UNCORRECTED PROOF
IMLET 4082 1–10
Immunology Le e s xxx (2004) xxx–xxx
Whole-cell pe ussis accine p o ec s agains
Bo de ella pe ussis exace ba ion o alle gic as hma
3
4
Da en P. Ennisaa, Joseph P. Cassidyb, Be na d P. Mahona,∗
5
aMucosal Immunology Labo a o y, Ins i u e o Immunology, NUI Maynoo h, I eland6bDepa men o Ve e ina y Pa hology, Uni e si y College Dublin, Dublin, I eland7
Recei ed 23 Augus 2004; ecei ed in e ised o m 1 Oc obe 2004; accep ed 4 Oc obe 2004
8
Abs ac 9
The p e alence o as hma and alle gic disease has inc eased in many coun ies and he e has been specula ion ha immuniza ion p omo es
alle gic sensi iza ion. Bo de ella pe ussis in ec ion exace ba es alle gic as hma ic esponses. We in es iga ed whe he whole-cell pe ussis
accine (Pw) enhanced o p e en ed B. pe ussis induced exace ba ion o alle gic as hma. G oups o mice we e immunized wi h Pw, in ec ed
wi h B. pe ussis and/o sensi ized o o albumin. Immunological, pa hological and physiological changes we e measu ed o assess he impac
o Pw immuniza ion on immune de ia ion and ai way unc ion. Pw immuniza ion modula ed o albumin-speci ic se um IgE p oduc ion,
and educed local and sys emic IL-13 and o he cy okine esponses o sensi izing alle gen. His opa hological examina ion e ealed Pw
immuniza ion educed he se e i y o ai way pa hology and dec eased b onchial hype eac i i y o me hacholine exposu e. Pw does no
enhance ai way IL-13 and consequen ly does no enhance bu p o ec s agains he exace ba ion o alle gic esponses. We ind no e idence
o Pw con ibu ing o alle gic as hma, bu a he p o ide e idence o a mechanism whe eby whole-cell pe ussis accina ion has a p o ec i e
ole.
10
11
12
13
14
15
16
17
18
19
© 2004 Else ie B.V. All igh s ese ed.
20
Keywo ds: Bo de ella pe ussis; Vaccine; As hma; IL-13; Alle gen
21
22
1. In oduc ion23
As hma is a ch onic disease o he espi a o y ac o in-24
c easing p e alence in de eloped socie ies [1]. The cu en 25
unde s andingo alle gicas hmais ha i esul s omab eak-26
down in he no mal ole ance o inhaled an igens, associa ed
27
wi h Th2 cy okine p oduc ion [2,3]. The in lamma o y e-28
sponse in as hma is igh ly associa ed wi h ai way hype e-29
sponsi eness, inc eased mucus p oduc ion and an in il a ion30
o he b onchial mucosa wi h CD4+ T-cells [4]. The e is e -31
idence o an al e ed local T-cell esponse in a ou o Th232
cy okine elease(IL-4,IL-5andIL-13) esul inginB-celliso-
33
ype swi ching o IgE, ec ui men o eosinophils, basophils
34
andmas cellsandp oduc iono in lamma o ymedia o s[5].
35
∗Co esponding au ho . Tel.: +353 1 7083835; ax: +353 1 7086337.
E-mail add ess: [email protected] (B.P. Mahon).
The mu ine OVA model o ai way hype esponsi eness ex- 36
hibi s many o he ea u es o human as hma, including ai - 37
way hype eac i i y, in lamma ion and inc eased se um IgE 38
le els [6,7]. Th2 cells sec e ing IL-4, IL-5, and IL-13 play a 39
cen al oleinini ia ingandsus aining heas hma ic esponse 40
in his model [8]. While Th2 cells p omo e ai way in lamma- 41
ion in as hma, i has been p oposed ha Th1 cells p o ec 42
agains alle gic disease by an agonizing Th2 ac i i y. In ec- 43
ious diseases ha induce Th1 ype esponses, migh hampe 44
he de elopmen o alle gen-speci ic Th2 cells and p e en 45
alle gy [9].46
Epidemiologicalandclinicals udiesha esugges edalink 47
be ween he ela i e absence o in ec ious diseases and he 48
inc ease in alle gic diso de s [10,11]; his is e e ed o as he 49
‘hygienehypo hesis’.I p edic s ha in ec ionsp e en hein- 50
duc ion o alle gen-speci ic Th2 cells h ough an agonism o 51
heinduc iono egula o yT-cells,pa icula lydu ingneona- 52
10165-2478/$ – see on ma e © 2004 Else ie B.V. All igh s ese ed.
