Interferon-g mediated immune effector mechanisms against Bordetella pertussis

Immunology Le e s 68 (1999) 213–217 In e e on-gmedia ed immune e ec o mechanisms agains Bo de ella pe ussis Be na d P. Mahon a, *, Kings on H.G. Mills b a Mucosal Immunology Labo a o y,Biology Depa men ,Na ional Uni6e si y o I eland,Maynoo h,Kilda e,I eland b In ec ion/Immuni y G oup,Biology Depa men ,Na ional Uni6e si y o I eland,Maynoo h,Kilda e,I eland Recei ed 4 Feb ua y 1999; accep ed 15 Feb ua y 1999 Abs ac The ole o IFN-gin educing he in acellula load o Bo de ella pe ussis in mu ine mac ophages in i o has been examined. The esul s demons a e ha exposu e o IFN-gcan educe bac e ial load in iable mac ophages and ha his is associa ed wi h p oduc ion o ni ic oxide (NO). These obse a ions p o ide a mechanism by which IFN-gmay media e i s an imic obial e ec and suppo an impo an ole o ac i a ed al eola mac ophages in he elimina ion o B.pe ussis om he espi a o y ac . Using in acellula i on chela ion, i is shown ha in acellula su i al o B.pe ussis is dependan on i on a ailabili y and sugges ha i on es ic ion may be an impo an mechanism by which IFN-gin luences bac e ial su i al wi hin mouse mac ophages. I is also shown ha IFN-gmay media e i s e ec h ough NO independen mechanisms and ha B.pe ussis is sensi i e o agen s ha s imula e he espi a o y bu s . Finally, i is shown ha he concen a ion o L - yp ophan may be a limi ing s ep in he in acellula su i al o B.pe ussis and ha he induc ion o yp ophan deg ading enzymes may be an addi ional mechanism h ough which IFN-gexe s i s an imic obial e ec s agains B.pe ussis. © 1999 Else ie Science B.V. All igh s ese ed. Keywo ds : Pe ussis; In e e on-g; Ni ic oxide; I on; Indoleamine 2,3-dioxygenase 1. In oduc ion In ec ion o he espi a o y ac by he g am-nega- i e bac e ium Bo de ella pe ussis esul s in whooping cough, an impo an cause o mo bidi y and mo ali y in human in an s. I is well known ha du ing coloniza- ion o he espi a o y ac his bac e ium can speci i- cally adhe e o cilia ed epi helium, howe e , i is now clea ha B.pe ussis can exploi bo h ex acellula and in acellula niches du ing in ec ion. Pe sis ence o B. pe ussis wi hin mu ine and abbi al eola mac ophages has been desc ibed [1,2], and he abili y o B.pe ussis o in ade and su i e wi hin human mac ophages and o he cell ypes has ecen ly been documen ed by a numbe o g oups [3–6], sugges ing ha in acellula localiza ion may be an impo an mechanism in he disease p ocess. P o ec i e immuni y induced by p e ious in ec ion o immuniza ion wi h whole cell pe ussis (Pw) and new gene a ion acellula pe ussis (Pa) accines can p o ec agains whooping cough. The oles o B cells, T cells, and IFN-gin immuni y o B.pe ussis using no mal mice o gene knockou mice ha e p e iously been ex- amined, and i was demons a ed ha p o ec i e immu- ni y in ol es bo h cell media ed and humo al mechanisms [7–11]. Adop i e ans e o CD4 + Th1 cells om con alescen mice con e ed p o ec ion agains B.pe ussis challenge in T cell de icien a hymic mice p o iding e idence ha T cells play a signi ican ole in p o ec ion. Ae osol challenge o nai e IFN-g ecep o knockou mice esul ed in an a ypical dissemi- na ed disease, demons a ing an impo an ole o his cy okine in con ining he bac e ium o he espi a o y ac du ing in ec ion o nai e animals. Fu he mo e, s udies in wild ype mice and in Ig −/− mice p io o o a e bac e ial challenge e ealed ha p o ec ion ea ly a e immuniza ion wi h Pa is media ed by an ibody agains mul iple p o ec i e an igens [10]. Howe e , he * Co esponding au ho . Tel.: +353-1-7083835; ax: +353-1- 7083845. E-mail add ess : [email p o ec ed] (B.P. Mahon) 0165-2478/99/$ - see on ma e © 1999 Else ie Science B.V. All igh s ese ed. PII: S0165-2478(99)00070-X B.P.Mahon,K.H.G.Mills / Immunology Le e s 68 (1999) 213–217 214 mo e comple e p o ec ion con e ed by p e ious in ec- ion o ollowing immuniza ion wi h Pw e lec s he induc ion o Th1 cells [9]. In he p esen s udy he in i o mechanisms by which IFN-gmay media e hese e ec s ha e been examined. 