UNCORRECTED PROOF
P io Bo de ella pe ussis in ec ion modula es alle gen p iming and he se e i y
o ai way pa hology in a mu ine model o alle gic as hma
D. P. Ennis
*
, J. P. Cassidywand B. P. Mahon
*
*
Mucosal Immunology Labo a o y, Ins i u e o Immunology, NUI Maynoo h, I eland and wDepa men o Ve e ina y Pa hology, Uni e si y College
Dublin, I eland
Summa y
Backg ound I has been p oposed ha T helpe (Th)2-d i en immune de ia ion in ea ly li e can be
coun e ed by Th1 inducing childhood in ec ions and ha such coun e - egula ion can p o ec agains
alle gic as hma.
Objec i e To es whe he Th1-inducing in ec ion wi h Bo de ella pe ussis p o ec s agains alle gic
as hma using well-cha ac e ized mu ine models.
Me hods G oups o mice we e sensi ized o o albumin (OVA) in he p esence o absence o
B. pe ussis, a well-cha ac e ized Th1 inducing espi a o y in ec ion. Immunological, pa hological
and physiological pa ame e s we e measu ed o assess he impac o in ec ion on immune de ia ion
and ai way unc ion.
Resul s We demons a e ha OVA sensi iza ion does no a ec he de elopmen o B. pe ussis-
specific immune esponses domina ed by IgG2a and IFN-gand does no impai Th1-media ed
clea ance o ai way in ec ion. In con as , B. pe ussis in ec ion a he ime o sensi iza ion modula ed
he esponse o OVA and significan ly educed o al se um and OVA-specific IgE. The pa e n o
cy okine esponses, in pa icula OVA-specific IL-5 esponses in he spleen was also modula ed.
Howe e , B. pe ussis did no cause global supp ession as IL-10 and IL-13 le els we e enhanced in
OVA-s imula ed spleen cell cul u es and in la age fluid om in ec ed co-sensi ized mice.
His opa hological examina ion e ealed ha B. pe ussis in ec ion p io o OVA sensi iza ion
esul ed in inc eased inflamma ion o b onchiola walls wi h accompanying hype plasia and mucous
me aplasia o lining epi helia. These pa hological changes we e accompanied by inc eased b onchial
hype - eac i i y o me hacholine exposu e.
Conclusion Con a y o he abo e p emise, a Th1 esponse induced by a common childhood
in ec ion does no p o ec agains b onchial hype - eac i i y, bu a he exace ba es he alle gic
as hma ic esponse, despi e modula ion o immune media o s.
Keywo ds alle gy, bac e ial, inflamma ion, lung, Th1/Th2 cells
Submi ed &&2003; e ised &&2004; accep ed 26 May 2003
In oduc ion
As hma is a ch onic disease o he espi a o y ac ha has
inc eased d ama ically in p e alence in wes e n socie y [1].
The inflamma o y esponse in as hma is igh ly associa ed
wi h ai way hype - esponsi eness (AHR), inc eased mucus
p oduc ion and an infil a ion o he b onchial mucosa wi h
CD4
1
T cells [2]. The e is e idence o an al e ed local T cell
esponse in a ou o T helpe (Th)2 cy okine elease (IL-4,
IL-5 and IL-13) esul ing in B cell iso ype swi ching o IgE,
mas cell, eosinophil and basophil ec ui men and p oduc-
ion o a wide ange o inflamma o y media o s [3]. The
esul ing pulmona y inflamma ion leads o b onchocons ic-
ion and ul ima ely o ai way emodelling [4].
The cu en unde s anding o he pa hophysiology o
alle gic as hma is ha i esul s om a b eakdown in he
no mal ole ance o inhaled an igens, associa ed wi h Th2
cy okine p oduc ion [5, 6]. The mu ine o albumin (OVA)
model o AHR exhibi s many o he ea u es o human
as hma, including ai way hype - eac i i y, ai way inflamma-
ion and inc eased se um IgE le els [7, 8]. This model has
been used ex ensi ely o p obe mechanisms o as hma [7, 9].
The inc eased incidence o as hma has been linked o
imp o ed sani a ion in indus ialized socie ies, which in u n
has educed he incidence o childhood in ec ions [10]. One
cu en a emp o explain hese obse a ions, loosely e med
he hygiene hypo hesis, s a es ha childhood as hma de elops
as a esul o dec eased exposu e o in ec ious agen s du ing
in ancy and ea ly childhood, which esul s in he pe sis ence
o he neona al Th1 defici , he eby p edisposing he child o
a opic disease [11]. While Th2 cells p omo e ai way inflam-
ma ion in as hma, i has been p oposed ha because Th1 cells
an agonize Th2 cell unc ion, immune de ia ion owa ds Th1
Jou nal: CEA HDisk used ED: Suma Copy ED: Guna Pgn by: su eshbabu
A icle : 2042 Pages: 10 Despa ch Da e: 19/7/2004 Scan: Roopa Colou : Fig. 6
Q2
Co espondence: B. P. Mahon, Mucosal Immunology Labo a o y,
Ins i u e o Immunology, Na ional Uni e si y o I eland Maynoo h,
Maynoo h, Co. Kilda e, I eland. E-mail: bpm[email p o ec ed]
Clin Exp Alle gy 2004 doi:10.1111/j.1365-2222.2004.02042.x
2004 Blackwell Publishing L d 1
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may be p o ec i e in as hma [12, 13]. One p edic ion a ising
om he abo e hypo hesis is ha a powe ul Th1-inducing
in ec ion du ing o p io o ai way sensi iza ion should
diminish o p o ec agains Th2-media ed alle gic as hma.
Bo de ella pe ussis is a G am-nega i e bac e ium ha
causes he se e e in an disease whooping cough. B. pe ussis
espi a o y challenge o mice is a well-cha ac e ized model o
ai way Th1-induced immuni y, which co ela es well o
immuni y in humans [14]. Reco e y om in ec ion is
associa ed wi h he de elopmen o B. pe ussis-specific Th1
cells in bo h humans and mice [15]. Th1 cells p oducing IFN-
gplay an essen ial, non- edundan ole in he clea ance o he
bac e ia om he espi a o y ac [16]. Mu ine espi a o y
challenge by ae osol adminis a ion o he bac e ia has been
used ex ensi ely o s udies o B. pe ussis immuni y and
pa hogenesis and al hough mice lack he cha ac e is ic cough,
in o he espec s he cou se o in ec ion and many o he
sys emic e ec s a e simila o hose obse ed in in an s [17].
In o de o es he abo e hypo hesis, we used he Th1 d i ing
B. pe ussis model, in combina ion wi h he mu ine OVA
model o alle gic as hma. Based on he p edic ion abo e,
p io in ec ion wi h B. pe ussis migh be expec ed o educe
OVA-induced Th2-media ed AHR and immunopa hology.
