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Vol 9 No 3
Resea ch a icle
Inhibi ion o indoleamine 2,3-dioxygenase-media ed yp ophan
ca abolism accele a es collagen-induced a h i is in mice
Sándo Szán ó1,2, Tamás Ko eny1, Ka alin Mikecz1, Tibo T Glan 1, Zol án Szekanecz2 and
John Va ga3
1Sec ion o Molecula Medicine, Depa men o O hopedic Su ge y, Rush Uni e si y Medical Cen e , Cohn Resea ch Building, Room 708, 1735 W.
Ha ison, Chicago, IL 60612, USA
2Ins i u e o Medicine, Di ision o Rheuma ology, Uni e si y o Deb ecen, Medical and Heal h Science Cen e , 22 Mó icz S ee , Deb ecen, H-4012,
Hunga y
3Di ision o Rheuma ology, No hwes e n Uni e si y Medical School, 303 Eas Chicago A e., Chicago, IL 60611, USA
Co esponding au ho : Sándo Szán ó, szan [email protected] e.hu
Recei ed: 13 Dec 2006 Re isions eques ed: 17 Jan 2007 Re isions ecei ed: 24 Ap 2007 Accep ed: 18 May 2007 Published: 18 May 2007
A h i is Resea ch & The apy 2007, 9:R50 (doi:10.1186/a 2205)
This a icle is online a : h p://a h i is- esea ch.com/con en /9/3/R50
© 2007 Szán ó e al.; licensee BioMed Cen al L d.
This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0),
which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
Abs ac
Indoleamine 2,3-dioxygenase (IDO) is one o he ini ial and a e-
limi ing enzymes in ol ed in he ca abolism o he essen ial
amino acid yp ophan. In cul u ed cells, he induc ion o IDO
leads o deple ion o yp ophan and yp ophan s a a ion.
Recen s udies sugges ha modula ion o yp ophan
concen a ion ia IDO plays a undamen al ole in inna e immune
esponses. Induc ion o IDO by in e e on-γ in mac ophages and
dend i ic cells esul s in yp ophan deple ion and supp esses
he immune-media ed ac i a ion o ib oblas s and T, B, and
na u al kille cells. To assess he ole o IDO in collagen-induced
a h i is (CIA), a model o heuma oid a h i is cha ac e ized by a
p ima ily Th1-like immune esponse, ac i i y o IDO was inhibi ed
by 1-me hyl- yp ophan (1-MT) in i o. The esul s showed
signi ican ly inc eased incidence and se e i y o CIA in mice
ea ed wi h 1-MT. Ac i i y o IDO, as de e mined by measu ing
he le els o kynu enine/ yp ophan a io in he se a, was
inc eased in he acu e phase o a h i is and was highe in
collagen-immunized mice ha did no de elop a h i is.
T ea men wi h 1-MT esul ed in an enhanced cellula and
humo al immune esponse and a mo e dominan pola iza ion o
Th1 in mice wi h a h i is compa ed wi h ehicle- ea ed a h i ic
mice. The esul s demons a ed ha de elopmen o CIA was
associa ed wi h inc eased IDO ac i i y and enhanced
yp ophan ca abolism in mice. Blocking IDO wi h 1-MT
agg a a ed he se e i y o a h i is and enhanced he immune
esponses. These indings sugges ha IDO may play an
impo an and no el ole in he nega i e eedback o CIA and
possibly in he pa hogenesis o heuma oid a h i is.
In oduc ion
Locally p oduced p oin lamma o y cy okines such as umo
nec osis ac o -α, in e leukin (IL)-1, and IL-6 play a pi o al ole
in he pa hology o heuma oid a h i is (RA). These cy okines,
by up egula ion o se e al genes, a e esponsible bo h o he
ec ui men and con inuous ac i a ion o he in lamma o y cells
and o inducing p oduc ion o he enzymes ha des oy bone
and ca ilage. Howe e , hese in lamma o y e en s need o be
balanced by he p oduc ion o endogenous inhibi o s, as
in lamma o y esponses a e gene ally localized and he conse-
quen des uc ion o a ec ed join s is less se e e [1].