2. Ma e ials and me hods 2 . 1 .G ow h o B.pe ussis B.pe ussis W28 phase I we e g own as p e iously desc ibed [10–12], all cul u es we e in exponen ial- phase g ow h a he ime o use. 2 . 2 .B.pe ussis in ec ion o mu ine mac ophages The mouse mac ophage-like cell line J774 in expo- nen ial g ow h phase was used in all s udies. Cells we e washed ou o cul u e medium and esuspended by agi a ion in a small olume o B.pe ussis anspo medium con aining eshly p epa ed bac e ia a a a io o 10 bac e ia pe mac ophage, a a io which did no induce mac ophage apop osis. A e a 1-h incuba ion, cells we e washed h ee imes in an ibio ic- ee RPMI 1640 (Gibco, Paisley, UK) o emo e unbound bac e- ia. Cells we e hen incuba ed o 40 min a 37°C in a Gen amycin (50 mg/ml)/Polymyxin B (20 mg/ml) solu- ion in RPMI 1640, which killed ex acellula bac e ia (5 log 10 CFU educ ion) bu was ound o ha e limi ed e ec on numbe s o in acellula bac e ia (0.5 log 10 CFU educ ion). In ec ed mac ophages we e hen washed a u he six imes be o e cul u ing o 48 h a 37°C, 5% CO 2 . In es cul u es di e en concen a ions o ecombinan mu ine IFN-g(a kind gi om A. Meaghe , NIBSC, He s, UK) we e added o mac ophage cul u es. A 48 h cells we e ha es ed and he bac e ial load assessed. Mac ophages emained 80% iable using his p o ocol excep when high con- cen a ions o IFN-gwe e used ( 400 ng/ml), his was impo an as loss o mac ophage iabili y can lead o a e ac s in bac e icidal assays [10]. Mac ophage iabil- i y was de e mined by ac idine o ange/e hidium b o- mide iable cell coun ing om sample wells. 2 . 3 .NO 2 − assay Cell ee supe na an s om mac ophage cul u es we e sampled 24 h a e es ablishmen o cul u es using he G eiss assay as p e iously desc ibed [13] 2 . 4 .E6alua ion o a ginine analogues on he bac e icidal ac i6i y o IFN-g In ec ed and unin ec ed mac ophage cul u es we e also pe o med in he p esence o 100 mMN- monome hyl- L -a ginine (L-NMMA) o N- monome hyl- D -a ginine (D-NMMA) (Calbiochem, No ingham, UK) added o cul u es a 0, 6 and 12 h a e in ec ion. 2 . 5 .E6alua ion o L - yp ophan,and i on chela ion on he bac e icidal ac i6i y o IFN-g In ec ed and unin ec ed mac ophage cul u es we e also es ablished in he p esence o ei he 1 mM L - yp- ophan (Sigma, Do se , UK); o 50 mM de e oxamine mesyla e (Sigma) an in acellula i on chela o [14]. Using hese concen a ions no dec ease was seen in mac ophage iabili y o e he cou se o he expe imen . 2 . 6 .Induc ion o he espi a o y bu s by me hylene blue Me hylene blue (Sigma), an agen ha induces he espi a o y bu s [15], was added o selec ed cul u es 1 h a e in ec ion o a concen a ion o 10 −4 M, a concen a ion which did no in luence mac ophage iabili y. 2 . 7 .Assessmen o bac e ial load Bac e ial load was assessed by emo al o 100 mlo lysed mac ophages om indi idual cul u es. These we e spo ed in iplica e on o each o h ee Bo de –Gengou aga pla es and he numbe o CFU was es ima ed a e 5 days o incuba ion. Resul s a e epo ed as he mean iable B.pe ussis om a leas h ee samples pe expe imen al g oup, and a e exp essed as CFU pe iable mac ophage. This a oided po en ial a e ac s due o loss o mac ophage iabili y du ing he cou se o bac e icidal assays [16]. 