2doi:10.1016/j.imle .2004.10.011
UNCORRECTED PROOF
IMLET 4082 1–10
2D.P. Ennisa e al. / Immunology Le e s xxx (2004) xxx–xxx
al and ea ly childhood de elopmen [10,12]. Howe e , he e
53
a e da a ha con ound his in e p e a ion, inc eased IFN-␥54
is seen in as hma ic pa ien s compa ed wi h no mal subjec s55
[3,13]. Alle gen-speci ic Th1 cells also ail o coun e ac ai -
56
way hype esponsi eness in mu ine models [14]. Fu he -57
mo e, se e al s udies ha e sugges ed ha i al/bac e ial in-58
ec ions do no p o ec bu exace ba e disease. Respi a o y
59
syncy ial i us, commonly associa ed wi h lowe lung in-60
ec ions in in ancy, is known o exace ba e as hma [15,16].61
As does Bo de ella pe ussis [17]. Consequen ly, compe ing
62
in e p e a ions o he pa hogenesis o as hma ha e been p o-63
posed [18,19].64
B. pe ussis is a G am-nega i e bac e ium and he
65
causa i e agen o pe ussis o “whooping cough”, a espi-
66
a o y disease ha emains a signi ican cause o mo bidi y67
and mo ali y in in an s wo ldwide. I is a highly con agious
68
disease, and can occu a any age, hough se e e illness is69
mo e common in young un-immunized child en. B. pe ussis
70
in ec ion induces Th1 esponses [20,21] and can be mod-71
elled by espi a o y challenge o mice, which co ela es well72
o esponses in humans [22].73
The e has been specula ion abou he possible p omo ion74
o alle gy by common childhood accina ions [23,24].A75
subs an ial p opo ion o child en p edisposed o alle gy and
76
as hma may no be ully immunized because o public app e-77
hension su ounding immuniza ion [25]. A numbe o s ud-
78
ies ha e analysed he p e alence o alle gic sensi iza ion and
79
a opic disease in ela ion o immuniza ion [24,26]. G ube e
80
al. ound ha child en wi h highe immuniza ion co e age
81
seemed o acqui e ansien p o ec ion agains de elopmen 82
o a opy in he i s yea s o li e [26]. In con as , Hu wi z83
and Mo gens e n sugges ed ha diph he ia/pe ussis/ e anus84
(DTP) immuniza ion appea ed o be associa ed wi h an85
inc eased isk o subsequen as hma o o he alle gies86
[24].
87
Two di e en ypes o pe ussis accine ha e been em-
88
ployed in in an immuniza ion p og ammes. The whole-cell
89
pe ussis accine (Pw) consis s o hea / o malin inac i a ed
90
i ulen whole bac e ia whe eas he pe ussis acellula ac-
91
cine (Pa) is composed o pu i ied componen s o he bac e ia92
(Pa), ypically including inac i a ed pe ussis oxin. Pw im-93
muniza ion has a high e icacy and is associa ed wi h he94
induc ion o an igen-speci ic Th1 cells [21,27,28], bu has95
been associa ed wi h eac ogenici y. In con as Pa immu-
96
niza ion induces a mixed Th1/Th2 esponse in child en and
97
in mu ine models, bu has educed eac ogenici y [29].I has
98
been sugges ed ha p omo ion o alle gy may occu di ec ly,
99
by adminis e ing po en ially p o-alle gic accines, o indi-100
ec ly, by hinde ing he Th1-p omo ing e ec o in ec ious101
agen s. Pe ussis accina ion ac s as an adju an o an igen-102
speci ic esponsesin labo a o yanimals[30];ac i epe ussis103
oxin, is known o enhance immunoglobulin E (IgE) o ma-104
ion in animal models [31] and has been linked wi h a shi 105
owa d Th2-like cy okines in humans [32,33].
106
In ec ionwi hB.pe ussismodula esalle genp imingand107
he se e i y o ai way pa hology in a mu ine model o al-
108
le gic as hma [17] and we ha e p e iously shown ha Pw 109
immuniza ion induces a simila immune esponse o in ec- 110
ion [34] and ha al hough a iables such as ou e, dose 111
and iming in luence T-cell esponses in animal models, Pw 112
is a consis en induce o Th1 esponses [20]. In o de o 113
es whe he immuniza ion wi h Pw exace ba ed as hma, we 114
employed a well-cha ac e ized mu ine model o whole-cell 115
pe ussis accina ion and B. pe ussis in ec ion in combina- 116
ion wi h he mu ine OVA model o ai way hype esponsi e- 117
ness. We show ha al hough Pw induces a Th1 ype im- 118
mune esponse o B. pe ussis in ec ion, i does no exac- 119
e ba e pa hology in a model o alle gic as hma. Ou ind- 120
ings demons a e ha Pw immuniza ion p e en s B. pe us- 121
sis enhancemen o OVA-induced IL-10 and IL-13, which 122
esul s in a subsequen dec ease in ai way hype esponsi e- 123
ness and pa hology. This s udy inds no e idence o a mech- 124
anism o suppo specula ion linking Pw immuniza ion and 125
as hma. 126
2. Ma e ials and me hods 127
2.1. Animals and expe imen al app oach 128
Six- o 8-week-old emale BALB/c (Ha lan, UK) mice 129
we e used unde he guidelines o he I ish Depa men 130
o Heal h and he esea ch e hics commi ee o he Na- 131
ional Uni e si y o I eland Maynoo h. The expe imen al 132
app oach is ou lined in Table 1, b ie ly g oups o mice 133
we e immunized wi h whole-cell pe ussis accine (Pw), 134
in ec ed wi h B. pe ussis, and hen sensi ised o o albu- 135
min (OVA) a he peak o in ec ion as de ailed below. Con- 136
ol mice ecei ed simila ea men in which 0.9% (w/ ) 137
(aq) NaCl (he ea e e med Saline) eplaced expe imen al 138
ea men . 139
2.2. Immuniza ion, sensi iza ion and ai way deli e y o 140
OVA 141
Fou g oups o a leas hi y- i e 6–8-week-old emale 142
BALB/c mice (Pw, PwBp, PwOVA and PwBpOVA) we e 143
immunized i.p. wi h 0.16I.U. o whole-cell pe ussis accine 144
(Pw) (Thi d In e na ional S anda d, 1998, pe ussis whole- 145
cell accine, NIBSC, UK), equi alen o 1/25 h o he hu- 146
man dose acco ding o he schedule ou lined in Table 1.A 147
0 day mice we e in ec ed wi h B. pe ussis, selec ed g oups 148
we e hen sensi ized wi h o albumin (OVA). Sensi iza ion 149
in ol ed 100␮g OVA (G ade V; Sigma, Do se , UK) emul- 150
si ied in Alhyd ogel®adju an (Supe os Biosec o , Swe- 151
den) (1mg/mouse aluminium hyd oxide) adminis e ed as 152
0.2ml i.p. a 10 and 24 days. Con ol g oup (C l) ecei ed 153
saline alone (i.p.). On 35, 36, and 37 days, PwOVA and 154
PwBpOVA sensi ized mice ecei ed 10␮l con aining 50␮g155
OVA in a-nasally (i.n.) whe eas emaining g oups ecei ed 156
saline only (Table 1). All expe imen s we e epea ed a leas 157
wice. 158
UNCORRECTED PROOF
IMLET 4082 1–10
D.P. Ennisa e al. / Immunology Le e s xxx (2004) xxx–xxx 3
Table 1
Expe imen al design
Time (days)
−42 −14 0 10 24 35/36/37
G oupa
Pw Pw Pw Saline ae osol Saline (i.p.) Saline (i.p. and i.n.) Saline (i.n.)
