incremento de il-21 pam y gw

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Increased frequency of circulating IL-21 producing Th-cells in patients withgranulomatosis with polyangiitis

Arthritis Research & Therapy2013,15:R70 doi:10.1186/ar4247

Wayel H Abdulahad ([email protected])Nikola Lepse ([email protected])

Coen A Stegeman ([email protected])Minke G Huitema ([email protected])

Berber Doornbos-van der Meer ([email protected])Henko H Tadema ([email protected])Abraham Rutgers ([email protected])

Pieter C Limburg ([email protected])Cees GM Kallenberg ([email protected])

Peter Heeringa ([email protected])

ISSN 1478-6354

Article type Research article

Submission date 16 January 2013

Acceptance date 18 June 2013

Publication date 24 June 2013

Article URL http://arthritis-research.com/content/15/3/R70

This peer-reviewed article can be downloaded, printed and distributed freely for any purposes (seecopyright notice below).

Articles inArthritis Research & Therapyare listed in PubMed and archived at PubMed Central.

Arthritis Research & Therapy
mailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]://arthritis-research.com/content/15/3/R70http://arthritis-research.com/content/15/3/R70mailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]


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Increased frequency of circulating IL-21 producing Th-cells in patients with granulomatosis

with polyangiitis (GPA

Wayel H Abdulahad1, Nikola Lepse

2, Coen A Stegeman

3, Minke G Huitema

1, e!be! "oo!nbos#$an de!

Mee!1, Henko %adema

1, Ab!aham &utge!s

1, 'iete! C Limbu!g

(, Cees GM )allenbe!g

1, and 'ete!

Hee!inga2*

1"epa!tment o+ &heumatology and Clinial -mmunology, .ni$e!sity o+ G!oningen, .ni$e!sity Medial

Cente! G!oningen, Han/eplein 1, 013 G, G!oningen, %he Nethe!lands*

2"epa!tment o+ 'athology and Medial iology, .ni$e!sity o+ G!oningen, .ni$e!sity Medial Cente!

G!oningen, Han/eplein 1, 013 G, G!oningen, %he Nethe!lands*

3"epa!tment o+ Neph!ology, .ni$e!sity o+ G!oningen, .ni$e!sity Medial Cente! G!oningen,

Han/eplein 1, 013 G, G!oningen, %he Nethe!lands*

("epa!tment o+ Labo!ato!y Mediine, .ni$e!sity o+ G!oningen, .ni$e!sity Medial Cente! G!oningen,

Han/eplein 1, 013 G, G!oningen, %he Nethe!lands*

Wayel H Abdulahad: [email protected]

Nikola Lepse: [email protected]

Coen A Stegeman: [email protected]

Minke G Huitema: [email protected]

e!be! "oo!nbos#$an de! Mee!: [email protected]

H k % d h t d @ l
mailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]


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A!stract

"!#ecti$es% %he p!esent study aimed to e5plo!e a possible !ole +o! -L#21 p!oduing %h#ells in the

immunopathogenesis o+ g!anulomatosis 6ith polyangiitis 7G'A8*

&ethods% 'e!iphe!al blood +!om (2 G'A#patients in !emission and 20 age#mathed healthy ont!ols

7HCs8 6e!e stimulated in vitro, and the +!e9uenies o+ -L#21 p!oduing %h ells 6e!e dete!mined by

+lo6 ytomet!y* Sine %h1#ells p!odue a lo6 le$el o+ -L#21, -L#1 6as also inluded in the analysis*

Gi$en that -L#21 is a hallma!k ytokine +o! % +olliula! helpe! ells 7%:H8, 6e ne5t e$aluated the

e5p!ession o+ thei! key t!ans!iption +ato! l#; by &%#'C& and +lo6 ytomet!y* %o in$estigate the

e++et o+ -L#21 on autoantibody#p!odution, 'MCs +!om G'A#patients 6e!e stimulated in vitro 6ith

A::


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Introduction

G!anulomatosis 6ith polyangiitis 7G'A8 is an autoimmune $asulitis o+ small# to medium#si/ed blood

$essels, assoiated 6ith the p!esene o+ i!ulating anti#neut!ophil ytoplasmi autoantibodies

