstrate that Tim1 and Tim3 are dispensable in orsubstantiallunginflammation.
regulatingAHRinamousemodelofallergicasthma. One striking observation made from our analysis of
Th2 cellresponses seem toremainlargely intactinthe na¨ıve mice, using Tim1-deficient mice as negative con-
absence of Tim1 and Tim3, although in Tim1(cid:3)/(cid:3) mice, trols for the cell surface staining of Tim1, was the
thereareminor,butstatisticallysignificant,reductionsin predominantexpressionofTim1onthesurfaceofaBcell
lung function measurements at low methacholine con- population,andnotTcellsduringlungallergy.Although
centrations,bloodeosinophilia,andlungcellinfiltration. theTim1transcripthasbeenreportedpreviouslyinBcell
It is unclear what underlies these small defects, but does cDNA from both the spleen and the lymph nodes in
suggestthatTim1islargelynotrequiredfortheresponse BALB/candC57BL/6mice,andTim1expressionhasbeen
to proceed. We were unable to detect a statistically notedonactivatedBcells,furthercharacterizationinvivo
significant deficit in OVA-specific IL-5 production from wasnotundertakeninthelung[17].WefoundthatTim11
thedraininglymphnodecells,orBALfluid,fromTim1(cid:3)/(cid:3) Bcellswerecharacterized asCD191CD861Bcells,prob-
mice.However,itremainspossiblethatlevelsofIL-5and ably representing a more activated B cell population.
eotaxin,orothergranulocytegrowthfactorssuchasGM- Moreover, following in vitro B cell activation with the
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1020 J.L.Barlowetal
polyclonal stimulator anti-IgM, Tim1 was found to be Ourresultsweresomewhatunexpected,givenprevious
expressedonalmostallproliferatingBcells,highlighting reportsregardingTim1,suggestingnotonlythatthegenes
the regulation of Tim1 expression on activated B cells encoding Tim1 and Tim3 within the Tapr locus, along
[17]. By contrast, we failed to observe the regulation of with their associated polymorphisms, may play a role in
Tim1 on T cells, although it is possible that Tim1 is regulatingpulmonaryfunction[7]butthatanti-Tim1and
expressed on T cells during other types of immune anti-Tim3 antibodies administered in vivo exerted wide-
response. ranging effects on T cell function during diabetes [20],
The role of up-regulated Tim1 in B cells remains transplant rejection [20], pulmonary inflammation [12],
uncertain, but the fact that it is induced by activation and EAE [21]. These differences mayreflect the variation
through the B cell receptor, but not via LPS stimulation in the biological effects that have been reported for the
[17], indicates that Tim1 is differentially regulated de- interaction of anti-Tim1 antibodies with agonistic and
pendingontheantigenencounteredandthatitmayplaya antagonisticepitopesonTim1[14].
roleinformingthesedivergentresponses.Followinglung Furthermore, it is possible that there is a functional
challenge,whileflowcytometricanalysisfailedtoidenti- redundancy between the Tim family members that also
fy Tim11 Tcells in the lungs, CD191Tim11 B cells were include Tim2, Tim4, Tim5, and Tim 6. Certainly, Tim2-
identifiedandthesewerealsoCD861.Thus,Tim1appears deficiency has been shown to exacerbate the allergic
to be a marker of a subset of activated B cells during in inflammatory response due to the up-regulation of type-
vivoimmuneresponsestoantigen.Despitetheexpression 2cytokinereleasebyTcells[22].However,therolesofthe
ofTim1onBcells,wefoundnoalterationinIgexpression otherfamilymembersinsuchresponsesareunknown.
orBcellproliferation.Wongandcolleaguesalsofailedto Genetic association studies in humans have indicated
find a role for Tim1 on B cells in antigen presentation in thatpolymorphismsinHAVCR1correlatewiththedevel-
thespleen. opmentofatopicdisease[8,23].Similarstudieshavealso
WealsofoundnomeasurableroleforTim3inthisOVA- failed tofindanysuchassociationinaJapanese popula-
induced model of allergic asthma, although unlike Tim1, tion [24]. We now demonstrate that although Tim1(cid:3)/(cid:3)
it was expressed on a population of CD41CD251 cells, miceshowaslightdecreaseinAHR,whichprobablyarises
which would classically be considered to be either T as a result of a broad-spectrum reduction in lung cell
regulatory cells or activated T cells. Our results contrast infiltrates, notably eosinophils, the absence of Tim1, or
with the report by Kearley et al. [16], in which OVA- indeedTim3,doesnotpreventtheallergiclungresponse,
induced airway inflammation was reduced following suggestingthattheymaynotbeclinicallyrelevanttargets
treatmentwithananti-Tim3antibodyandcorrelatedwith fornewtherapies.
a decrease in IL-5 expression. However, these differences
wereonlyobservedinamodelinvolvingthetransferofin
vitrogeneratedTh2cellsintona¨ıverecipients,andnotina
Acknowledgements
more conventional OVA-sensitization model of airway
inflammation [16]. Thus, although the modulation of WewouldliketothanktheSABU/CBSstaff,andparticu-
Tim3canhaveanimpactonatype-2response,itislikely larly Tracey Butcher, for their technical assistance. All
that the effects of inhibiting Tim3 vary considerably authors were supported by the MRC and declare no
dependingonthemodelused. conflictsofinterest.
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SupportingInformation
typeandTim3(cid:3)/(cid:3)Th1andTh2cells.Con,vectorcontaining
Additional Supporting Information may be found in the fulllengthTim3transcript;Neg,negativecontrol.(D)Flow
onlineversionofthisarticle: cytometricanalysisofTim3expressiononlungcellsuspen-
sionsfromOVAsensitisedwildtypeandTim3(cid:3)/(cid:3)mice.
Fig. S1. Inactivation of Havcr2 (Tim3) by homologous
recombination (A) The structure of the Tim3 locus, the Please note: Wiley-Blackwellis notresponsiblefor the
targetingvector,andpredictedhomologousrecombination content or functionality of any supporting materials
event. (B) Genotyping of genomic DNA from Tim3 gene- supplied by the authors. Any queries (other than missing
targetedmicebyPCRanalysis.(C)RT-PCRanalysisofTim3 material) should be directed to the corresponding author
expression,assessedusingexon-specificprimersetsinwild forthearticle.
(cid:2)c2011BlackwellPublishingLtd,Clinical&ExperimentalAllergy,41:1012–1021