Pw and B. pe ussis in ec ion
(PwBp) Pw Pw B. pe ussis ae osol in ec ion Saline (i.p.) Saline (i.p. and i.n.) Saline (i.n.)
Pw and OVA sensi iza ion
(PwOVA) Pw Pw Saline ae osol OVA (i.p.) OVA (i.p and i.n.) OVA (i.n.)
Pw, B. pe ussis in ec ion and
OVA sensi iza ion (PwBpOVA) Pw Pw B. pe ussis ae osol in ec ion OVA (i.p.) OVA (i.p and i.n.) OVA
G oupb
Con ol (C l) Saline (i.p.) Saline (i.p.) Saline ae osol Saline (i.p.) Saline (i.p. and i.n.) Saline (i.n.)
B. pe ussis in ec ion (Bp) Saline (i.p.) Saline (i.p.) B. pe ussis ae osol in ec ion Saline(i.p.) Saline (i.p. and i.n.) Saline (i.n.)
OVA sensi iza ion (OVA) Saline (i.p.) Saline (i.p.) Saline ae osol OVA (i.p.) OVA (i.p and i.n.) OVA (i.n.)
B.pe ussis in ec ionandOVAsen-
si iza ion (BpOVA) Saline (i.p.) Saline (i.p.) B. pe ussis ae osol in ec ion OVA (i.p.) OVA (i.p and i.n.) OVA (i.n.)
aG oups o 6–8-week-old emale BALB/c mice we e immunized (i.p.) and boos ed wi h whole-cell pe ussis accine (Pw) a −42 and −14 days. On 0 day,
mice we e ei he sham in ec ed o in ec ed wi h B. pe ussis (Bp) by ae osol. A 10 and 24 days selec ed g oups we e sensi ised o OVA by i.p alone o i.p and
i.n. ou es, espec i ely. On 35, 36 and 37 days mice we e exposed o ei he saline o OVA by he i.n. ou e (i.e. 25, 26, and 27 days pos -OVA p iming and a e
bac e ial clea ance). Bac e ial bu dens in he ai ways we e measu ed be ween 0 and 37 days. All o he eadou s, including ple hysmog aphy we e pe o med
a 37 days.
bFo compa ison, u he g oups o unimmunized (sham immunized) mice we e ea ed as ollows: con ol (C l) mice we e sham in ec ed on 0 day and
sham sensi ised wi h saline. A second g oup (Bp) we e in ec ed wi h B. pe ussis a 0 day and sham sensi ized. The hi d g oup (OVA) we e sham in ec ed
bu sensi ized wi h OVA (100␮g, i.p.) a 10 and 24 days and again (50␮g i.n.) a 24, 35, 36, and 37 days. A sepa a e g oup (BpOVA) we e in ec ed wi h
B. pe ussis on 0 day, and sensi ized as abo e. Each expe imen was epea ed a leas wice, on each occasion n>35 mice pe g oup, bac e ial bu dens we e
measu ed be ween 0 and 37 days, all o he eadou s we e a 37 days.
2.3. B. pe ussis ae osol in ec ion
159
Respi a o y in ec ion was ini ia ed by ae osol challenge160
wi h B. pe ussis s ain W28, ollowing g ow h unde agi a-161
ion condi ions a 37◦C in S aine -Schol e liquid medium.162
Bac e ia om a log-phase cul u e we e esuspended a a con-163
cen a ion o 2×1010 CFU/ml in 1% (w/ ) casein in 0.9%164
(w/ ) saline. The challenge inoculum was adminis e ed o165
g oups o mice on 0 day (Bp, PwBp and PwBpOVA g oups).166
Adminis a ion was by ae osol o e a pe iod o 15min using
167
a nebulize . G oups o ou o mo e mice we e killed a a i-
168
ous ime poin s a e ae osol challenge o assess he numbe
169
o iable B. pe ussis in he lungs. Remaining mice ecei ed
170
a simila ae osol o s e ile saline alone.