7ANCA8 that a!e mainly di!eted against p!oteinase 3=1#3>* Histopathologially, G'A is ha!ate!i/ed

by g!anulomatous in+lammation and paui#immune $asulitis, inluding ne!oti/ing !esenti

glome!uloneph!itis*

Although the p!odution o+ ANCA is di!etly att!ibutable to auto!eati$e #ells, the!e is e5tensi$e

e$idene that %#ells play a !itial !ole in G'A as 6ell* %he -gG sublass dist!ibution o+ ANCA, 6ith a

p!eponde!ane o+ the -gG1 and -gG( sublasses, suggests a % ell?dependent immune !esponse=(>*

-n+ilt!ating %#ells in g!anulomatous lesions as 6ell as pe!sistent %#ell ati$ation ha$e been obse!$ed

in G'A#patients=@, ;>* -n addition, an abe!!ant %#ell phenotype and impai!ed !egulato!y %#ell

+untion a!e also !epo!ted in G'A#patients in !emission=#0>, suggesting that e$en du!ing !emission,

the immune system is dys!egulated* Mo!eo$e!, %#helpe! ell pola!i/ation 6ith an in!ease in %h1

ells has been demonst!ated=1, 11>* %h1 ells and thei! ytokine -L#1 ha$e been sho6n to play a

!itial !ole in many in+lammato!y diseases* esides -L#1, %h1 ells an p!odue -L#21, a ytokine

that is la!gely !esponsible +o! ell lass s6ithing and antibody p!odution, and 6hih indues

di++e!entiation o+ ells to6a!ds plasma ells by syne!gi/ing 6ith #ell ati$ating +ato! 7A::8=12,

13>* -t is the!e+o!e onei$able that -L#21 may ont!ibute to the p!odution o+

pathogeni autoantibodies in G'A*

l l d l d l d l l + h h +


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diabetes=1;, 1>* -nte!estingly, !eent genome#6ide assoiation studies ha$e p!o$ided on$ining

e$idene that geneti $a!iants in the !egion on h!omosome (92 that ha!bo! the -L#21 and -L#2

genes a!e assoiated 6ith h!oni in+lammato!y diso!de!s, inluding SL, -" and pso!iasis=1D#2>*

%hus, -L#21 seems to play an impo!tant !ole in autoimmune diseases in gene!al and ould onstitute a

no$el ta!get +o! the!apy*

-L#21 is p!odued mainly by ati$ated C"(E %h#ells* &eent studies ha$e demonst!ated that -L#21,

besides its p!odution by %h1 ells, is p!edominantly se!eted by a distint %h ell lineage, te!med

+olliula! helpe! %#ells 7%:H8 that e5p!ess the t!ans!iption +ato! l#; and a!e onside!ed to be

speiali/ed p!o$ide!s o+ ell help=21>* 5pansion o+ i!ulating % ells !esembling %:Hells has been

!epo!ted in patients 6ith SL and in patients 6ith !heumatoid a!th!itis=22#2(>* %o date, no study has

in$estigated the !ole o+ -L#21 p!oduing %h#ells in G'A* %he!e+o!e, this study aimed to assess the

+!e9ueny o+ -L#21 p!oduing %h#ells, and to e$aluate 6hethe! %:Hells o! %h1 ells a!e the maBo!

sou!e o+ -L#21 in G'A#patients* :o! this pu!pose, 6e e5amined the e5p!ession o+ both -L#21 and -L#1

in i!ulating C"(E %#ells o+ patients 6ith G'A* %o imp!o$e ou! unde!standing o+ the !ole o+ -L#21

p!oduing %h ells in autoantibody p!odution 6e assessed thei! +!e9uenies in ANCA#positi$e and

ANCA#negati$e patients, and studied e++ets o+ -L#21 on immunoglobulin and ANCA p!odution in

vitro*


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&ethods

+tudy population, :o!ty#t6o patients 6ith G'A and 20 age# and se5#mathed healthy ont!ols 71D

males, 11 +emales, mean age @; =S" F13> yea!s, !ange 2;#2 yea!s, P *1;8 6e!e inluded in this

study* %he diagnosis o+ G'A 6as established ao!ding to the de+initions o+ the Chapel Hill Consensus

Con+e!ene and patients +ul+illed the lassi+iation !ite!ia o+ the Ame!ian College o+ &heumatology

7AC&8=2@, 2;>* nly patients 6ithout linial signs and symptoms o+ ati$e $asulitis and onside!ed

to be in omplete !emission, as indiated by a so!e o+ /e!o on the i!mingham Iasulitis Ati$ity

