S-27
1Dipa imen o Biomedico di Medicina
In e na e Specialis ica, Chai and
Di ision o Rheuma ology, Pale mo
Uni e si y School o Medicine;
2Dipa imen o Biomedico di Medicina
In e na e Specialis ica, Chai and Di ision
o Gas oen e ology, Pale mo Uni e si y
School o Medicine, Pale mo, I aly.
Angelo Fe an e, MD, PhD
F ancesco Ciccia, MD, PhD
Al onso P incipa o, MD
Anna Ri a Gia dina, MD, PhD s uden
Rosalia Impas a o, MD
Se gio Pe al a, MD
Gio anni T iolo, MD
This s udy was suppo ed by a g an om
he Minis e o della Uni e si à e della
Rice ca Scien ifica, I aly.
Please add ess co espondence o:
P o esso Gio anni T iolo,
Uni à Ope a i a di Reuma ologia,
Piazza delle Cliniche 2,
90127 Pale mo, I aly.
E-mail: [email p o ec ed]
Recei ed on Janua y 11, 2010; accep ed in
e ised o m on Ma ch 5, 2010.
Clin Exp Rheuma ol 2010; 28 (Suppl. 60):
S27-S30.
© Copy igh CLINICAL AND
EXPERIMENTAL RHEUMATOLOGY 2010.
Compe ing in e es s: none decla ed.
ABSTRACT
Objec i es. Behçe ’s disease has been
his o ically classified as a Th1 disease.
The ecen ly desc ibed IL-17/IL-23
pa hway seems o play an impo an
ole in many inflamma o y diseases and
in he in es inal abno mali ies o AS
and CD. The aim o he p esen s udy
was o e alua e he IL-17/IL-23 axis in
pa allel wi h Th1 and IL-27 esponse
in he in es ine o pa ien s wi h BD and
gas oin es inal abno mali ies.
Me hods. Quan i a i e TaqMan e-
e se ansc ip ase-polyme ase chain
eac ion (RT-PCR) was u ilised o all
de e mina ions on ileal biopsy speci-
mens ob ained om BD, AS and CD
pa ien s. The se um le els o Th1 and
Th17 cy okines we e e alua ed by en-
zyme-linked immunoso ben assay.
Resul s. A Th1 bu no a Th17 esponse
is p esen in he gas oin es inal in-
ol emen o Behçe ’s disease.
Conclusions. Al hough BD sha es
clinical mani es a ions wi h bo h CD
and AS, he immunologic abno mali ies
seen in he in es ine a e qui e di e en ,
indica ing ha o he immune mecha-
nisms should be aken in o accoun .
In oduc ion
Behçe ’s disease (BD) is a a e ascu-
li is cha ac e ised by ecu en o al and
geni al ulce s, eye lesions, skin lesions
and posi i e pa he gy es . O al ulce s,
e y hema nodosum, u ei is and a h i-
is a e common mani es a ions o BD
oge he wi h in es inal inflamma ion
bu also o ankylosing spondyli is (AS)
and C ohn’s disease (CD).
We ha e ecen ly p oduced e idence
ha AS and CD sha e some clinical and
immunological simila i ies a gas oin-
es inal le el (1).
Ac i a ion o T cells and monocy es/
mac ophages is ega ded as an impo -
an ac o in he pa hogenesis o BD
as well as AS and CD. His o ically BD
has been conside ed o be a ypical Th1
disease, cha ac e ised by inc eased le -
els o Th1 cy okines such as IFN-γ, IL-
2 and TNF-α (2, 3).
The IL-17/IL-23 pa hway seems o
play an impo an ole in many in-
flamma o y diseases (4) and in he in-
es inal abno mali ies o AS and CD
(1). Inc eased IL-17 le els ha e been
ound in he se a o BD pa ien s (5), i
is no ye clea , howe e , whe he he
IL-17/IL-23 pa hway is in ol ed in he
pa hogenesis o he in es inal BD. Da a
a ailable, in ac , on cy okine p oduc-
ion in he gu o BD pa ien s a e lim-
i ed and indica e a small impai men o
Th1 cy okines (6).
The aim o he p esen s udy was o
e alua e he IL-17/IL-23 axis in pa al-
lel wi h Th1 esponse in he in es ine o
pa ien s wi h BD and gas oin es inal
abno mali ies.