171
2.4. Enume a ion o iable bac e ia in he lungs
172
Lungs we e emo ed asep ically in o 1ml o s e ile phys-173
iological saline wi h 1% casein. Hund ed mic oli es o se i-174
ally dilu ed homogena e om indi idual lungs we e placed175
on o iplica e Bo de -Gengou aga pla es and he numbe o 176
CFU de e mined a e incuba ion a 37◦C o 4 days. Re-177
sul s a e epo ed as he mean numbe o B. pe ussis CFU178
(±S.E.M.) o indi iduallungs,eachde e minedin iplica e,179
om ou o mo e mice pe ime poin . All expe imen s we e180
epea ed wice.
181
2.5. B onchoal eola la age
182
B onchoal eola la age luids (BALF) we e ob ained by183
cannula ion o he achea ollowed by epea adminis a ion184
and aspi a ion o 0.5ml PBS pe mouse. This was pooled 185
om i e mice ( o al 2.5ml) pe expe imen al g oup. All ex- 186
pe imen s we e pe o med a leas wice. Dilu ed BALF was 187
assessed o he p esence o cy okines. 188
2.6. Measu emen o OVA and B. pe ussis-specific 189
an ibody 190
OVA and B. pe ussis-speci ic IgG1, 2a, 2b, and 3 p esen 191
in collec ed se a we e measu ed on day 37 by ELISA as p e- 192
iously desc ibed [35,36]. To al and OVA-speci ic IgE was 193
measu ed using a a an i-mouse IgE monoclonal an ibody 194
(BD, Pha mingen, San Diego, CA, USA). The IgE concen- 195
a ion was exp essed as ␮g/ml a e compa ison o mu ine 196
IgE s anda ds. 197
2.7. T-cell p oli e a ion assays 198
Spleen cells (2×106/ml) om in ec ed, sensi ized and 199
con ol mice (n=4 o mo e pe g oup) we e es ed o 200
in i o p oli e a ion agains hea -inac i a ed B.pe ussis 201
(1×104CFU/ml), OVA (20␮g/ml), Concana alin A (Con 202
A) (5␮g/ml, posi i e con ol), o medium alone (nega i e 203
con ol). A e 72h, cell p oli e a ion was assessed by liquid 204
scin illa ion coun ing o [3H]- hymidine inco po a ion and 205
esul swe eexp essedasmeanCPMo iplica ewells±S.E. 206
A he 72h ime poin , cul u e supe na an s we e sampled o 207
cy okine analysis, al hough he kine ics o cy okine p oduc- 208
ion a ies his ime poin has p e iously p o ed accep able 209
o de ec ion o mos cy okines [22].210
UNCORRECTED PROOF
IMLET 4082 1–10
4D.P. Ennisa e al. / Immunology Le e s xxx (2004) xxx–xxx
2.8. Cy okine measu emen
211
Concen a ions o IL-5, IL-10, IL-13 and IFN-␥ om
212
spleen, and BALF we e assessed by ELISA (BD, Pha min-213
gen, San Diego, CA, USA). Cy okine concen a ions we e
214
calcula ed by compa ison wi h known cy okine s anda ds as215
p e iously desc ibed [35], all de e mina ions we e made in216
iplica e, esul s a e p esen ed as mean cy okine concen a-217
ion (±S.E.M.).
218
2.9. Whole body ple hysmog aphy
219
Ai way esponsi enesson37dayswasassessedbyme ha-
220
choline (MCh) induced ai low obs uc ion om conscious221
mice using whole-body ple hysmog aphy (Buxco Elec on-222
ics, Sha on, CT, USA) as p e iously desc ibed [37]. Pul-
223
mona yai lowobs uc ionwasmeasu edbyenhancedpause224
(PenH), a alue de e mined om he a io o expi a o y ime225
and elaxa ion ime o peak expi a o y low and peak inspi-226
a o y low and hough o co ela e wi h ai way esponsi e-227
ness. Measu emen s we e ob ained a e exposu e o mice228
o 3min o PBS (baseline) ollowed by inc emen al doses
229
(3.3mg–50mg/ml) o MCh deli e ed by ae osol [38].
230
2.10. Respi a o y ac his ology231
Animals (n=5 pe g oup pe expe imen ) we e sac i iced232
a 37 days. Lungs we e emo ed, ixed in a pa a o malde-233
hyde/lysine/pe ioda e ixa i e, pa a in embedded, sec ioned234
and s ained using he haema oxylin and eosin (H&E), Dis-235
combes (iden i ica ion o eosinophils), alcian blue (iden-
236
i ica ion o mucus), PAS (assessmen o basemen mem-
237
b ane hickness), azu e-A (iden i ica ion o mas cells) and
238
Van Gieson (iden i ica ion o ib osis) me hods. His opa ho-
239
logical changes e iden we e g aded acco ding o a semi-240
quan i a i e sco ing sys em as mild, mode a e o se e e by241
wo esea che s wi hou p io knowledge o he ea men 242
g oup using a p e iously es ablished sco ing sys em [17].243
All expe imen s we e pe o med a leas wice.244
2.11. S a is ical me hods
245
Resul s a e exp essed as he mean±S.E.M. o he indi-246
ca ednumbe o animals.AS uden ’s - es wasused ode e -
247
mine signi icance among he g oups. A alue o P<0.05 was248
conside ed signi ican . Analyses we e pe o med using he249
G aph-Pad P ismTM so wa e (G aphPad, San Diego, CA).250
3. Resul s
251
3.1. O albumin sensi iza ion does no impai
252
accine-media ed clea ance o B. pe ussis253
The mu ine OVA model o ai way hype esponsi eness
254
induces a powe ul Th2 esponse [7] whe eas bo h B. pe -255
Fig. 1. Cou se o B. pe ussis in ec ion in expe imen al and con ol mice.