3. Resul s 3 . 1 .IFN-g educes mac ophage bac e ial load in 6i o. The mechanisms unde lying he es ablished ole o IFN-gin limi ing B.pe ussis in ec ion we e in es i- ga ed in i o. The mu ine mac ophage-like cell line J774 was in ec ed wi h i ulen B.pe ussis a a a io o 10 bac e ia pe mac ophage and exposed o a ying concen a ions o mu ine IFN-g. Fig. 1 shows ha a concen a ions be ween 3.2 and 80 ng/ml IFN-g e- duced he in acellula load o B.pe ussis o e 48 h o in i o cul u e. A high concen a ions (400 ng/ml and abo e) he bac e ial load appea ed o inc ease, how- e e , his was accompanied by a loss o mac ophage iabili y and p esumably bac e ial elease in o he cul- u e supe na an . B.P.Mahon,K.H.G.Mills / Immunology Le e s 68 (1999) 213–217 215 Fig. 1. IFN-g educes mac ophage bac e ial load in i o. Bo de ella pe ussis-in ec ed mac ophages we e cul u ed wi h ecombinan mu ine IFN-ga concen a ions anging be ween 0 and 400 ng/ml, in he absence ( e ical shading) o p esence o 100 mMN-monome hyl- L -a ginine ( L -NMMA) (ho izon al shading) o 100 mMN- monome hyl- D -a ginine ( D -NMMA) (open ba s). Fig. 3. IFN-gac s h ough mo e han one mechanism. Bo de ella pe ussis-in ec ed mac ophages we e cul u ed in he absence (open ba s) o p esence (ho izon al shading) o 50 ng/ml ecombinan mu ine IFN-g. To de e mine he mechanisms o IFN-gac ion mac ophages we e ea ed wi h medium (con ol), o wi h exogenous 1mM L - yp ophan, he in acellula i on chela o de e oxamine mesyla e (i on chela ion) o me hylene blue an induce o he espi a- o y bu s . ND, no de e mined. 3 . 2 .IFN-ginduces ni ic oxide ( NO ) elease om B. pe ussis in ec ed cells in 6i o Exposu e o B.pe ussis in ec ed mac ophages o IFN-g esul ed in he p oduc ion o NO as measu ed by NO 2 − elease (Fig. 2), which was supp essed by addi- ion o he compe i i e inhibi o L -NMMA, bu no by D -NMMA [17]. Addi ion o hese compounds o cul- u es o in ec ed mac ophages in he p esence o ab- sence o IFN-g(Fig. 1) showed ha D -NMMA had li le o no e ec on bac e ial load. Inco po a ion o L -NMMA esul ed in an inc ease in bac e ial load compa ed o con ols, howe e , i did no ully supp ess he e ec o IFN-g. These esul s sugges ha while he p oduc ion o NO may con ibu e o B.pe ussis killing he e a e likely o be o he bac e icidal mechanisms in ope a ion. 3 . 3 .IFN-gmay also ac h ough NO-independen mechanisms. T ea men o mac ophages wi h me hylene blue, an elec on ca ie and an inhibi o o guanyla e cyclase [15], enhanced he killing o in acellula B.pe ussis (Fig. 3). Howe e , addi ion o ca alase, supe oxide dismu ase o manni ol, inhibi o s o he e ec s o su- pe oxide anions, hyd ogen pe oxide o hyd oxyl adi- cals had no e ec on bac e ial eco e y (da a no shown). T ea men o mouse mac ophages wi h he in acellula i on chela o , de e oxamine mesyla e, in- hibi ed he in acellula g ow h o B.pe ussis (Fig. 3). Taken oge he hese esul s indica e ha he in acellu- la su i al o B.pe ussis is dependan on i on a ailabili y and sugges ha i on es ic ion may be an impo an mechanism by which IFN-gin luences bac e- ial su i al wi hin mouse mac ophages. IFN-gis also known o induce in acellula L - yp ophan deple ion by ac i a ion o indoleamine 2,3-dioxygenase [18,19]. Addi ion o L - yp ophan o in ec ed mac ophage cul- u es in he absence o IFN-g, esul ed in an inc eased bac e ial load compa ed o con ols, al hough his was educed in he p esence o IFN-g. This sugges s ha he concen a ion o L - yp ophan may be a limi ing s ep in Fig. 2. IFN-ginduces ni ic oxide (NO) elease om Bo de ella pe ussis-in ec ed cells in i o. NO 2 − was measu