Pa ien s and me hods
In es inal biopsy specimens we e ob-
ained, a e in o med consen , om
11 pa ien s (7 males and 4 emales, age
ange 25–58 yea s) wi h BD and in es-
inal complain s. Two adjacen mucos-
al biopsy samples om he ileum we e
ob ained om each subjec , independ-
en ly o he p esence o mac oscopic
in ol emen . All pa ien s ulfilled he
in e na ional c i e ia o disease classi-
fica ion and we e conside ed o ha e an
ac i e disease (7). Ileal specimens om
10 heal hy subjec s, 8 AS and 7 CD pa-
ien s who unde wen ileocolonscopy
o ou ine clinical e alua ion we e
also included as heal hy and disease
con ols, espec i ely. The AS g oup
consis ed o 5 men and 3 women wi h
a median age o 41 yea s ( ange 38–65
yea s) who we e diagnosed as ha ing
AS acco ding o he modified New
Yo k c i e ia (8). Disease ac i i y was
e alua ed using he Ba h AS Disease
Ac i i y Index (BASDAI) (9), wi h a
A Th1 bu no a Th17 esponse is p esen in he gas oin es inal
in ol emen o Behçe ’s disease
A. Fe an e1, F. Ciccia1, A. P incipa o1, A.R. Gia dina1, R. Impas a o1,
S. Pe al a2, G. T iolo1
S-28
Thl esponse in he gu o BD pa ien s / A. Fe an e e al.
BASDAI sco e o >4 indica ing ac i e
disease. All pa ien s we e HLA–B27
posi i e. A he ime mucosal biopsy
specimens we e ob ained, he mean ±
SD BASDAI sco e was 6.7±3.4. The
CD g oup consis ed o 5 men and 2
women wi h a median age o 50 yea s
( ange 48–60 yea s). The diagnosis o
CD was made acco ding o clinical pa-
ame e s and findings o adiog aphic,
endoscopic, and his opa hologic analy-
ses. Disease ac i i y was e alua ed us-
ing he CD Ac i i y Index (10), wi h a
sco e >150 indica ing ac i e disease,
and endoscopic and his opa hologic
da a. A he ime o sample collec ion,
he mean ± SD CD Ac i i y Index sco e
was 253±51.5, and none o he pa ien s
had ecei ed s e oids and/o cy o oxic
d ugs, immunosupp essi e agen s, o
an ibio ics. The con ol g oup (NHS)
consis ed o 6 men and 4 women wi h
a median age o 45 yea s ( ange 41–58
yea s) who we e unde going ileo-
colonoscopy o diagnos ic pu poses.
The s udy was app o ed by he e hics
commi ee and he ins i u ional e iew
boa d o he Uni e si y o Pale mo.
The quan i a i e TaqMan e e se as-
c ip ase-polyme ase chain eac ion
(RT-PCR) was u ilised o all de e -
mina ions as ex ensi ely desc ibed in
e e ence 1. To al RNA was ex ac ed
using he Qiagen RNeasy Mini ki
(Qiagen, Cha swo h, CA), wi h on-
column DNase I diges ion. A o al o 1
μg o RNA was e e se- ansc ibed o
complemen a y DNA (cDNA) using a
The moSc ip Fi s -S and cDNA Syn-
hesis ki (In i ogen, Ca lsbad, CA).
Samples we e un in iplica e a 20 ng
o cDNA pe well and de ec ed using
an ABI P ism 7900HT ins umen . Re-
sul s we e analysed using ABI P ism
7900HT Sequence De ec ion Sys em
e sion 2.1 so wa e. Rela i e quan-
ifica ion was assessed using he C
me hod.
IL-12, IL-17, IFN-γ, TNF-α se um
le els we e analysed wi h Human
Au oimmune Response Mul i-Ana-
ly e ELISA ay ki s (SABiosciences
Co p, F ede ick, Md) acco ding o he
manu ac u e ’s ins uc ions. IL-23 and
IL-27 se um le els we e e alua ed us-
ing comme cially a ailable ki s (R&D
Sys ems, Minneapolis, MN).
S a is ics
Resul s a e exp essed as mean ± SD.
S a is ical analysis o quan i a i e
a iables was pe o med using he
K uskal-Wallis nonpa ame ic es ,
wi h Dunn’s pos es when analysing
mo e han 2 g oups and he Mann-
Whi ney U- es when analysing 2
g oups. P- alues less han 0.05 we e
conside ed significan .
Resul s
Six pa ien s complain abdominal pain,
4 dia hoea and one bo h. A mo phol-
Fig. 1. IL-23, IL-17, IL-1β, IL-27, INF-γ, TNF-
α, IL-12, STAT-3 and supp esso cy okine sig-
nalling 3 (SOCS-3) – ela ed gene exp ession
assessed by TaqMan eal- ime polyme ase chain
eac ion in ileal biopsy specimens ob ained om
pa ien s wi h Behçe ’s disease (BD), ankylosing
spondyli is (AS), C ohn’s disease (CD), and no -
mal con ols (HC).
Ba s show he mean and SD.