G oups o mice we e sac i iced 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 imen s and a e p e-
sen ed as mean (±S.E.M.) CFU in he lungs, de e mined indi idually om
ou mice a each ime poin , and o each expe imen al g oup. Da a o C l
and OVA g oups ha e been o se om ze o o cla i y.
ussis in ec ion and Pw immuniza ion induce a powe ul Th1 256
esponse [29]. In o de o examine immune c oss- egula ion 257
and po en ial in e e ence wi h immuniza ion, we es ed he 258
e ec o OVA sensi iza ion upon he de elopmen o a p o- 259
ec i e esponse o in ec ion in Pw immunized and non- 260
immunized mice. Mice ecei ed combina ions o OVA sen- 261
si iza ion, Pw immuniza ion and ae osol challenge wi h i - 262
ulen B. pe ussis (Table 1). G oups o mice in ec ed wi h 263
B. pe ussis (Bp and BpOVA) showed simila kine ics o 264
bac e ial clea ance (Fig. 1), indica ing ha OVA sensi iza- 265
ion does no in luence bac e ial clea ance a es. Likewise, 266
OVA sensi ized and non-sensi ized mice ha had been im- 267
munized p io o bac e ial challenge (PwBpOVA and PwBp, 268
espec i ely) showed iden ical kine ics o clea ance. No bac- 269
e ia we e eco e ed om he OVA sensi ised o con ol 270
(C l) g oups, which we e unin ec ed bu ecei ed saline by 271
ae osol (Fig. 1). The bac e ial bu den in he Bp and BpOVA 272
g oups peaked a 10 days and declined he ea e . Pw im- 273
munized mice clea ed subsequen in ec ion by B. pe us- 274
sis by 7 days. In con as unimmunized mice (Bp and he 275
BpOVA g oups) only showed comple e bac e ial clea ance 276
by 35 days (Fig. 1). The e o e sensi iza ion wi h OVA did 277
no impai accine-media ed clea ance o B. pe ussis in his 278
model. 279
3.2. OVA-specific IgE p oduc ion is modula ed by Pw 280
immuniza ion 281
The goal o his s udy was o examine he in luence o 282
Pw immuniza ion on esponses associa ed wi h alle gic sen- 283
si iza ion. Al hough OVA-induced sensi iza ion does no im- 284
pai accine-media ed clea ance o B. pe ussis, i was pos- 285
sible ha Pw in luenced alle gic sensi iza ion. OVA-speci ic 286
IgG was no de ec ed om mice in ec ed wi h B. pe ussis 287
UNCORRECTED PROOF
IMLET 4082 1–10
D.P. Ennisa e al. / Immunology Le e s xxx (2004) xxx–xxx 5
Fig. 2. Se um IgE and IgG subclasses elici ed by Pw accina ion, bac e ial in ec ion and alle gic sensi iza ion. (A) B. pe ussis o (B) OVA-speci ic se um
an ibody esponses by IgG subclasses elici ed in whole-cell B. pe ussis (Pw), accina ed plus Bp in ec ion (PwBp), accina ed plus sensi ized (PwOVA),
o in mice accina ed hen in ec ed wi h B. pe ussis p io o sensi iza ion (PwBpOVA), esponse om mice ha ecei ed OVA sensi iza ion alone (OVA)
o combined wi h in ec ion (BpOVA) a e shown o compa ison. (C) B. pe ussis-speci ic se um IgE and (D) OVA-speci ic se um IgE p esen om each
expe imen al g oup. Resul s a e ep esen a i e o wo expe imen s exp essed as geome ic mean i e o ng/ml o an ibody (±S.E.M.) om ou animals each
de e mined independen ly in iplica e. *P<0.05 compa ed o he PwOVA ea ed g oup.
only; simila ly B. pe ussis-speci ic IgG could no be de-288
ec ed in OVA sensi ized animals, sugges ing no signi ican 289
c oss- eac ion be ween he wo immunogens (Fig. 2A and
290
B). An analysis o he se um an ibody subclasses e oked291
e ealed ha Pw p edominan ly induced pe ussis-speci ic292
IgG2a (Fig. 2A) as p e iously epo ed [28,34]. IgG2a e-293
mained he dominan subclass o pe ussis-speci ic an i-
294
body when Pw immuniza ion was combined wi h OVA sen-295
si iza ion (PwOVA) o B. pe ussis in ec ion (PwBp) al-
296
hough IgG1 and IgG3 was de ec ed in he la e g oups297
(Fig. 2A). Pw immuniza ion p io o OVA sensi iza ion298
(PwOVA) did no signi ican ly al e he dominan OVA-299
speci ic IgG1 esponse. In ec ion o hese mice wi h B. pe -300
ussis (PwBpOVA) did no b oaden he subclasses o OVA-301
speci ic an ibody de ec ed al hough he i e was inc eased302
(Fig. 2B). Taken oge he , hese da a indica e ha Pw im-303
muniza ion does no modula e se um IgG subclasses in-304
duced by alle gic sensi iza ion. In con as o he conse -305
a i e e ec s on se um IgG subclasses, signi ican di e -306
ences we e obse ed in he induc ion o IgE (Fig. 2C and
307
D). Pw induces li le B. pe ussis-speci ic IgE, and his is
308
no al e ed by OVA sensi iza ion o in ec ion (Fig. 2C).