Ou findings demons a e ha al hough dampening o he
Th2 esponse was seen a he local and sys emic le el, OVA
sensi iza ion du ing B. pe ussis in ec ion led o enhanced
p oduc ion o ai way IL-10 and IL-13, coupled wi h a
subsequen inc ease in AHR and pa hology. This sugges s
ha al hough IL-10 may be ega ded as a egula o y
cy okine, i has b oade unc ions ha may no always
p o ec agains inflamma o y disease. These da a ha e
implica ions wi h ega d o he alidi y o he hygiene
hypo hesis and aise conce ns ega ding he apies based on
he con e sion o Th2-domina ed alle gic inflamma o y
esponses in o Th1-domina ed esponses based on p o ec i e
e ec s o Th1 cells in alle gy and as hma.
Ma e ials and me hods
Animals
Six- o eigh -week old emale BALB/c (Ha lan, UK) mice
we e used unde he guidelines o he I ish Depa men o
Heal h and he esea ch e hics commi ee o he Na ional
Uni e si y o I eland Maynoo h.
Ae osol in ec ion
Respi a o y in ec ion was ini ia ed by ae osol challenge wi h
B. pe ussis s ain W28, ollowing g ow h unde agi a ion
condi ions a 37 1C in S aine –Schol e liquid medium.
Bac e ia om a log-phase cul u e we e esuspended a a
concen a ion o 2 10
10
CFU/mL in 1% (w/ ) casein in
0.9% (w/ ) saline. The challenge inoculum was adminis e ed
o wo g oups o mice on 0 day (Bp and BpOVA g oups).
Adminis a ion was by ae osol o e a pe iod o 15 min using
a nebulize . G oups o ou o mo e mice we e killed a
a ious ime poin s a e ae osol challenge o assess he
numbe o iable B. pe ussis in he lungs. Remaining mice
ecei ed a simila ae osol o s e ile saline alone.
Immuniza ion and ai way deli e y o OVA
Two g oups o 6–8-week-old emale BALB/c mice (OVA and
BpOVA) we e sensi ized by i.p. injec ion o 100 mg OVA
(G ade V; Sigma, Do se , UK) emulsified in 2% Alhyd ogel
s
adju an (Supe os Biosec o , Sweden) a 10 and 24 days a e
bac e ial o saline challenge. Con ol g oups (C l and Bp)
ecei ed saline alone (i.p.). On 35, 36 and 37 days, OVA and
BpOVA-sensi ized mice ecei ed 50 mg OVA in a–nasally,
whe eas C l and Bp g oups ecei ed saline only.
Enume a ion o iable bac e ia in he lungs
Lungs we e emo ed asep ically in o 1 mL o s e ile
physiological saline wi h 1% casein. One hund ed mic o-
lio es o se ially dilu ed homogena e om indi idual lungs
we e placed on o iplica e Bo de –Gengou aga pla es and
he numbe o CFU de e mined a e incuba ion a 37 1C o
4 days. Resul s a e epo ed as he mean numbe o B.
pe ussis CFU o indi idual lungs om ou o mo e mice.
B onchoal eola la age
B onchoal eola la age (BAL) fluids we e ob ained by epea
adminis a ion and aspi a ion o 0.5 mL olumes ( o al 5 mL)
o phospa e-bu e ed saline (PBS) ia cannula ion o he
achea o mice om h ee expe imen s (n55). Cells om he
la age fluid we e eco e ed by cen i uga ion a 300 g o
6 min and esuspended in PBS; o al leucocy es we e coun ed
and cy ospin p epa a ions we e s ained wi h a combined
Alcian blue/Discombe’s s ain o de e mine he di e en ial cell
coun . Supe na an s we e collec ed o cy okine analysis and
s o ed a 80 1C.
Measu emen o o albumin- and Bo de ella pe ussis-
speci ic an ibody
OVA- and B. pe ussis-specific-IgG1, IgG2a, IgG2b and IgG3
p esen in collec ed se a we e measu ed by ELISA as
p e iously desc ibed [16, 18]. B iefly, pla es we e coa ed wi h
OVA p o ein (5 mg/mL) o sonica ed B. pe ussis an igen (1 mg/
mL) o e nigh a 4 1C. A e blocking and he addi ion o
se um samples, alkaline phospha ase-labelled a an i-mouse
IgG1, IgG2a, IgG2b and IgG3 (Pha mingen) we e used o
de ec OVA- and B. pe ussis-specific an ibody as p e iously
desc ibed [19]. To al and OVA-specific IgE was measu ed
using a a an i-mouse IgE monoclonal an ibody (Pha min-
gen). The IgE concen a ion was exp essed as mic og ams pe
millili e a e compa ison wi h mu ine IgE s anda ds.
T cell p oli e a ion assays
Spleen cells (2 10
6
/mL) om in ec ed, sensi ized and
con ol mice (n54 o mo e pe g oup) we e es ed o
in i o p oli e a ion agains hea -inac i a ed B.pe ussis
(1 10
4
CFU/mL), OVA (20 mg/mL), Concana alin A (Con
A) (5 mg/mL, posi i e con ol), o medium alone (nega i e
con ol). A e 72 h, cell p oli e a ion was assessed by liquid
scin illa ion coun ing o [
3
H]-Thymidine inco po a ion and
esul s we e exp essed as mean CPM o iplica e wells SE.
A he 72-h ime-poin , cul u e supe na an s we e sampled o
cy okine analysis, al hough he kine ics o cy okine p oduc-
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2D. P. Ennis e al.
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ion a ies his ime-poin has p e iously p o ed accep able
o de ec ion o mos cy okines [14].
Cy okine measu emen
Concen a ions o IL-4, IL-5, IL-10, IL-13 and IFN-g om
spleen and b oncho-al eola la age fluid (BALF) we e
assessed by ELISA (Pha mingen). Cy okine concen a ions
we e calcula ed by compa ison wi h known cy okine s an-
da ds as p e iously desc ibed [16].
Whole-body ple hysmog aphy
Ai way esponsi eness was assessed by me hacholine (MCh)-
induced ai flow obs uc ion om conscious mice using whole-
body ple hysmog aphy (Buxco Elec onics, USA) as p e-
iously desc ibed [20]. Pulmona y ai flow obs uc ion was
measu ed by enhanced pause (PenH), a alue de e mined
om he a io o expi a o y ime and elaxa ion ime o peak
expi a o y flow and peak inspi a o y flow and hough o
co ela e wi h ai way esponsi eness. Measu emen s we e
ob ained a e exposu e o mice o 3 min o PBS (baseline)
ollowed by inc emen al doses (3.3–50 mg/mL) o MCh
deli e ed by ae osol [21].
Respi a o y ac his ology
Animals (n55 pe g oup pe expe imen ) we e killed a 37
days. Lungs we e emo ed, fixed in a pa a o maldehyde/
lysine/pe ioda e fixa i e, pa a fin embedded, sec ioned and
s ained using he haema oxylin and eosin (H&E), Discombe’s
(iden ifica ion o eosinophils)/Alcian blue (iden ifica ion o
mucus), Pe iodic Acid-Schi (assessmen o basemen mem-
b ane hickness), azu e-A (iden ifica ion o mas cells) and
Van Gieson (iden ifica ion o fib osis) me hods. His opa ho-
logical changes e iden we e g aded acco ding o a semi-
quan i a i e sco ing sys em as mild, mode a e o se e e by
wo esea che s wi hou p io knowledge o he ea men
g oup (Table 2). All expe imen s we e pe o med a leas
wice (n55) on each occasion.