The ubiqui ously exp essed heme enzyme indoleamine 2,3-
dioxygenase (IDO) ca alyzes he non-hepa ic oxida i e deg a-
da ion o yp ophan, he ini ial and a e-limi ing s ep in yp-
ophan me abolism, esul ing in deple ion o his leas
abundan essen ial amino acid. T yp ophan s a a ion enables
he hos o es ic he g ow h o in acellula pa hogens [2].
The exp ession o IDO in heal hy issues is gene ally qui e low
bu is ma kedly up egula ed in esponse o in ec ion and
in lamma ion. Recen s udies ha e es ablished an en i ely
no el impo an biological unc ion o IDO. These s udies indi-
ca e ha IDO-media ed yp ophan deple ion in i o esul s in
inhibi ion o ma ix-deg ading me allop o einase enzyme p o-
duc ion, supp ession o in lamma o y esponses, and
1-MT = 1-me hyl- yp ophan; CFA = comple e F eund's adju an ; CIA = collagen-induced a h i is; CII = ype II collagen; CTLA-4 = cy o oxic T lym-
phocy e-associa ed an igen-4; ELISA = enzyme-linked immunoso ben assay; IDO = indoleamine 2,3-dioxygenase; IFN-γ = in e e on-γ; IgGAM =
immunoglobulins G, A, and M; IL = in e leukin; i.p. = in ape i oneally; K = kynu enine; NK = na u al kille ; RA = heuma oid a h i is; T = yp ophan.
A h i is Resea ch & The apy Vol 9 No 3 Szán ó e al.
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p omo ion o immune ole ance [3,4]. Acco dingly, an eme g-
ing immunological pa adigm ega ds IDO as a key egula o y
con ol enzyme in bo h inna e and adap i e immune
esponses. Indeed, modula ion o cellula IDO exp ession and/
o ac i i y is now inc easingly implica ed in in lamma ion and
au oimmuni y, o gan ansplan a ion and e al ejec ion, and
e asion o immune su eillance by umo cells. Fu he mo e,
agen s ha inhibi IDO a e unde ac i e in es iga ion as po en-
ial adju an s o umo immuno he apy [5].
IDO-media ed yp ophan ca abolism esul s in deple ion o
yp ophan wi h concomi an gene a ion o kynu enine and ul i-
ma ely NAD (nico inamide adenine dinucleo ide). Exp ession
o he IDO gene is induced in mos cell ypes in esponse o
in ec ion wi h mic obial agen s ia ac i a ion o oll-like ecep-
o s. Fu he mo e, in e e on-gamma (IFN-γ), IL-10, and cy o-
oxic T lymphocy e-associa ed an igen-4 (CTLA-4) ha e also
been shown o s imula e IDO [4,6]. In con as , IL-4, IL-13, and
ans o ming g ow h ac o -β a e supp esso s o IDO [7]. In
p e ious s udies, we ha e shown ha IFN-γ caused ime-
dependen induc ion o IDO gene exp ession and ac i i y in
cul u ed no mal ib oblas s [4]. The consequen yp ophan
ca abolism and ensuing yp ophan s a a ion in hese cul u es
we e associa ed wi h p o ound supp ession o collagenase
and s omelysin gene exp ession induced by IL-1β. Ab oga ion
o IL-1β-induced ma ix me allop o einase s imula ion upon
ac i a ion o IDO in hese ib oblas s was di ec ly due o he
educ ion o local yp ophan concen a ions a he han he
accumula ion o kynu enine and o he yp ophan me aboli es.
These obse a ions led us o p opose he hypo hesis ha ,
h ough i s abili y o induce ansien yp ophan s a a ion,
IDO ep esen ed an impo an endogenous an i-in lamma o y
mechanism and ha dis up ion o IDO induc ion o unc ion
would be associa ed wi h exagge a ed in lamma o y
esponses.