309
OVA sensi iza ion induces high le els o OVA-speci ic IgE
310
bu his is signi ican ly educed by p io Pw immuniza ion
311
(Fig. 2D). Howe e , a combina ion o immuniza ion and in-
312
ec ion p io o OVA sensi iza ion esul ed in a signi ican e-313
duc ion o IgE (P<0.05) (Fig. 2D) compa ed o he PwOVA
314
g oup.315
3.3. Pw immuniza ion p e en s B. pe ussis 316
enhancemen o OVA-induced IL-10 and IL-13 317
B. pe ussis in ec ion enhances OVA-induced IL-10 and 318
IL-13 [17]. Pw immuniza ion has hi he o been ega ded as 319
inducing essen ially simila immune esponses o hose in- 320
duced by in ec ion [22]. In o de o dissec he in luence o 321
immuniza ion on ai way hype esponsi eness, we examined 322
cell-media edimmune esponses in he a iouss udy g oups. 323
Pw immuniza ion alone o in combina ion wi h B. pe ussis 324
in ec ion (Pw o PwBp) induced e y li le IL-5 bu s ong 325
IFN-␥ esponses (Fig. 3A and B). This was consis en wi h 326
he p o ec ion obse ed ea lie (Fig. 1) and p e ious da a 327
[34]. Pw immuniza ion educed le els o IL-5, IL-13 and 328
IFN-␥(Fig. 3A–C) in all immunized g oups sugges ing ha 329
Pw immuniza ion p e en s li e B. pe ussis enhancemen o 330
hese indices; mi o ing he educ ion in OVA-speci ic IgE 331
(Fig. 2D). In e es ingly, p e ious esul s ha e shown ha B. 332
pe ussis in ec ion induced speci ic IL-10 as well as IL-13 333
esponses [17]. He e we demons a e ha in con as o in- 334
ec ion, Pw-immuniza ion esul ed in signi ican ly educed 335
le els o IL-10, and IL-13 (Fig. 3A–D). 336
To ex end hese indings, we examined he le els o cy- 337
okines p esen in b onchoal eola la age luid (BALF) om 338
each g oup o mice. Pw immuniza ion alone induced li le o 339
no de ec able cy okines in BALF. As expec ed, OVA sensi i- 340
za ion induced high le els o IL-5, -10 and -13 bu he le els 341
o IL-10 and -13 in pa icula , known o ise in in ec ed mice, 342

UNCORRECTED PROOF
IMLET 4082 1–10
6D.P. Ennisa e al. / Immunology Le e s xxx (2004) xxx–xxx
Fig.3. Cellmedia edimmune esponses omspleen,elici edbyPw accina ion,bac e ialin ec ionandalle gicsensi iza ion.IL-5(A),IFN-␥(B),IL-13(C)and
IL-10 (D) esponses om spleen cell cul u es s imula ed wi h medium alone (− e con ol, ho izon al shading), hea inac i a ed B. pe ussis a 1×104CFU/ml
(ha ched ba ), OVA (open ba ) o Con A (+ e con ol, black ba ). Responses a e ep esen a i e o duplica e expe imen s each pe o med in iplica e on
indi idual samples om ou mice pe g oup and a e exp essed as mean (±S.E.M.). *P<0.05 compa ed o he PwOVA ea ed g oup.
we e educed in immunized mice ha had been in ec ed wi h343
B. pe ussis (PwBpOVA) (Fig. 4A–D).344
3.4. Pw immuniza ion p io o B. pe ussis in ec ion345
dec eases b onchial hype esponsi eness o sensi izing346
an igen
347
I has been p oposed ha p io Th1 esponses o bac e-
348
ial in ec ions p o ec agains alle gic disease howe e , Th1-349
inducing B. pe ussis in ec ion exace ba es ai way hype e-350
sponsi eness in OVA sensi ized mice. I migh be p edic ed351
ha Pw, which induces a e y simila immune esponse o B.352
pe ussiswouldha easimila exace ba ingin luence.In ac ,
353
his is no he case. We used whole body ple hysmog aphy as
354
a su oga e measu e o ai way eac i i y in mice immunized
355
wi h Pw and in ec ed wi h B. pe ussis p io o OVA sensi i-
356
za ion(Fig.5).P io immuniza ionwi hPwdoesno enhance
357
bu p o ec s agains B. pe ussis exace ba ed ai way hype e-358
sponsi eness in compa ison o con ols. S a is ical analysis
359
using wo-way analysis o a iance (ANOVA) showed ha 360
mice accina ed wi h Pw, and sensi ized o OVA ollowing
361
B.pe ussisin ec ion(PwBpOVA)displayedsigni ican ly e-362
duced b onchial hype eac i i y compa ed o BpOVA sensi-
363
ized animals (P<0.05) (Fig. 5D). Thus demons a ing ha 364
accina ion wi h Pw p o ec s agains B. pe ussis exace ba- 365
ion o alle gic as hma. 366
B. pe ussis in ec ion is known o modula e he quali y 367
o he in lamma o y in lux o he espi a o y ac , wi h a 368
ma ked educ ion in eosinophil numbe s accompanied by 369