Resul s
O albumin sensi iza ion does no impai T helpe 1-
media ed mechanisms o bac e ial clea ance
In o de o examine he e ec s o immune c oss- egula ion we
es ed he e ec o OVA sensi iza ion upon he de elopmen
o a p o ec i e Th1 esponse o in ec ion. G oups o mice
we e sensi ized o OVA in he p esence o absence o a p io
B. pe ussis in ec ion (Table 1). The kine ics o bac e ial
clea ance om he lungs o expe imen al animals we e
moni o ed by pe o ming colony coun s on whole-lung
homogena es a di e en imes pos -bac e ial challenge. Mice
ecei ed ei he saline (C l and OVA) o iable B. pe ussis
(Bp and BpOVA) by ae osol. This was ollowed by OVA
(OVA and BpOVA) o sham sensi iza ion (Table 1). G oups
in ec ed wi h B. pe ussis (Bp and combined BpOVA g oups)
showed simila kine ics o bac e ial clea ance. No bac e ia
we e eco e ed om he OVA o C l g oup (Fig. 1), which
we e unin ec ed bu ecei ed saline by ae osol. Bac e ial
bu den in he Bp and BpOVA g oups peaked a 10 days and
declined he ea e . By 35 days-pos -challenge, bo h he Bp
and he BpOVA g oups showed comple e bac e ial clea ance
(Fig. 1). The e o e, sensi iza ion wi h OVA does no impai
he e ec o unc ion associa ed wi h he Th1-media ed
clea ance o a bac e ial disease.
Table 1.Expe imen al design
Desc ip ion
*
Time (days)
0 10 24 35/36/37
Con ol Saline ae osol Saline (i.p.) Saline (i.p. and i.n.) Saline (i.n.)
Bp in ec ion Bp ae osol in ec ion Saline (i.p.) Saline (i.p. and i.n.) Saline (i.n.)
OVA sensi iza ion Saline ae osol OVA (i.p.) OVA (i.p and i.n.) OVA (i.n.)
Bp in ec ion and OVA sensi iza ion (BpOVA) Bp ae osol in ec ion OVA (i.p.) OVA (i.p and i.n.) OVA (i.n.)
*
G oups o 6–8-week-old emale BALB/c mice (n54 o mo e pe eplica e pe ime-poin ) we e ea ed as ollows: Con ol (C l) mice we e sham in ec ed on 0 day
and sham sensi ized a 10 days and 24 days. A second g oup (Bp) we e in ec ed wi h Bo de ella pe ussis a 0 day and sham sensi ized a 10 day and 24 days. The
hi d g oup (OVA) we e sham in ec ed bu sensi ized wi h OVA (100 mg, i.p.) a 10 and 24.days and hen again (50 mg i.n.) a 24, 35, 36, and 37 days. The inal g oup
(BpOVA) we e in ec ed wi h B. pe ussis on 0 day, and sensi ized as abo e.
Table 2.His ological assessmen o ai way pa hology
T ea men g oup
Epi helial mucous
me aplasia
Epi helial
hype plasia
Smoo h muscle
hype ophy
Pe i-ai way in lamma ion
*
O e all E N L M F
Con ol     
Bp 11 1111
OVA 11 11 11 11 1 11 11 
BpOVA 111 111 11 111 11 111 111 
A semi-quan i a i e sco e ( , absen ; 1, mild; 11, mode a e; 111, se e e) was assigned o ea u es o ai way pa hology obse ed.
*
Pe i-ai way in lamma ion assessed in e ms o o e all deg ee and o numbe s o in il a ing eosinophils (E), neu ophils (N), lymphocy es, plasma cells and
mac ophages (L), mas cells (M) and in e ms o ci cumsc ibing ib osis (F). Obse a ions a e ep esen a i e o a leas wo expe imen s whe e n55 o mo e in
each case.
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Bo de ella pe ussis in ec ion 3
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Bo de ella pe ussis supp esses o albumin-speci ic
humo al immune esponses
Al hough OVA-induced sensi iza ion does no impai Th1
clea ance o B. pe ussis, we wan ed o examine he influence
o B. pe ussis in ec ion on esponses associa ed wi h OVA
Th2 sensi iza ion. OVA-specific IgG was no de ec ed om
mice in ec ed wi h B. pe ussis only; simila ly B. pe ussis-
specific IgG could no be de ec ed in OVA-sensi ized animals,
sugges ing no significan c oss- eac ion be ween he wo
immunogens occu ed (Figs 2a and b). An analysis o
an ibody subclasses e ealed ha in ec ion induced g ea e
se um i es o B. pe ussis-specific IgG2a han IgG1 (Figs 2a
and b), consis en wi h ou p e ious findings [19]. Sensi iza-
ion o in ec ed mice wi h OVA (BpOVA) did no signifi-
can ly al e his p ofile. OVA sensi iza ion in he absence o
in ec ion esul ed in s ong an ibody esponses almos
exclusi ely o he IgG1 subclass, consis en wi h a Th2 model
o p iming. In con as o he minimal influence o OVA on
he esponse o in ec ion (Fig. 2a), bac e ial B. pe ussis
in ec ion supp essed o modula ed immuni y o OVA. Fo
example, OVA-specific IgG1 i es we e significan ly lowe
(Po0.001) in he combined g oup (Fig. 2b). Supp ession was
no confined o IgG subclasses. To al se um and OVA-
specific IgE was also significan ly educed (Po0.001) be ween
he OVA and he Bp/OVA g oups (Figs 2c and d), suppo ing
a ole o B. pe ussis in supp essing o egula ing he immune
esponse o an igen exposu e du ing in ec ion.
O albumin sensi iza ion du ing Bo de ella pe ussis
in ec ion enhances ai way In e leukin-10 and In e leukin-13
In o de o dissec he na u e o B. pe ussis supp ession, cell-
media ed immune esponses we e examined om spleen
cul u es. As p e iously epo ed [22], B. pe ussis in ec ion
induced s ong splenic p oli e a i e and IFN-g esponses bu
e y li le IL-5 (Figs 3a–c). This was consis en wi h he
obse ed p o ec ion (Fig. 1) and he an ibody subclass da a
(Fig. 2). Again, IFN-gwas no significan ly educed by OVA
sensi iza ion (Fig. 3c). As expec ed, OVA sensi iza ion alone
induced significan IL-5 bu no IFN-g(Figs 3a and c).
Howe e B. pe ussis did influence cy okine esponses o
OVA. Reduced le els o IL-5 we e de ec ed in he combina-
ion g oup sugges ing ha B. pe ussis supp essed Th2
esponses o OVA, mi o ing he educ ion in i e o specific
an ibody de ec ed. In e es ingly B. pe ussis in ec ion did
induce specific IL-10 as well as IL-13 esponses (Figs 3b and
d). While OVA sensi iza ion had li le o he e ec on he
immune esponse induced by in ec ion, i significan ly
enhanced he le els o IL-10 and IL-13 (Po0.01 and
o0.01, espec i ely) p oduced in esponse o he bac e ium
(Figs 3b and d).
