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]. The esul s desc ibed
he ein, p o ide a mechanism by which IFN-gmay
media e his 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 .
Acknowledgemen s
The au ho s acknowledge he echnical help o Ms
Fiona G i in. This wo k was suppo ed by g an s om
he Wellcome T us .
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P obable e asion s a egy Re e encesImmune e ec o
mechanism
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