ed by G eiss assay om 24 h supe na an s sampled om B.pe ussis-in ec ed mac ophages, cul u ed wi h ecombinan mu ine IFN-ga concen a- ions anging be ween 0 and 400 ng/ml (open squa es). Simila samples we e aken om non-in ec ed con ol cul u es (solid squa es) o in ec ed cul u es incuba ed in he p esence o 100 mMN- monome hyl- L -a ginine ( L -NMMA) (open cicles) o 100 mMN- monome hyl- D -a ginine ( D -NMMA) (solid ci cles). B.P.Mahon,K.H.G.Mills / Immunology Le e s 68 (1999) 213–217 216 he in acellula su i al o B.pe ussis and ha his may be an addi ional mechanism h ough which IFN-g exe s i s an imic obial e ec . 4. Discussion Like mos success ul bac e ial pa hogens, B.pe ussis appea s o ha e adap ed ad e sa ial s a egies o delay, o e come o e ade mos o he immune e ec o mecha- nisms deployed by he hos (Table 1). P e iously he au ho s and o he s ha e desc ibed an impo an ole o IFN-gin he immune esponse o pe ussis [7– 11,20], in he p esen s udy he po en ial mechanism by which hese e ec s may be media ed in i o ha e been examined. The demons a ion ha IFN-g educed he in acellula load o B.pe ussis o e 48 h o in i o cul u e is consis en wi h indings by o he s who ha e desc ibed an an imic obial e ec o IFN-g[20–23]. I has been shown ha his e ec was associa ed wi h he p oduc ion o NO, a po en an ibac e ial agen , as measu ed by ni i e p oduc ion. The esul s using an in acellula i on chela o sugges ha he in acellula su i al o B.pe ussis is dependan on i on a ailabil- i y. I is known ha eac i e ni ogen in e media es cause in acellula i on loss by inhibi ing a ious i on- dependen enzymes, i may be ha i on es ic ion is an impo an mechanism by which IFN-gin luences bac e- ial su i al wi hin mu ine mac ophages. Whils he associa ion be ween he educ ion o bac e ial load and NO in his s udy, and be ween NO and Pw by o he s [24,25], sugges s an an ibac e ial ole o eac i e ni o- gen in e media es, o he IFN-ginduced mechanisms a e also likely o be in ope a ion. Inco po a ion o L - NMMA, a compe i i e inhibi o o NO p oduc ion [17], bu no he analogue D -NMMA, d ama ically educed ni i e concen a ions in in ec ed mac ophage cul u es bu did no comple ely abolish he bac e icidal e ec s o IFN-g. Fu he mo e p oduc ion o high con- cen a ions o NO in esponse o high le els o IFN-g, esul ed in educed iabili y o cul u ed mac ophages. This sugges s ha NO may be inducing mac ophage dea h. In e es ingly ecen epo s ha e implica ed NO in B.pe ussis-media ed pa hology o he espi a o y epi helium [26]. Me hylene blue, an elec on ca ie and an inhibi o o guanyla e cyclase, known o s imula e he espi a o y bu s [15] enhanced he killing o in acellula B.pe us- sis. Howe e , addi ion o inhibi o s o he e ec s o supe oxide anions, hyd ogen pe oxide o hyd oxyl adi- cals, impo an media o s o he espi a o y bu s , had no e ec on bac e ial eco e y. Ano he mechanism h ough which IFN-gcan media e i s an imic obial e ec is by ac i a ion o indoleamine 2,3-dioxygenase which deple es in acellula L - yp ophan [18,19]. Addi- ion o L - yp ophan o B.pe ussis-in ec ed cul u es esul ed in enhanced bac e ial load indica ing ha he concen a ion o L - yp ophan may be a limi ing s ep in he in acellula su i al o B.pe ussis. This may be an addi ional mechanism h ough which IFN-gexe s i s an imic obial e ec s agains his pa hogen. Recen s udies ha e shown ha deple ion o al eola mac ophages esul s in inc eased numbe s o B.pe us- sis in he lungs du ing he ea ly phase o in ec ion [27] and a c i ical ole o IFN-gdu ing his pe iod has p e iously been desc ibed [11]. 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