To ex end hese findings, we examined he le els o
cy okines p esen in BALF. OVA sensi iza ion, bu no
in ec ion, induced IL-4 (Fig. 4a); howe e , p io in ec ion
wi h B. pe ussis supp essed his. B. pe ussis in ec ion
induced local IFN-g, which was no educed by OVA
sensi iza ion (Fig. 4d). In e es ingly, while OVA sensi iza ion
induced IL-10 and IL-13 de ec able in BALF, his was
significan ly (Po0.01 and o0.01, espec i ely) enhanced i
sensi iza ion ollowed in ec ion (Figs 4b and c).
A T-helpe 1 in ec ion in he espi a o y ac does no
p o ec , bu exace ba es he alle gic as hma ic esponse
I has been p oposed ha p io Th1 esponses o bac e ial
in ec ions p o ec agains alle gic disease by dampening he
ac i i y o Th2 e ec o cells. I migh also be p edic ed ha
he supp essi e o modula o y e ec s o B. pe ussis ou lined
abo e would p o ec agains Th2-d i en pa hology. We used
whole-body ple hysmog aphy in o de o measu e ai way
eac i i y in mice in ec ed wi h B. pe ussis p io o OVA
sensi iza ion in compa ison wi h con ols (Fig. 5). We ound
ha con a y o he abo e p emise, a Th1 esponse induced
by in ec ion in he espi a o y ac did no p o ec agains
b onchial hype - eac i i y bu a he exace ba ed he alle gic
as hma ic esponse. S a is ical analysis using wo-way ANOVA
showed ha mice sensi ized o OVA ollowing B. pe ussis
in ec ion displayed significan ly g ea e b onchial hype -
eac i i y compa ed wi h OVA sensi ized alone (Po0.001)
(Fig. 5). This demons a es ha in ec ion wi h B. pe ussis o
he espi a o y ac p io o sensi iza ion esul s in inc eased
ai way eac i i y and exace ba es he alle gic esponse.
B. pe ussis in ec ion also modula ed he quali y o he
inflamma o y influx o he espi a o y ac . The e was a
ma ked educ ion in eosinophil numbe s obse ed in BpOVA
compa ed wi h OVA-sensi ized ai ways (Table 3). His ologi-
cal examina ion o lung issue showed ha pa hological
changes we e la gely ocussed on b onchioles and adjacen
pe ib onchiola blood essels wi h a ying deg ees o ai way
wall inflamma ion and smoo h muscle hype ophy accom-
panied by a ying deg ees o epi helial hype plasia and
mucous me aplasia (Fig. 6). A semi-quan i a i e his opa ho-
logical sco ing sys em was used o acili a e compa isons
be ween g oups (Table 2). Minimal changes we e obse ed in
0 7 14 21 28 35
0
1
2
3
4
5
6
Bp
BpOVA
C l
OVA
Days a e challange
Bac e ial bu den (Log10 CFU/lung)
Fig. 1.Cou se o Bo de ella pe ussis in ec ion in expe imen al and con ol
(C l) mice. G oups o mice we e killed a in e als a e challenge and he
numbe o iable bac e ia es ima ed by pe o ming colony coun s on
indi idual lung homogena es. Resul s a e ep esen a i e om wo expe i-
men s and a e p esen ed as mean CFU in he lungs de e mined indi idually
pe g oup om ou mice a each ime poin . Da a o C l and o albumin
(OVA) g oups ha e been o se om ze o o cla i y.
4D. P. Ennis e al.
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mice challenged wi h B. pe ussis only, as in ec ion had
esol ed by he 37 days ime-poin . OVA sensi iza ion
esul ed in ypical inflamma ion o ai way walls wi h
infil a ion o eosinophils, neu ophils and lymphocy es
(Fig. 6c). Howe e , BpOVA mice displayed mo e se e e
ai way wall inflamma ion wi h a g ea e deg ee o bo h
epi helial hype plasia and mucous me aplasia han Bp, OVA
o con ol mice (Fig. 6d). Gi en ha ai way esis ance (R)is
in e sely p opo ional o he ou h powe o he ai way
luminal adius (
4
) e en minimal na owing caused by
p ocesses such as ansmu al inflamma ion, epi helial hype -
plasia o mucus exuda ion subsequen o epi helial mucous
me aplasia can p o oundly inc ease pulmona y esis ance as
indica ed by ple hysmog aphy (Fig. 5).
Discussion
The p esen s udy demons a es ha p io in ec ion wi h
iable B. pe ussis modula es he immune esponse induced by
alle gen sensi iza ion. In ec ion supp esses an ibody and cell-
C l Bp OVA BpOVA
0
1000
2000
3000
B. pe ussis-speci ic IgG
C l Bp OVA BpOVA
0
1000
2000
3000 IgG1
IgG2a
IgG2b
IgG3
OVA-speci ic IgG
C l Bp OVA BpOVA
0
10
20
30
*
To al se um IgE (µg/mL)
C l Bp OVA BpOVA
0
250
500
750
1000 *
OVA-speci ic IgE (ng/mL)
(a) (b)
(c) (d)
Fig. 2.Se um and o albumin
(OVA)-speci ic IgE and IgG sub-
classes elici ed by bac e ial in ec-
ion and alle gic sensi iza ion. (a)
Bo de ella pe ussis and (b) OVA-
speci ic se um an ibody esponses
by IgG subclass elici ed in con ol
(C l), in ec ed (Bp), sensi ized
(OVA) mice, o in mice in ec ed wi h
B. pe ussis p io o sensi iza ion
(BpOVA); exp essed as geome ic
mean i e o an ibody (SE). (c)
To al IgE and (d) OVA-speci ic IgE
p esen in se a om each expe i-
men al g oup exp essed as mg/mL
o ng/mL, espec i ely. OVA-speci-
ic IgE (ng/mL) is a ela i e mea-
su e de e mined by adap a ion o a
s anda d IgE ELISA. Resul s a e
ep esen a i e o h ee expe imen s
om ou animals pe o med inde-
penden ly in iplica e.
*
S a is ical
signi icance, Po0.001 compa ed
wi h BpOVA- ea ed g oup.
IL-5
C l Bp OVA BpOVA
0
250
500
750
1000
1250
1500
1750
2000
(a)
Conc. IL-5 (pg/mL)
Conc. IL-13 (pg/mL)
IL-13
C l Bp OVA BpOVA
0
250
500
750
1000
C
Bp
OVA
+
(b)
IFN - γ
C l B
p
OVA B
p
OVA
0
200
400
600
800
1000
(c)
Conc. IFN - γ
γ
(pg/mL)
IL-10
C l Bp OVA BpOVA
0
200
400
600
800
1000
(d)
Conc. IL-10 (pg/mL)
Fig. 3.Cell-media ed immune
esponses om spleen, elici ed by
bac e ial in ec ion and alle gic sensi-
iza ion. IL-5 (a), IL-13 (b), IFN-g(c),
and IL-10 (d) esponses om spleen
cell cul u es s imula ed wi h medium
alone (nega i e con ol, e ical
shading), hea inac i a ed Bo de ella
pe ussis sonica e a 1 10
4
CFU/
mL (ha ched ba ), o albumin (OVA)
(20 mg/mL) (open ba ) o Con A
(posi i e con ol, black ba ). Re-
sponses a e ep esen a i e o ipli-
ca e expe imen s each assay was
pe o med in iplica e on indi idual
samples om ou mice pe g oup
and esul s a e exp essed as mean
(SE).