Collagen-induced a h i is (CIA) is gene a ed in gene ically
suscep ible mouse o a s ains by immuniza ion wi h ype II
collagen (CII) dissol ed in comple e F eund's adju an (CFA).
CIA highly esembles RA acco ding o o e lapping immun-
opa hogenic pa hways and simila his opa hologic ea u es
[8]. Among o he easons, he cen al ole o class II majo his-
ocompa ibili y complex [9], he in ol emen o CD4+ T cells in
he immunopa hogenesis o he disease, he p edominan ly
Th1 ype immune esponse o CII in he induc ion phase
[10,11], as well as he cy okine p o ile h oughou he e olu-
ion o CIA [12] make his expe imen al animal model one o
he mos adequa e models o RA. To add ess he ques ion o
whe he IDO plays a nega i e egula o y ole in an animal
model o a h i is, we assessed IDO ac i i y in he se a o mice
a di e en s ages o a h i is and he e ec o 1-me hyl- yp-
ophan (1-MT), a speci ic inhibi o o IDO, on a h i is de elop-
men and an igen-speci ic immune esponses in CIA.
Ma e ials and me hods
Immuniza ion and assessmen o collagen-induced
a h i is in mice ecei ing ei he 1-me hyl- yp ophan o
ehicle
Mice we e housed and b ed unde s anda d condi ions a he
Compa a i e Resea ch Cen e o Rush Uni e si y (Chicago,
IL, USA). The Ins i u ional Animal Ca e and Use Commi ee
app o ed all animal expe imen s. DBA/1 male mice, 6 o 8
weeks o age, we e pu chased om The Jackson Labo a o y
(Ba Ha bo , ME, USA). Mice we e immunized by a s anda d
immuniza ion p o ocol. B ie ly, 100 μg o human CII was emul-
si ied in CFA (Di co Labo a o ies Inc., now pa o Bec on
Dickinson and Company, F anklin Lakes, NJ, USA) and
injec ed in o he p oximal ail o mice. A second injec ion o he
same dose and adju an was gi en in ape i oneally (i.p.) on
day 21. Mice ha did no de elop a h i is wi hin 3 weeks o
he second an igen injec ion we e boos ed wi h a hi d injec-
ion adminis e ed in equally di ided doses i.p. and in o he
p oximal ail. All mice we e sac i iced 8 weeks a e he i s
injec ion. A e he second immuniza ion, all mice we e exam-
ined o swelling and e y hema o dis al join s. Paws we e con-
side ed o ha e a h i is when swelling and e y hema we e
no ed in a leas wo digi s and/o o he join s. The clinical
se e i y o a h i is was g aded on a scale o 0 o 4 o each
paw, acco ding o swelling and edness. Speci ically, sco ing
was pe o med as ollows: 0 = heal hy; 1 = mild swelling and
e y hema; 2 = mode a e swelling and e y hema; 3 = mo e
in ense e y hema, swelling, and edness a ec ing a g ea e
p opo ion o he paw; 4 = se e e e y hema, swelling, and ed-
ness a ec ing he en i e paw. A cumula i e sco e anging om
0 o 16, based on indi idual paw sco es o 0 o 4, was
assigned o each animal.
Table s con aining slow elease o D, L-1-MT (Inno a i e
Resea ch o Ame ica, Sa aso a, FL, USA) o ehicle we e su -
gically inse ed unde he do sal skin o mice on days 22 and
29 a e he i s immuniza ion. The able s eleased 10 mg/day
1-MT o a pe iod o 7 o 10 days.
Measu emen o an ibody p oduc ion and T-cell
esponse
An ibodies o he immunizing human and mouse (sel ) ca i-
lage CII we e de e mined by enzyme-linked immunoso ben
assay (ELISA), as desc ibed elsewhe e [13-15]. B ie ly, Max-
iso p 96-well pla es (Nalge Nunc, Nape ille, IL, USA) we e
coa ed wi h 0.1 μg o human o mouse ca ilage-de i ed CII in
100 μl o coa ing bu e . An ibodies we e de e mined in se ial
dilu ions o se a (1:500 o 1:62,500) using pe oxidase-conju-
ga ed goa an i-mouse immunoglobulins G, A, and M (IgGAM)
seconda y an ibodies (Zymed Labo a o ies Inc., now pa o
In i ogen Co po a ion, Ca lsbad, CA, USA). Se um an ibody
le els we e exp essed in millig ams pe li e using mouse
IgGAM as con ol (In i ogen Co po a ion).