S-29
Thl esponse in he gu o BD pa ien s / A. Fe an e e al.
ogy pa ien s showed ch onic non-spe-
cific inflamma o y abno mali ies and
in some cases enule asculi is and
ap hous ulce s. Pa ien s we e un ea ed
(n=3) o ea ed wi h colchicine (n=4)
low dose p ednisone (n=2) o bo h
(n=2). None was unde immunosup-
p essan s. All CD pa ien s had an ac-
i e disease. O he 8 pa ien s wi h ac-
i e AS who unde wen colonoscopy
(mean BASDAI sco e 6.7), e idence
o subclinical in es inal inflamma ion
was obse ed in 5 (one wi h acu e in-
flamma ion and 4 wi h ch onic inflam-
ma ion). Resul s a e shown in Figu e 1.
In con as wi h esul s ob ained in AS
and CD pa ien s, no mal le els o IL-
23 mRNA we e ound in BD. STAT-3,
which media es IL-23 signalling upon
IL-23 ecep o liga ion, and IL-17A
mRNA we e also no mal. Samples
om BD pa ien s displayed SOCS-3
o e exp ession a le els compa able
o hose obse ed in CD bu no in
AS pa ien s. Le els o TNF-α, IFN-γ,
IL-12p35 mRNA in BD specimens
we e significan ly highe han hose o
heal hy con ols and AS and compa a-
ble o hose ound in CD. In e es ingly
IL-27, a Th1 cy okine which inhibi s
de no o Th17 de elopmen om nai e
T cells (11) was ound o be ma kedly
up- egula ed a le els simila o hose
ound in CD.
Analysis o se um cy okines showed
inc eased concen a ions o IL-12,
TNF-α and IFN-γ in BD pa ien s when
compa ed o NHS (Fig. 2). A end o-
wa d an inc ease in IL-17, IL-23 and
IL-27, (al hough no s a is ically sig-
nifican ) was obse ed in BD pa ien s
e sus NHS.
Discussion
The p esence o a sha ed clinical be-
ha iou be ween BD, AS and CD is
sugges ed by e idence o simila a -
h opa hy, in es inal inflamma ion and
muco-cu aneous abno mali ies in pa-
ien s wi h ei he disease.
Based on ecen findings, i is becom-
ing inc easingly clea ha IL-23 ex-
e s an essen ial pa hogene ic ole in
p omo ing au oimmuni y and ch onic
inflamma ion in se e al models o au-
oimmune diseases, such as expe i-
men al inflamma o y a h i is (12) and
expe imen al coli is (13). In his and
ou p e ious s udy (1) we ha e demon-
s a ed a s ong and significan up- eg-
ula ion o IL-23p19 ansc ip s in he
e minal ileum o pa ien s wi h AS and
pa ien s wi h CD. Unlikely CD and AS,
in BD pa ien s IL-23 was no up- egu-
la ed and he e was no in ol emen o
he IL-17/IL-23 pa hway. On he o he
hand, in es inal BD seems o be cha -
ac e ised by a Th1 esponse. Le els o
TNF-α, IFN-γ, IL-12p35 mRNA we e,
in ac , significan ly highe han hose
o heal hy con ols and AS and compa-
able o hose ound in CD. IL-27 was
ound also ma kedly up- egula ed and
may accoun o he limi ed Th-17 cell
pool in he BD in es ine (11). Analy-
sis o se um cy okines concen a ion
by ELISA ays showed a ma ked and
significan inc ease o Th1, bu no
Th17 cy okines in BD pa ien s when
compa ed o NHS. The Th1 p e alen
esponse in BD could be ela ed o a
de ec i e egula o y T cell esponse as
sugges ed by a ecen s udy showing
ha TGF-be a1 and IL-10 gene poly-
mo phism may a ec hos suscep ibil-
i y o BD (14).
A p esen we do no know whe he
mRNA le els ound in ou pa ien s a e
pa alleled by hei co esponding p o-
ein le els. Howe e , he BD popula-
ion in his s udy was no la ge enough
o consen defini e conclusions. Some
conside a ions, howe e , may be done.
In pa icula , al hough BD sha es clini-
cal mani es a ions wi h bo h CD and AS,
he immunologic abno mali ies seen in
he in es ine a e qui e di e en indica -
ing ha o he immune mechanisms,
possibly in ol ing gamma/del a T cells
(15), should be aken in o accoun .
Fig. 2. IL-12 (A), IFN-γ (B), TNF-α (C), IL-17 (D) IL-23 (E), and IL-27 (F) we e measu ed in se um
samples om pa ien s wi h Behçe ’s disease (BD) and heal hy con ols (HC) by ELISA ays. A-D da a
a e p esen ed as abso bance alues a 450 nm. IL-23 and IL-27 le els a e exp essed as pg/ml.
S-30
Thl esponse in he gu o BD pa ien s / A. Fe an e e al.
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