Bo de ella pe ussis in ec ion 5
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media ed esponses agains OVA locally and sys emically,
while enhancing he le els o he egula o y cy okine IL-10
and IL-13. Despi e his modula ion, B. pe ussis exace ba es
OVA-induced ai way pa hology, leading o he de elopmen
o mo e p onounced alle gen-induced ai way inflamma ion as
well as he induc ion o enhanced AHR.
As hma is a ch onic inflamma o y disease o he ai ways,
he p e alence o which has inc eased subs an ially in ecen
decades [24, 25]. The explana ion ha has a ac ed mos
a en ion is he hygiene hypo hesis, which sugges s ha he
inc ease in alle gic disease is caused by a cleane en i onmen
and ewe childhood in ec ions [26]. The goal o his s udy
was o es he hypo hesis ha a powe ul Th1-media ed
in ec ion such as B. pe ussis would diminish o p o ec in a
mu ine model o alle gic as hma. Zuany-Amo im e al. [27]
used a simila model, bu employed hea -killed Mycobac e -
ium accae ha was e ec i e in blocking alle gic inflamma-
ion bu by a mechanism independen o IFN-g. The same
au ho s wen on o find ha mycobac e ia induce IL-10-
p oducing egula o y T cells. In iguing ecen da a om
McGui k e al. [15] ha e shown ha he filamen ous
haemaglu inin componen o B. pe ussis beha es in a simila
way. Ou obse a ion o IL-10 in la age fluid and ollowing in
i o s imula ion o spleen cells suppo s he la e finding. In
he case o he p o ec ion gene a ed by mycobac e ial
exposu e, i is p oposed ha IL-10 has an essen ial ole in
modula ing he immune sys em by inducing a shi om an
alle gen-specific Th2 esponse [28]. In he p esen s udy, we
obse e a simila modula ion bu his does no esul in
p o ec ion agains ai way hype - eac i i y, implying ha
al hough he es o a ion o a pu a i e balance be ween Th1
and Th2 is an a ac i e heo y, i is unlikely o p o ide a
uni e sal explana ion o he pa hogenesis o as hma.
S udies wi h Th1 and Th2 cells in diabe es melli us and
au oimmune encephalomyeli is indica e ha c oss- egula ion
does no always ope a e and in some ins ances can be
unexpec edly ha m ul [29]. Fo example, Genain e al. [30]
showed in a model o mul iple scle osis ha a shi in cy okine
p oduc ion om a Th1 o a Th2 pa e n inc eased
concen a ions o pa hogenic au oan ibodies and in some
ins ances exace ba ed au oimmune disease. Pakala e al. [31]
also showed ha immune de ia ion owa ds a Th2 esponse
did no educe, bu a he exace ba ed pa hology and disease.
In he p esen s udy, he Th2-associa ed e ec o OVA
sensi iza ion had li le influence on he Th1-media ed
clea ance o a bac e ial in ec ion o he ai ways. In con as ,
B. pe ussis in ec ion supp esses IgG and IgE esponses
associa ed wi h OVA sensi iza ion. This p o ides compelling
IL-4
C l Bp OVA BpOVA
0
10
20
30
40
50
60
(a)
Conc. IL- 4 (pg/mL)
IL-10
C l Bp OVA BpOVA
0
100
200
300
400
500 *
(b)
Conc. IL-10 (pg/mL)
IL-13
C l Bp OVA BpOVA
0
100
200
300
400
500
600
700 *
(c)
Conc. IL-13 (pg/mL)
IFN-γ
C l Bp OVA BpOVA
0
250
500
750
(d)
Conc. IFN-γ
γ
(pg/mL)
Fig. 4.Bo de ella pe ussis in ec ion modula es he
local cy okine esponse o alle gen. G oups o mice
we e ea ed as desc ibed in he legend o Fig. 1. A
37 days, dilu ed b onchoal eola la age was pooled
om i e mice pe g oup and concen a ions o IL-4
(a), IL-10 (b), IL-13 (c) and IFN-g(d) esponses
de e mined by enzyme immunoassay. Resul s a e
ep esen a i e o iplica e expe imen s, assays we e
pe o med in iplica e, alues a e exp essed as mean
SE be ween expe imen al and con ol g oups.
*
Po0.01
1 10 10
0
0
1
2
3
4
5
C l
Bp
OVA
BpOVA
MCh Conc. (m
g
/mL)
Ai way eac i i y (PenH)
Fig. 5.Bo de ella pe ussis exace ba es b onchial hype - esponsi eness
o sensi izing an igen. G oups o mice we e ea ed as desc ibed in he
legend o Fig. 1. A 37 days, ai way hype - eac i i y in esponse o
inc easing concen a ions o inhaled me hacholine (MCh) was measu ed by
whole-body ple hysmog aphy. Resul s a e ep esen a i e o h ee expe i-
men s (n54 pe g oup) and alues a e exp essed as mean enhanced
pause (PenH) SE.
6D. P. Ennis e al.
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e idence ha B. pe ussis powe ully modula es he esponse
o he hi d pa y an igens. These da a a e again consis en
wi h s udies in which M. accae injec ion o OVA-immunized
mice significan ly supp essed se um IgE [32] bu a e in di ec
con as o Zuany-Amo im e al. [27] in which hey ailed o
see any e ec on IgG2a le els o se um IgE. Al hough
M. accae and B. pe ussis p o oke simila immune
esponses, adically di e en pa hologies a e induced. The
esolu ion o his pa adox may lie in he di e en ae iologies
o bo h M. accae and B. pe ussis. Respi a o y challenge by
M. accae causes minimal epi helial damage and limi ed
ai way pa hology. In s a k con as B. pe ussis causes
significan damage o he epi helial lining o he ai ways
[33]. This is media ed h ough a bac e ial i ulence ac o
called acheal cy o oxin, which induces IL-1band eac i e
ni ogen in e media es ha b ing abou cilios asis, ollowed
by ai way emodelling [34, 35]. Thus, while M. accae and
B. pe ussis induce e y simila immune esponses, i may be
ha du ing B. pe ussis in ec ion he e is a combina ion o
epi helial damage, IFN-g, IL-10 and IL-13 p oduc ion ha
has p o ound influences on he epi helium and i s local
en i onmen , which se es o exace ba e a he han p o ec
agains as hma.