a ying deg ees o epi helial hype plasia, mucus me aplasia, 370
and ai way pa hology [17]. Lung issue was assessed his o- 371
logically (Table 2). Minimal pa hology was obse ed in mice 372
immunized wi h Pw o hose immunized and in ec ed wi h 373
B. pe ussis (PwBp) (Fig. 6A and B). Pw and OVA sensi ized 374
(PwOVA) mice illus a ed mode a e mu al and pe i-ai way 375
in lamma ionwi haccompanyingmildmucusme aplasiaand 376
mode a e hype plasia o he epi helium (Fig. 6C). The com- 377
bina iono Pwimmuniza ion,B.pe ussisin ec ionandOVA 378
sensi iza iondid no showenhancedpa hology bu onlymod- 379
e a e mucus me aplasia and mode a e hype plasia o he ep- 380
i helium (Fig. 6D). Gi en ha p e ious wo k has shown ha 381
B. pe ussis in ec ion in combina ion wi h OVA sensi iza ion 382
(BpOVA) displayed mo e se e e ai way in lamma ion wi h 383
a g ea e deg ee o bo h epi helial hype plasia and mucous 384
me aplasia, i can be clea ly seen he e ha Pw immuniza- 385
ion educes he se e i y o ai way pa hology (Fig. 6D) and 386
UNCORRECTED PROOF
IMLET 4082 1–10
D.P. Ennisa e al. / Immunology Le e s xxx (2004) xxx–xxx 7
Fig. 4. Pw immuniza ion modula es he local cy okine esponse o B. pe ussis in ec ion and OVA sensi iza ion. Dilu ed BALF (0.5ml pe mouse) was pooled
om i e mice pe g oup and concen a ions o IL-5 (A), IFN-␥(B), IL-13 (C) and IL-10 (D) we e de e mined by EIA. Resul s a e ep esen a i e o duplica e
expe imen s. Cy okine concen a ions we e assayed in iplica e wi h alues exp essed as mean cy okine concen a ion in dilu ed BALF (±S.E.M.). *P<0.05
compa ed o he PwOVA ea ed g oup.
educes pulmona y esis ance as indica ed by ple hysmog a-
387
phy (Fig. 5D).
388
4. Discussion389
The p esen s udy demons a es ha Pw immuniza ion
390
p o ec s agains B. pe ussis exace ba ion o OVA-induced391
Table 2
His ological assessmen o ai way pa hology
T ea men g oup Mucous me aplasia o
ai way epi helium Hype -plasia o ai way
epi helium Smoo h muscle
hype ophy o ai way wall Pe i-ai way/ ascula
in lamma iona
O e all deg ee E N L M F
C l −−−−−−−−−
Bp −+++−+++−−
O a++++++++
b+++++
c−−
BpOVA +++ +++ ++ +++b++ +++ +++c−−
Pw −−−−−−−−−
PwBp −++−−−−−−
PwOVA + ++ + ++b++ ++ ++c−−
PwBpOVA ++ ++ ++ ++b++ ++ ++c−−
Asemi-quan i a i e sco e(−absen , + mild, ++ mode a e,+++ se e e)wasassigned o ea u es o ai way pa hologyobse ed acco ding o p e iously desc ibed
c i e ia [17].
aPe i-ai way/ ascula in lamma ion was 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).
bIn lamma ion ex ending in o su ounding pulmona y in e s i ium and al eola spaces.
cMac ophage gian cells o m pa o in lamma o y exuda es wi hin su ounding al eola spaces.
ai way hype esponsi eness in a mu ine model. Pw immu- 392
niza ion supp esses an ibody and cell media ed esponses 393
agains OVA sensi iza ion in combina ion wi h B. pe ussis 394
in ec ion a bo h he local and sys emic le el, coupled wi h 395
a subsequen educ ion in ai way eac i i y and pa hology. 396
I has been p e iously shown ha B. pe ussis exace ba es 397
OVA-induced ai way pa hology leading o he de elopmen 398
o mo e p onounced alle gen-induced ai wayin lamma ion
UNCORRECTED PROOF
IMLET 4082 1–10
8D.P. Ennisa e al. / Immunology Le e s xxx (2004) xxx–xxx
Fig. 5. Pw immuniza ion p io o B. pe ussis in ec ion dec eases b onchial hype esponsi eness o sensi izing an igen. 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. (A) Con ol and Bp in ec ed mice (B), Pw and
PwBp (C), OVA and PwOVA (D), BpOVA and PwBpOVA g oups, espec i ely. Resul s a e ep esen a i e o wo expe imen s (n=4) and alues a e exp essed
as mean enhanced pause (PenH)±S.E.M., in g oups whe e no e o s a e isible, e o ba s a e sho e han he size o he da a poin symbol.
[17]. We demons a e ha Pw immuniza ion p o ec s agains 399
his as well as educing ai way hype esponsi eness.400
The p e alence o as hma and alle gic disease has in-
401
c eased in many coun ies [39,40] and he e has been spec-
402
ula ion as o possible causes [41,42], including he possible
403
oleo immuniza ioninp omo ingalle gicsensi iza ion[43].