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An igen-speci ic T-cell esponses, including IL-2 p oduc ion
and T-cell p oli e a ion, we e measu ed in quad uplica e sam-
ples o spleen cells (3 × 105 cells pe well) cul u ed in he
p esence o 100 μg o collagen p o ein pe millili e . IL-2 was
measu ed in supe na an s ha es ed on day 2 by he p oli e a-
ion o he IL-2-dependen CTLL-2 cell line. An igen-speci ic T-
cell p oli e a ion was assessed on day 5 by he inco po a ion
o 3 [H] hymidine. In bo h cases, he an igen-speci ic esponse
was exp essed as s imula ion index, which is a a io o inco -
po a ed 3 [H] hymidine (coun s pe minu e) in sil e -s imula ed
cul u es ela i e o coun s pe minu e in non-s imula ed cul-
u es [13,14]. An igen-speci ic IFN-γ and IL-4 p oduc ion by T
cells was de e mined in cul u e condi ions iden ical o hose
desc ibed o T-cell p oli e a ion in 4-day-old condi ioned
medium (2.5 × 106 mononuclea cells pe millili e ) using cap-
u e ELISAs (R&D Sys ems, Inc., Minneapolis, MN, USA).
High-pe o mance liquid ch oma og aphy analysis
Se um yp ophan (T) and kynu enine (K) concen a ions we e
measu ed simul aneously by high-pe o mance liquid ch oma-
og aphy, as p e iously desc ibed [16]. As ela i ely high ol-
umes o se a a e needed o he de e mina ion o K and T,
pooled se a we e used, K and T concen a ions we e de e -
mined, and hen K/T a ios we e calcula ed.
S a is ical analysis
Analyses o he a h i is sco e and disease incidence a di e -
en ime poin s we e ca ied ou using he non-pa ame ic
Mann-Whi ney U es and chi-squa e con ingency analysis,
espec i ely. S uden es was used o s a is ical analysis o
all o he da a. Analyses we e pe o med using SPSS e sion
7.5 so wa e package (SPSS Inc., Chicago, IL, USA). Signi i-
cance was se a a p alue o less han 0.05.
Resul s
Clinical and his ological ea u es o a h i is: inc ease o
bo h he incidence and se e i y in mice ea ed wi h 1-
me hyl- yp ophan
Mice we e immunized wi h CII and moni o ed o he de elop-
men o a h i is o 54 days a e he p ima y immuniza ion.
Clinical signs o a h i is ypically appea ed wi hin 4 o 8 days
a e he second and hi d injec ions, which we e adminis e ed
on days 21 and 42 a e he p ima y immuniza ion, espec-
i ely. T ea men wi h 1-MT adminis e ed by he inse ion o
able s con aining 1-MT (o ehicle) on days 22 and 29 a e
he i s injec ion esul ed in an inc eased incidence o a h i is
(Figu e 1a). In addi ion o he highe disease incidence, he
se e i y indica ed by he cumula i e a h i is sco e was signi i-
can ly inc eased in mice ecei ing 1-MT as compa ed o hose
ecei ing ehicle be ween days 37 and 47 a e he p ima y
immuniza ion (Figu es 1b and 2). His ological analysis o he
ankle, me a a sophalangeal, and in e phalangeal join s o 1-
MT- ea ed and ehicle- ea ed mice showed ypical a h i is
cha ac e ized by ex ensi e leukocy e in il a ion, syno ial p oli -
e a ion, pannus o ma ion, and e osions. The se e i y o in lam-
ma o y cell in il a ion and des uc ion o ca ilage and bone
e lec ed he clinical s a e, bu no quali a i e di e ences could
be obse ed in mice ecei ing 1-MT compa ed o con ol mice.