In as hma, he b onchial epi helium is highly abno mal
wi h s uc u al changes in ol ing sepa a ion o columna cells
om hei basal a achmen s and unc ional changes esul ing
in inc eased exp ession and elease o p o-inflamma o y
cy okines and g ow h ac o s [36]. Benea h he damaged and
dys unc ional epi helium lie inc eased numbe s o subepi he-
lial myofib oblas s ha deposi in e s i ial collagens causing
hickening o he basemen memb ane [4]. These e ec s a e
seen in he p esen s udy in he OVA g oup (Fig. 6c) bu a e
mo e p ominen in he combina ion g oup ha ecei ed
B. pe ussis p io o OVA sensi iza ion (Fig. 6d). E idence
sugges s ha he epi helium should no be iewed in isola ion,
as ai way smoo h muscle cells con ibu e o he pe pe ua ion
o ai way inflamma ion and ai way emodelling [37]. G un-
s ein e al. [38] has sugges ed ha IL-10 may play an
impo an ole in alle gic as hma by ac ing di ec ly on he
sensi ized ai way smoo h muscle i sel . The p esen s udy
sugges s ha al hough IL-10 may be a egula o y cy okine, i
has b oade unc ions ha may no always p o ec agains
inflamma o y disease, pa icula ly i he e has been damage
o he supe ficial epi helium. Lee e al. [39] ha e also
demons a ed ha IL-10 induces IL-13 p oduc ion in i o
and ha his induc ion was esponsible o he mucus, bu no
he inflamma o y and fib o ic e ec s o IL-10. This would
appea o be consis en wi h ou own da a (Figs 3b and d, 4b
and c) whe e we see an inc ease in bo h IL-10 and IL-13 a he
sys emic and local le el. The dec ease in eosinophilia (Table
Table 3.Leucocy es p esen in BAL luid
G oup
To al leucocy es
(10
4
)
Eosinophils
(10
4
)
Mac ophages
(10
4
)
Lymphocy es
(10
4
)
Neu ophils
(10
4
)
Con ol 5.0 0.5 o0.005 3.9 2.0 0.2 0.1 o0.005
Bp 5.0 0.4 o0.005 4.6 0.9 1.1 0.7 0.2 0.1
OVA 8.0 1.7 4.1 0.9 2.9 1.2 0.6 0.1 0.5 0.2
BpOVA 5.0 0.8 2.4 0.8
*
2.1 0.8 0.4 0.1 0.5 0.3
G oups o mice we e killed (37 days), b onchoal eola la age (BAL) cells we e collec ed, coun ed, and cy ospin p epa a ions s ained o ob ain he di e en ial
leucocy e coun . Da a ep esen mean (SE) alues; n55–8 mice pe expe imen .
*
Po0.05 s. OVA g oup.
Fig. 6.Bo de ella pe ussis inc eases he se e i y o
ai way pa hology o sensi izing an igen. Pho omic o-
g aphs a–d illus a e ep esen a i e mo phological
changes in ans e se sec ions o b onchioles a 37 days
(n55 pe g oup). (a) Con ol g oup; (b) B. pe ussis
in ec ed g oup (mild mu al and pe i-ai way in lamma ion
e iden ); (c) o albumin (OVA)-sensi ized g oup illus a ing
mode a e mu al and pe i-ai way in lamma ion wi h ac-
companying mode a e mucus me aplasia (blue s aining
goble cells) and hype plasia o epi helium; (d) Combined
B. pe ussis/OVA- ea ed g oup illus a ing se e e mu al
and pe i-ai way in lamma ion, mode a e epi helial hype -
plasia and se e e mucous me aplasia wi h accompanying
mucus plugging o he lumen (P). All sec ions s ained wi h
a combined Discombes/Alcian blue s ain. O iginal mag-
ni ica ion 400.
Bo de ella pe ussis in ec ion 7
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2) obse ed in he BpOVA g oup is mos likely because o he
modula ion o IL-5 esponses in hese mice (Fig. 3a). IL-5 is a
cy okine necessa y o he egula ion o eosinophil g ow h,
di e en ia ion, ac i a ion and su i al and plays a c i ical
ole in he ec ui men o eosinophils o he lung [40]. Simila
esponses we e obse ed by Wu e al. [41] using mu ine
cy omegalo i us in ec ion in conjunc ion wi h he mu ine
model o OVA-induced alle gic ai way disease. P e ious
s udies in humans ha e demons a ed ha IL-13 mRNA and
p o ein le els a e ele a ed in he lungs o a opic and non-
a opic as hma ics [42, 43], sugges ing ha o e p oduc ion o
IL-13 may p edispose owa d he de elopmen o bo h ypes
o as hma [43]. Wal e e al. [44] showed ha an OVA-specific
Th2 line gene a ed om IL-13
/
mice, which p oduced
high le els o IL-4 and IL-5, bu no IL-13, ailed o induce
AHR, demons a ing he essen ial ole o IL-13 in he
de elopmen o AHR. IL-13 may also inc ease AHR di ec ly.
IL-13 induces smoo h muscle p oli e a ion in i o [45] and
can aid con ac ions o acheal smoo h muscle [46]. Ai way
smoo h muscle cells ha e also been shown o exp ess IL-13
ecep o s, including bo h componen s o he IL-13R complex
[47]. Am ani e al. [48] ha e sugges ed ha inc eased le els o
IFN-gin as hma ic indi iduals may p omo e AHR and
exace ba e as hma by di ec ly modula ing con ac ile e-
sponses. OVA sensi iza ion o in ec ed mice in he combina-
ion g oup induced significan le els o IFN-gde ec able in
he BALF (Fig. 4d), sugges ing ha IFN-gmay con ibu e o
he obse ed exace ba ion o pa hology (Fig. 6d). This
co ela es well wi h s udies in humans whe e inc eased IFN-g
was seen in as hma ic pa ien s compa ed wi h no mal subjec s
[49, 50]. I has also been shown ha AHR can mani es
independen ly o pulmona y inflamma ion [51] al hough his
was no seen in his s udy. Respi a o y syncy ial i us (RSV),
commonly associa ed wi h lowe lung in ec ions in in ancy is
also known o exace ba e as hma [52, 53]. Ma suse e al. [54]
ound ha he e ec o RSV in ec ion a ies depending upon
he inflamma o y con ex o he lung. Bo h p ima y and
ecu en RSV in ec ions augmen ongoing alle gic inflam-
ma ion, howe e , in he absence o alle gic sensi iza ion, he
e ec s o RSV we e ansien . Compa able wi h ou own
s udy wi h B. pe ussis, Lukacs e al. [55] ound ha an ini ial
RSV in ec ion can ini ia e a p o-as hma ic en i onmen ha
p omo es a mo e se e e as hma ic esponse, e en when he
alle gic esponse is ini ia ed a a ime a e clea ance o he
RSV-induced eac ions. Likewise, OVA sensi iza ion o mice
in ec ed in a enously wi h Lis e ia monocy ogenes, con e s
a non-le hal in ec ion o a le hal disease. In ha model IL-10
plays a c i ical ole in he supp ession o an i-lis e ial
esis ance in OVA-immunized mice [56]. O he g oups ha e
shown ha hea -killed Mycobac e ium bo is-BCG supp essed
he de elopmen o OVA-induced ai way eosinophila [57, 58].
In many o hese s udies he iming o sensi iza ion and
in ec ion influence he ou come, howe e , he exace ba ion
media ed by B. pe ussis appea s o be pe sis en and long
li ed (da a no shown). Taken oge he , hese s udies sugges
ha pa hogens may induce an al e ed cy okine en i onmen
in he con ex o ai way emodelling ha ul ima ely p o ides
o an exace ba ed as hma ic- ype esponse [55].