404
Fo example, pe ussis accina ion ac s as an adju an o
405
an igen-speci ic esponses in labo a o y animals [30,33];a406
speci ic IgE esponse o pe ussis oxin i sel has been iden-407
i ied in child en ecei ing pe ussis immuniza ion [44]; and408
accina ion wi h some o he o ganisms such as Haemophilus409
influenzae enhances his amine elease in labo a o y animals
410
[45]. Ac i e pe ussis oxin has a simila e ec [46].Inad-
411
di ion, wo s udies ha e sugges ed ha pe ussis in ec ion
412
inc eased he isk o a opy [47,48]. I is he e o e heo e i-
413
cally possible ha Pw immuniza ion migh con ibu e o he414
de elopmen o alle gic disease.415
The goal o his s udy was o es whe he immuniza ion416
wi h whole-cell pe ussis (Pw) accine would p o ec agains 417
B. pe ussis exace ba ion o alle gic as hma. B. pe ussis in-418
ec ionmodula esalle genp imingand hese e i yo ai way419
pa hology in a mu ine model[17]. I has been p oposed ha
420
IL-10 plays an essen ial ole in modula ing he immune e-421
sponses by inducing owa ds egula o y T-cell esponses[49]
422
howe e Leee alha edemons a ed ha IL-10inducesIL-13 423
p oduc ion in i o and ha his is esponsible o he mucus, 424
bu no he in lamma o y/ ib o ic e ec s o IL-10 [50].In 425
he p esen s udy, we obse e ha Pw immuniza ion p e en s 426
induc ion o IL-10 and IL-13 and p o ec s agains ai way hy- 427
pe eac i i y. Al hough IL-10 is known o ac in an immune 428
egula o y manne , we and o he s ha e sugges ed ha i has 429
b oade unc ions ha mayno alwaysp o ec agains in lam- 430
ma o y disease [17,51]. Fo example, G uns ein e al. ha e 431
sugges ed ha IL-10 may play an impo an ole in alle gic 432
as hma by ac ing di ec ly on he ai way smoo h muscle i - 433
sel [51]. P e ious s udies in humans ha e demons a ed ha 434
IL-13 mRNA and p o ein le els a e ele a ed in he lungs o 435
a opic and non-a opic as hma ics [52] sugges ing ha o e 436
exp ession o IL-13 may p edispose owa d he de elopmen 437
o bo h ypes o as hma [53]. The educ ion in bo h IL-10 438
and IL-13 a he sys emic and local le els sugges s ha by 439
emo ing hedamaginge ec so pe ussisin ec ionandcon- 440
sequen ly educing IL-10 and IL-13, Pw exe s a p o ec i e 441
e ec .In e es ingly, e y ecen wo kbyKim e al has shown 442
ha componen s o B. pe ussis can inhibi ai way hyepe e- 443
sponsi eness [54]. Tha s udy demons a ed ha unme hy- 444
la ed CpG sequences om B. pe ussis DNA inhibi ed Th2 445
cy okines in he ai ways ia a TLR9 in e ac ion [54]. Ou 446
UNCORRECTED PROOF
IMLET 4082 1–10
D.P. Ennisa e al. / Immunology Le e s xxx (2004) xxx–xxx 9
Fig. 6. Whole-cell B. pe ussis accine educes he se e i y o ai way pa hology o sensi izing an igen bo h in he p esence and absence o B. pe ussis
in ec ion. Rep esen a i e mo phological changes a 37 days in ans e se sec ions o b onchioles om (A) Pw immunized mice showing no changes e iden ;
(B) Pw/Bp mice, minimal changes e iden ; (C) Pw/OVA ea ed mice, illus a ing mode a e mu al and pe i-ai way in lamma ion wi h accompanying mild
mucous me aplasia (blue s aining goble cells) and mode a e hype plasia o epi helium; (D) Combined Pw/Bp/OVA ea ed g oup, illus a ing a mode a e mu al
andpe i-ai wayin lamma ion, mode a emucousme aplasiaandmode a eepi helial hype plasia; (E)OVAsensi izedg oup illus a ing mode a emu al andpe i-
ai way in lamma ion wi h accompanying mode a e mucus me aplasia (blue s aining goble cells) and hype plasia o epi helium; (F) 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. All sec ions a e ep esen a i e o g oups o i e mice pe expe imen , pe o med a leas wice. Sec ions s ained wi h a combined
Discombe’s/Alcian blue s ain, o iginal magni ica ion 400×.
obse a ion o educed in lamma ion and OVA-speci ic IgE
447
(Figs. 2 and 6) in PwBpOVA mice is consis en wi h ha
448
inding. TLR9 media ed educ ion in Th2 cy okines can be449
in oked in his si ua ion, because al hough hese mice will be450
exposed obac e ialDNAinacon ex whe ep io immuniza-451
ion will limi issue damage o he o he immunomodula o y452
e ec s o iable B. pe ussis [17,54].453
The mos common o mula ion o he hygiene hypo hesis454
is based upon a lack o immunological s imuli in ea ly in-455
ancy ha esul s in abe an Th2 esponses [10,12]. Clea ly456
in an pe ussis immuniza ion o in ec ion could po en ially457
in luence his p ocess. S udies in neona al mice suppo ou 458
indings ha Pwisas ongTh1induce ,bu i is lesse ec i e
459
ininducingan ibody esponses du ing his pe iod [28,55,56].460
A s udy by G ube e al. e ealed no e idence o an alle gy 461
p omo inge ec o commonchildhood accinesinap ospec- 462
i ely ollowed a opy isk-enhanced bi h coho [26]. Mo e- 463
o e , hey ound ha child en wi h a be e accina ion co - 464
e age seemed o be be e p o ec ed agains he de elopmen 465
o a opy in hei second and hi d yea s o li e. In pa icu- 466
la , measles/mumps, pe ussis, and diph he ia/ e anus immu- 467
niza ion we e associa ed wi h a ansien educ ion o a opy, 468
whe eas immuniza ion agains polio and H. influenzae had 469
no e ec . Fu he mo e, immuniza ion o child en wi h Pw 470
down egula ed he IgE esponse o co-adminis e ed diph he- 471
ia and e anus oxoids [57]. In con as , a s udy in ol ing he 472