Cellula and humo al immune esponses
Because o he di e ences in incidence and se e i y o a h i is
be ween mice ecei ing 1-MT o ehicle, i seemed o be p u-
den o de ec immune esponses o CII. Signi ican inc eases
we e ound in bo h CII-speci ic au oan ibody and he e oan i-
body i e s in 1-MT- ea ed mice compa ed o he ehicle
g oup; howe e , hese di e ences disappea ed by he end o
he ollow-up pe iod (day 55) (Figu e 3a,b). When assessing
T-cell p oli e a ion in he wo g oups a he end o ollow-up, no
Figu e 1
Incidence and se e i y o collagen-induced a h i is (CIA) in mice ea ed wi h 1-me hyl- yp ophan (1-MT) o ehicleIncidence and se e i y o collagen-induced a h i is (CIA) in mice
ea ed wi h 1-me hyl- yp ophan (1-MT) o ehicle. A h i is was i s
de ec ed on day 26 a e immuniza ion wi h ype II collagen on days 0,
21, and 42 (solid a ows) in mice ea ed wi h 1-MT o ehicle (open
a ows). (a) Incidence o CIA exp essed as he pe cen age o a h i ic
animals. (b) Disease se e i y exp essed as he cumula i e a h i is
sco e in a ec ed animals. S a is ically signi ican di e ences in a h i is
sco es we e ound be ween days 37 and 47 (p < 0.05). Values a e p e-
sen ed as he mean and s anda d e o o he mean o 25 1-MT- ea ed
and 20 ehicle- ea ed DBA/1 mice pe g oup and ep esen wo inde-
penden expe imen s.
A h i is Resea ch & The apy Vol 9 No 3 Szán ó e al.
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di e ences in T-cell esponses o he e ologous CII, and only
mode a ely inc eased T-cell p oli e a ion o au ologous CII in
he 1-MT g oup, could be de ec ed. Simila ly o T-cell p oli e -
a ion, IL-2 concen a ions in he supe na an s o spleen cells
s imula ed wi h mouse o human CII we e sligh ly (bu non-sig-
ni ican ly) highe in he 1-MT- ea ed g oup han in ehicle-
ea ed g oup a he end o he ollow-up (da a no shown).
Spleen cell supe na an cy okine le els in mice wi h
collagen-induced a h i is ea ed wi h 1-me hyl-
yp ophan o ehicle
The concen a ions o IFN-γ and IL-4 we e de e mined in he
supe na an s o spleen cells om mice wi h CIA and ea ed
wi h 1-MT o ehicle, espec i ely, a he end o he ollow-up.
Cy okine p oduc ion o cells was assessed ei he wi hou s im-
ula ion o a e challenging wi h mouse o human CII. A lowe
concen a ion o IL-4 was de ec ed in supe na an s o spleen
cells om mice ea ed wi h 1-MT in compa ison o ea ed
hose wi h ehicle, bu he di e ence was signi ican only
when challenging he cells wi h human CII (p < 0.05) (Figu e
4a). In con as , sligh ly (bu non-signi ican ly) highe concen-
a ions o IFN-γ could be measu ed in supe na an s o spleen
cells om 1-MT- ea ed mice compa ed o hose om ehicle-
ea ed animals (Figu e 4b).
Kynu enine/ yp ophan a ios in collagen-induced
a h i is mice ea ed wi h 1-me hyl- yp ophan o
ehicle
To es ima e he enzyma ic ac i i y o IDO in di e en s ages o
a h i is, K and T concen a ions we e measu ed and K/T a ios
we e calcula ed in he pooled se a o mice ea ed wi h 1-MT
o ehicle. Inc eased K and dec eased T concen a ions we e
measu ed, and hus inc eased K/T a io was de ec ed in mice
wi h acu e a h i is, bu his a io was e en highe in immunized
and immedia e p e-a h i ic mice (day 33). Thus, on day 33,
animals ha will no subsequen ly de elop a h i is ha e highe
K/T a ios in compa ison o hose ha will de elop a h i is. As
was expec ed, ea men wi h 1-MT dec eased he K/T a io in
he se um o a h i ic and immedia e p e-a h i ic mice com-
pa ed o he ehicle- ea ed ones. A he end o ollow-up, he
K/T a io o mice was as low as ha o he immunized mice. 1-
MT ea men did no ha e any e ec on K/T a io (Figu e 5).