Immune coun e - egula ion based on Th1/Th2 mechanisms
o e en egula o y T cells sec e ing IL-10 ha e been sugges ed
as mechanisms ha could p o ec agains as hma. We show
he e ha his esponse mus be iewed in he b oade con ex
o he hos –pa hogen in e ac ion. B. pe ussis ulfils many o
he c i e ia o a po en immunomodula o ha should
p o ec agains as hma. Howe e , he po en influence on
ai way emodelling du ing in ec ion means ha his bac e -
ium has he opposi e e ec . This s udy clea ly shows ha
while he hygiene hypo hesis is an a ac i e heo y, i is o
limi ed alidi y as cu en ly s a ed. Fu he mo e, ou esul s
aise conce ns ega ding immunomodula o y he apies aimed
a he con e sion o Th2-domina ed alle gic inflamma o y
esponses in o Th1-domina ed esponses based on coun e -
egula ion ha may be o limi ed e ficacy, o e en ha m ul.
Acknowledgemen s
We hank M s Sheila Wo ell, M Joseph B ady and M s
Be nade e Ruane o hei expe echnical assis ance. This
wo k was suppo ed by g an s om he I ish HEA PRTL
p og amme (Da en Ennis). Be na d Mahon is a Wellcome
T us /HRB new blood ellow (GR 054236).
Re e ences
1 Ume su DT, McIn i e JJ, Akba i O, Macaubas C, DeK uy RH.
As hma: an epidemic o dys egula ed immuni y. Na Immunol
2002; 3:715–20.
2 Wilde JA, Collie DD, Wilson BS, Bice DE, Lyons CR, Lipscomb
MF. Dissocia ion o ai way hype esponsi eness om immuno-
globulin E and ai way eosinophilia in a mu ine model o alle gic
as hma. Am J Respi Cell Mol Biol 1999; 20:1326–34.
3 Ying S, Humbe M, Ba kans J e al. Exp ession o IL-4 and IL-5
mRNA and p o ein p oduc by CD41and CD81T cells,
eosinophils, and mas cells in b onchial biopsies ob ained om
a opic and nona opic (in insic) as hma ics. J Immunol 1997;
158:3539–44.
4 Holga e ST, Da ies DE, Lackie PM, Wilson SJ, Puddicombe SM,
Lo dan JL. Epi helial–mesenchymal in e ac ions in he pa hogen-
esis o as hma. J Alle gy Clin Immunol 2000; 105:193–204.
5 Del P e e G, Maggi E, Pa onchi P e al. IL-4 is an essen ial ac o
o he IgE syn hesis induced in i o by human T cell clones and
hei supe na an s. J Immunol 1988; 140:4193–8.
6 Swain SL, Weinbe g AD, English M, Hus on G. IL-4 di ec s he
de elopmen o Th2-like helpe e ec o s. J Immunol 1990;
145:3796–806.
7 Haczku A, Takeda K, Hamelmann E e al. CD23 deficien mice
de elop alle gic ai way hype esponsi eness ollowing sensi iza ion
wi h o albumin. Am J Respi C i Ca e Med 1997; 156:1945–55.
8 Pad id PA, Ma hu M, Li X e al. CTLA4Ig inhibi s ai way
eosinophilia and hype esponsi eness by egula ing he de elop-
men o Th1/Th2 subse s in a mu ine model o as hma. Am J
Respi Cell Mol Biol 1998; 18:453–62.
9 He z U, Lumpp U, Da Palma JC e al. The ele ance o mu ine
animal models o s udy he de elopmen o alle gic b onchial
as hma. Immunol Cell Biol 1996; 74:209–17.
10 S achan DP. Family size, in ec ion and a opy: he fi s decade o
he hygiene hypo hesis. Tho ax 2000; 55 (Suppl. 1):S2–10.
11 Tan isi a KG, Weiss ST. Childhood in ec ions and as hma: a he
c oss oads o he hygiene and Ba ke hypo heses. Respi Res 2001;
2:324–7.
12 Lipscomb MF, Wilde JA. Immune dys egula ion as a cause o
alle gic as hma. Cu Opin Pulm Med 1999; 5:10–20.
8D. P. Ennis e al.
CEA : 2042
2004 Blackwell Publishing L d, Clinical and Expe imen al Alle gy
BWUK CEA 2042.PDF 19-Jul-04 19:1 844323 By es 10 PAGES
UNCORRECTED PROOF
13 Mosmann TR, Co man RL. TH1 and TH2 cells: di e en pa e ns
o lymphokine sec e ion lead o di e en unc ional p ope ies.
Annu Re Immunol 1989; 7:145–73.
14 Mills KH, Ryan M, Ryan E, Mahon BP. A mu ine model in which
p o ec ion co ela es wi h pe ussis accine e ficacy in child en
e eals complemen a y oles o humo al and cell-media ed
immuni y in p o ec ion agains Bo de ella pe ussis. In ec
Immunol 1998; 66:594–602.
15 McGui k P, McCann C, Mills KH. Pa hogen-specific T egula o y
1 cells induced in he espi a o y ac by a bac e ial molecule ha
s imula es in e leukin 10 p oduc ion by dend i ic cells: a no el
s a egy o e asion o p o ec i e T helpe ype 1 esponses by
Bo de ella pe ussis. J Exp Med 2002; 195:221–31.
16 Mahon BP, Sheahan BJ, G i fin F, Mu phy G, Mills KH. A ypical
disease a e Bo de ella pe ussis espi a o y in ec ion o mice wi h
a ge ed dis up ions o in e e on-gamma ecep o o immunoglo-
bulin mu chain genes. J Exp Med 1997; 186:1843–51.
17 Sa o Y, Izumiya K, Sa o H, Cowell JL, Mancla k CR. Ae osol
in ec ion o mice wi h Bo de ella pe ussis. In ec Immunol 1980;
29:261–6.
18 Mo oka a T, Ishikawa J, Ida K, Yamada T. C57BL/6 mice a e
mo e suscep ible o an igen-induced pulmona y eosinophilia han
BALB/c mice, i espec i e o sys emic T helpe 1/T helpe 2
esponses. Immunology 1999; 98:345–51.
19 Mahon BP, B ady MT, Mills KH. P o ec ion agains Bo de ella
pe ussis in mice in he absence o de ec able ci cula ing an ibody:
implica ions o long- e m immuni y in child en. J In ec Dis 2000;
181:2087–91.
20 Hamelmann E, Gel and EW. Animal models o ai way sensi iza-
ion. Cu P o ocols Immunol 1999.
21 Hamelmann E, Schwa ze J, Takeda K e al. Nonin asi e
measu emen o ai way esponsi eness in alle gic mice using
ba ome ic ple hysmog aphy. Am J Respi C i Ca e Med 1997;
156:766–75.
22 Mahon BP, Ryan MS, G i fin F, Mills KH. In e leukin-12 is
p oduced by mac ophages in esponse o li e o killed Bo de ella
pe ussis and enhances he e ficacy o an acellula pe ussis accine
by p omo ing induc ion o Th1 cells. In ec Immunol 1996;
64:5295–301.