Discussion
RA and expe imen al in lamma o y join diseases ha e a p o-
g essi e cha ac e wi h in ol emen o inc easing numbe s o
join s; howe e , he ini ial and agg essi e acu e phase in
a ec ed join s slows down o e ime and he in lamma o y
p ocesses bu n ou . Se e al lines o e idence indica e ha he
e ec o mechanism ha ini ially a acks he join s is T cell-
d i en in esponse o he e ec o p oin lamma o y cy okines,
bu he mechanisms esponsible o he limi a ion o acu e
in lamma o y p ocesses a e much less unde s ood. The no el
inding o ou s udy is ha IDO ac i i y is up egula ed in he
acu e phase o CIA e lec ed by he inc eased K/T a io in he
se um. Fu he mo e, we could also demons a e ha inhibi ion
o IDO in his expe imen al model augmen s he incidence and
se e i y o he disease and inc eases he immune esponses
o he au oan igens and alloan igens. These da a sugges ha
IDO plays a cen al ole in he nega i e egula o y eedback o
immunological mechanisms in in lamma o y join diseases.
CIA, like RA in humans, is cha ac e ized by he accumula ion
o T cells, plasma cells, mac ophages, B cells, mas cells, na -
u al kille (NK) cells, and dend i ic cells in he syno ial sublin-
ing [17,18]. Fu he mo e, in lamma o y cells in il a ing he
syno ial issue in RA and in he acu e phase o CIA exhibi a
p edominan ly Th1 pa e n o cy okine exp ession [10,11]. By
p iming he Th1- ype in lamma o y cell esponses, IFN-γ is one
o he mos impo an p oin lamma o y ac o s in he induc ion
o T cell-d i en au oimmune a h i is, such as CIA. Howe e ,
IFN-γ plays an ambiguous ole in au oimmuni y. A e he ac i-
a ion o sel - eac i e lymphocy e clones and o bys ande and
accesso y cells in he acu e phase, IFN-γ down egula es he
au oimmune p ocesses [19]. Indeed, CIA and CFA de eloped
mo e eadily in IFN-γ ecep o -de icien mice han in wild- ype
Figu e 2
Hind paw images o DBA miceHind paw images o DBA mice. (a) Hind paws o heal hy non-immunized DBA mice. (b) Type II collagen-immunized (a h i ic) DBA mice ea ed wi h
1-me hyl- yp ophan 6 o 7 days a e he onse o collagen-induced a h i is. (c) Type II collagen-immunized (a h i ic) DBA mice ea ed wi h ehicle
6 o 7 days a e he onse o collagen-induced a h i is. Acco ding o ou sco ing sys em (see Ma e ials and me hods), he h ee s ages ha e been
assigned sco es o 0 (a), 4 (b), and 3 (c).
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li e ma es [20]. As a possible explana ion, i has eme ged ha
CFA elici s s ong myelopoiesis and expansion o Mac-1+
cells, which play a c ucial ole in disease pa hogenesis, and
his p ocess is down egula ed by IFN-γ [20]. Howe e , he
exac mechanism esponsible o he e ec o IFN-γ in CIA has
no been ully elucida ed.
One o he mos likely mechanisms o he down egula ion o
CIA by IFN-γ is he inc eased exp ession o IDO by non-T cells
[21]. In ib oblas s [22], mac ophages [23], and dend i ic cells
[3], IFN-γ s imula es he enzyme IDO, which deg ades he
amino acid yp ophan o o m kynu enine, esul ing in he inhi-
bi ion o au oimmune p ocesses. Acco ding o his assump-
ion, we demons a ed an ele a ed K/T a io, indica ing high
IDO ac i i y du ing he acu e phase o CIA. Mo eo e , he K/T
a io was e en highe in mice ha ecei ed CII and CFA bu
de eloped no clinical and his ological signs o a h i is. These
da a sugges ha IDO ac s as a nega i e eedback in his
model, and he onse and se e i y o expe imen al a h i is a e
in e sely p opo ional o IDO ac i i y.
To con i m he egula o y ole o IDO in CIA, we used 1-MT, a
known compe i i e inhibi o o IDO. 1-MT did no in luence
IFN-γ, bu i signi ican ly supp essed IL-4 p oduc ion by spleen
cells, esul ing in an inc eased Th1/Th2 esponse. In 1-MT-
ea ed mice, we could demons a e a signi ican dec ease o
K/T a io in he immedia e p e-a h i ic and acu e phase o
a h i is compa ed o ehicle- ea ed animals, sugges ing he
high ac i i y o IDO only in hese s ages o in lamma o y p oc-
esses. In o he wo ds, he low blocking ac i i y o 1-MT ei he
in he p e-a h i ic phase o in he ch onic phase o a h i is
deno es he an i-in lamma o y e ec o IDO only in he case o
Figu e 3
Humo al immune esponses in DBA mice immunized wi h ype II colla-gen (CII) and comple e F eund's adju an (CFA) and ea ed wi h 1-me hyl- yp ophan (1-MT) o ehicleHumo al immune esponses in DBA mice immunized wi h ype II colla-
gen (CII) and comple e F eund's adju an (CFA) and ea ed wi h 1-
me hyl- yp ophan (1-MT) o ehicle. (a) Concen a ions o an ibodies
( o al concen a ions o IgGAM [immunoglobulins G, A, and M]) o he -
e ologous (human) CII we e de e mined in he se um o DBA/1 mice
immunized wi h CII and CFA and ea ed wi h 1-MT o ehicle. (b) Con-
cen a ions o an ibodies ( o al concen a ions o IgGAM) o au ologous
(mouse) CII we e de e mined in he se um o DBA/1 mice immunized
wi h CII and CFA and ea ed wi h 1-MT o ehicle. Se a we e ob ained
on days 5, 22, 33, and 54. Values a e p esen ed as he mean and
s anda d e o o he mean o 25 1-MT- ea ed and 20 ehicle- ea ed
DBA/1 mice pe g oup and ep esen wo independen expe imen s. *p
< 0.05 be ween ehicle and 1-MT- ea ed mice on he co esponding
days.
Figu e 4
Concen a ions o in e leukin-4 (IL-4) and in e e on-γ (IFN-γ) in he supe na an s o spleen cells ha es ed om mice wi h collagen-induced a h i is and ea ed wi h 1-me hyl- yp ophan (1-MT) o ehicleConcen a ions o in e leukin-4 (IL-4) and in e e on-γ (IFN-γ) in he
supe na an s o spleen cells ha es ed om mice wi h collagen-
induced a h i is and ea ed wi h 1-me hyl- yp ophan (1-MT) o ehi-
cle. The elease o IL-4 (a) and IFN-γ (b) in o he supe na an s o spleen
cells in esponse o human o mouse ype II collagen (CII) was de e -
mined a he end o he expe imen . Values a e p esen ed as he mean
and s anda d e o o he mean o 16 animals pe g oup (*p < 0.01).

A h i is Resea ch & The apy Vol 9 No 3 Szán ó e al.
Page 6 o 7
(page numbe no o ci a ion pu poses)
up egula ion o in lamma o y p ocesses, especially Th1
esponses.