23 Magnan AO, Mely LG, Camilla CA e al. Assessmen o he Th1/
Th2 pa adigm in whole blood in a opy and as hma. Inc eased IFN-
gamma-p oducing CD8(1) T cells in as hma. Am J Respi C i
Ca e Med 2000; 161:1790–6.
24 Ge gen PJ, Weiss KB. Changing pa e ns o as hma hospi aliza ion
among child en: 1979 o 1987. JAMA 1990; 264:1688–92.
25 Hansen G, Be y G, DeK uy RH, Ume su DT. Alle gen-specific Th1
cells ail o coun e balance Th2 cell-induced ai way hype eac i i y bu
cause se e e ai way inflamma ion. J Clin In es 1999; 103:175–83.
26 Bousque J, Je e y PK, Busse WW, Johnson M, Vignola AM.
As hma. F om b onchocons ic ion o ai ways inflamma ion and
emodeling. Am J Respi C i Ca e Med 2000; 161:1720–45.
27 Zuany-Amo im C, Manlius C, T ifilie A e al. Long- e m
p o ec i e and an igen-specific e ec o hea -killed Mycobac e ium
accae in a mu ine model o alle gic pulmona y inflamma ion.
J Immunol 2002; 169:1492–9.
28 Zuany-Amo im C, Sawicka E, Manlius C e al. Supp ession o
ai way eosinophilia by killed Mycobac e ium accae-induced
alle gen-specific egula o y T-cells. Na Med 2002; 8:625–9.
29 La aille JJ, Kee e FV, Hsu AL e al. Myelin basic p o ein-specific
T helpe 2 (Th2) cells cause expe imen al au oimmune encephalo-
myeli is in immunodeficien hos s a he han p o ec hem om
he disease. J Exp Med 1997; 186:307–12.
30 Genain CP, Abel K, Belma N e al. La e complica ions o immune
de ia ion he apy in a nonhuman p ima e. Science 1996; 274:2054–7.
31 Pakala SV, Ku e MO, Ka z JD. T helpe 2 (Th2) T cells induce
acu e panc ea i is and diabe es in immune-comp omised nonobese
diabe ic (NOD) mice. J Exp Med 1997; 186:299–306.
32 Wang CC, Rook GA. Inhibi ion o an es ablished alle gic esponse
o o albumin in BALB/c mice by killed Mycobac e ium accae.
Immunology 1998; 93:307–13.
33 Mahon BP, Mills KH. In e e on-gamma media ed immune
e ec o mechanisms agains Bo de ella pe ussis. Immunol Le
1999; 68:213–7.
34 Flak TA, Goldman WE. Au o oxici y o ni ic oxide in ai way
disease. Am J Respi C i Ca e Med 1996; 154:S202–6.
35 Luke KE, Collie JL, Kolodziej EW, Ma shall GR, Goldman WE.
Bo de ella pe ussis acheal cy o oxin and o he mu amyl pep ides:
dis inc s uc u e-ac i i y ela ionships o espi a o y epi helial
cy opa hology. P oc Na l Acad Sci USA 1993; 90:2365–9.
36 Holga e ST. Epi helial damage and esponse. Clin Exp Alle gy
2000; 30 (Suppl. 1):37–41.
37 Lazaa AL, Pane ie i RA J . Ai way smoo h muscle as an
immunomodula o y cell: a new a ge o pha maco he apy? Cu
Opin Pha macol 2001; 1:259–64.
38 G uns ein MM, Hakona son H, Lei e J e al. Au oc ine signaling
by IL-10 media es al e ed esponsi eness o a opic sensi ized
ai way smoo h muscle. Am J Physiol Lung Cell Mol Physiol 2001;
281:L1130–7.
39 Lee CG, Home RJ, Elias JA e al. T ansgenic o e exp ession o
in e leukin (IL)-10 in he lung causes mucus me aplasia, issue
inflamma ion, and ai way emodeling ia IL-13-dependen and -
independen pa hways. J Biol Chem 2002; 277:35466–74.
40 Lopez AF, Sande son CJ, Vadas MA e al. Recombinan human
in e leukin 5 is a selec i e ac i a o o human eosinophil unc ion.
J Exp Med 1988; 167:219–24.
41 Wu CA, Pudding on L, Th all RS. Mu ine cy omegalo i us
in ec ion al e s Th1/Th2 cy okine exp ession, dec eases ai way
eosinophilia, and enhances mucus p oduc ion in alle gic ai way
disease. J Immunol 2001; 167:2798–807.
42 Nasee T, Minshall EM, Hamid Q. Exp ession o IL-12 and IL-13
mRNA in as hma and hei modula ion in esponse o s e oid
he apy. Am J Respi C i Ca e Med 1997; 155:845–51.
43 Humbe M, Du ham SR, Kay AB e al. Ele a ed exp ession o
messenge ibonucleic acid encoding IL-13 in he b onchial mucosa
o a opic and nona opic subjec s wi h as hma. J Alle gy Clin
Immunol 1997; 99:657–65.
44 Wal e DM, McIn i e JJ, Ume su DT. C i ical ole o IL-13 in he
de elopmen o alle gen-induced ai way hype eac i i y. J Immu-
nol 2001; 167:4668–75.
45 Wei LH, Jacobs AT, Mo is SM J , Igna o LJ. IL-4 and IL-13
up egula e a ginase I exp ession by cAMP and JAK/STAT6
pa hways in ascula smoo h muscle cells. Am J Physiol Cell
Physiol 2000; 279:C248–56.
46 Tliba O, Deshpande D, Am ani Y. L-13 enhances agonis -e oked
calcium signals and con ac ile esponses in ai way smoo h muscle.
B J Pha macol 2003; 140:1159–62.
47 Heinzmann A, Mao XQ, Shi akawa T e al. Gene ic a ian s o
IL-13 signalling and human as hma and a opy. Hum Mol Gene
2000; 9:549–59.
48 Am ani Y, Moo e PE, Ho man R, Sho e SA, Pane ie i RA J .
In e e on-gamma modula es cys einyl leuko iene ecep o -1
exp ession and unc ion in human ai way myocy es. Am J Respi
C i Ca e Med 2001; 164:2098–101.
49 Cho SH, S anciu LA, Begishi ili T, Ba es PJ, Holga e ST, Johns on
SL. Pe iphe al blood CD41and CD81T cell ype 1 and ype 2
cy okine p oduc ion in a opic as hma ic and no mal subjec s. Clin
Exp Alle gy 2002; 32:427–33.
50 Cemb zynska-Nowak M, Szkla z E, Inglo AD, Teodo czyk-
Injeyan JA. Ele a ed elease o umo nec osis ac o -alpha
and in e e on-gamma by b onchoal eola leukocy es om
pa ien s wi h b onchial as hma. Am Re Respi Dis 1993;
147:291–5.
51 Makela MJ, Kanehi o A, Bo ish L e al. IL-10 is necessa y o he
exp ession o ai way hype esponsi eness bu no pulmona y
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