As a yp ophan-ca abolizing enzyme, IDO can induce he
pe iphe al ole ance and educe he pe sis en immune ac i a-
ion. On one hand, IDO dec eases he yp ophan concen a-
ion in he mic oen i onmen o in lamma o y cells. Al hough
yp ophan is an essen ial amino acid indispensable o he
biosyn hesis o p o eins, he low yp ophan concen a ion
esul s in he a es o cell p oli e a ion in he mid-G1 a es
poin . T cells a e speci ically sensi i e o yp ophan dep i a-
ion [2,5] and hus IDO ac i i y can block he po en ial ha m ul
au oimmune esponse. On he o he hand, Zhu and colleagues
[24] p oposed ha syno ial T cells de i ed om RA syno ial
luids a e esis an o IDO-media ed yp ophan dep i a ion.
This may be one mechanism by which au o eac i e T cells a e
sus ained in i o in pa ien s wi h a h i is [24]. In addi ion,
selec ed me aboli es on he yp ophan-kynu enine pa hway
a e able o supp ess p oli e a ion o allogeneic T cells and, o
a lesse ex en , B and NK cells [25]. Mo eo e , some o he
kynu enine de i a es can induce in i o he selec i e apop o-
sis o Th1 cells, bu no Th2 cells [7]. In acco dance wi h hese
esul s, 1-MT ea men in ou s udy esul ed in an inc eased,
mainly Th1 cell-media ed immune esponse o he CII and con-
sequen ly in he signi ican wo sening o se e i y and inc ease
o onse o CIA media ed by Th1 esponse.
In his ega d, ecen s udies wi h he CIA model o a h i is
using an an ibody o he cos imula o y molecule CD137, a
membe o he umo nec osis ac o ecep o supe amily, a e
o g ea in e es . These esul s showed ha ea men o mice
wi h he an i-CD137 an ibody esul ed in induc ion o IDO in
i o, which was associa ed wi h signi ican amelio a ion o he
se e i y o CIA [26]. Fu he mo e, pha macological inhibi ion
o IDO e e sed he e ec s o he an i-CD137 an ibody and
agg a a ed he a h i is in his model.
The yp ophan-IDO pa hway may ha e impo an ele ance
o he biological he apy o RA. In a s udy o Boasso and col-
leagues [27], CTLA-4-Fc ea men o human pe iphe al blood
CD4+ T cells esul ed in inc eased IDO exp ession by hese
cells. This e ec was no obse ed in CD8+ T cells. Thus,
aba acep (CTLA4-Ig) he apy may ac , a leas in pa , by he
s imula ion o IDO p oduc ion.
Conclusion
The impo ance o IDO ac i i y in he egula ion o CIA sup-
po s he hypo hesis ha IDO exp ession by an igen-p esen -
ing cells is esponsible o supp ession o undesi able Th1
cells in human in lamma o y join diseases, as yp ophan deg-
ada ion could be demons a ed in pa ien s wi h RA [28].
These indings heo e ically aise he possibili y ha he local
o sys ema ic induc ion o IDO ac i i y could be es ed in
in lamma o y join diseases.
Compe ing in e es s
The au ho s decla e ha hey ha e no compe ing in e es s.
Au ho s' con ibu ions
SS pe o med he expe imen al wo k and p epa ed he manu-
sc ip . TK pe o med he expe imen al wo k. ZS ad ised on he
s udy. KM and TTG a e he heads o labo a o y, supe ised he
expe imen al wo k, and ad ised on he s udy. JV is he senio
esea che and supe iso o he expe imen al wo k and
ad ised on he s udy. All au ho s ead and app o ed he inal
manusc ip .
Acknowledgemen s
The au ho s acknowledge Kuniaki Sai o, o he Na ional Ins i u es o
Heal h (NIH), o pe o ming he se um kynu enine assays and he con-
ibu ions o many colleagues in he Sec ion o Biochemis y and Molec-
ula Biology, Rush Uni e si y and Sec ion o Rheuma ology, Uni e si y o
Illinois College o Medicine. This wo k was suppo ed by a g an om he
NIH (AR047163).
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Kynu enine/ ypophan (K/T) a ios in se a o DBA/1 mice immunized wi h ype II collagen (CII) and comple e F eund's adju an (CFA) and ea ed wi h 1-me hyl- yp ophan (1-MT) o ehicleKynu enine/ ypophan (K/T) a ios in se a o DBA/1 mice immunized
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