So!e 7IAS8, 6e!e inluded in the study=2>* Se!ostatus +o! ANCA 6as +ollo6ed +o! se$e!al months

in all patients, and patients 6ith a stable se!ostatus +o! ANCA 7positi$e o! negati$e8 +o! at least 3

months 6e!e inluded in this study* ased on these !ite!ia, 23 patients 6e!e positi$e +o! '&3#ANCA,

6he!eas 10 6e!e ANCA#negati$e* %6enty#se$en patients 6e!e onside!ed to ha$e a histo!y o+

generalized disease inluding !enal in$ol$ement, and 1@ patients localized disease, in 6hih the

disease had been on+ined to the uppe! and


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&easurement of AA titres and specificity, Anti#neut!ophil ytoplasmi antibody 7ANCA8 tite!s

6e!e measu!ed by indi!et immuno+luo!esene 7--:8 on ethanol#+i5ed human g!anuloytes ao!ding

to standa!d p!oedu!es as p!e$iously des!ibed=2D>* ANCA tite!s highe! than 1( 6e!e onside!ed

positi$e* ANCA antigeni spei+iity 6as dete!mined using an in#house aptu!e en/yme#linked

immunoso!bent assay 7L-SA8 as des!ibed be+o!e=20, 3>* !ie+ly, a 0;#6ell plate 6as oated 6ith

goat#anti#mouse -g +o! (D hou!s* A+te! 6ashing, plates 6e!e inubated 6ith mouse monolonal

antibody against human '&3 +o! 2 hou!s* A+te! 6ashing, the plate 6as inubated o$e!night at ( JC

6ith an e5t!at o+ human a/u!ophili g!anules 6hih 6e!e isolated +!om neut!ophils o+ healthy

dono!s* :u!the!, se!ial dilutions o+ se!um 76ith a sta!ting dilution o+ 118 6e!e inubated +o! 1

hou!* %he plate 6as 6ashed, and the aptu!ed antibodies 6e!e deteted 6ith pu!i+ied :7ab8 2goat#

anti#human -gG onBugated to alkaline phosphatase* '#nit!ophenyl#phosphate disodium 6as used as

a subst!ate, and the optial density 6as measu!ed at (@ nm*

Anti!odies used in flow cytometry, %he +ollo6ing onBugated antibodies 6e!e used in +lo6

ytomet!y Allophyoyanin 7A'C8#onBugated anti#C"3 7lone .CH%18, pe!idin hlo!ophyll p!otein

7'e!C'8#onBugated anti#C"D 7lone S)18, 'hyoe!yth!in 7'8#onBugated anti#-L#21#!eepto! 7lone

1A128, and 'e!C'#onBugated anti#C"10 7lone (G8, all pu!hased +!om eton K "ikinson

7Amste!dam, %he Nethe!lands8 '#onBugated anti#-L#21 7lone eio3A3#N28, Ale5a :luo! (DD

7A(DD8#onBugated anti#-L#1 7lone eio;("C18, and A(DD#onBugated anti#:o5'3 7lone 'CH118,

all pu!hased +!om eiosiene 7San "iego, CA, .SA8 '#onBugated anti#CL; 7lone -C@(;'8 6as


8/34


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CL; and :o5'3 ao!ding to the manu+atu!e!Qs inst!utions 7eiosiene staining set +o!

t!ans!iption +ato!s8* !ie+ly, 'MCs 6e!e adBusted to 1 5 1;

ells in 1L and inubated 6ith

app!op!iate onent!ation o+ A'C#onBugated anti#C"3 and 'e!C'#onBugated anti#C"D +o! 3

minutes at (oC in the da!k, +ollo6ed by +i5ation and pe!meabili/aion in :i5


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Se9uene "etetion System, Applied iosystems, :oste! City, CA, .SA8 6ith spei+i %a9man

p!ime!s and GA'"H =Hs 000000@m1>, Applied iosystems8*

Ampli+iation 6as pe!+o!med using standa!d onditions and alulations o+ +old indution 6e!e

pe!+o!med* We no!mali/edgene e5p!ession to GA'"H and e5p!essed $alues !elati$e to ont!ol

using

the C% method*

ell stimulation and total IgG production,

'MCs !eo$e!ed +!om the g!adient inte!+ae 6e!e 6ashed t6ie in 'S and adBusted to 1;ells* !ie+ly, Costa! 0;#6ell L-SA plates 6e!e oated 6ith 2

mg


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&easurement of invitroproduction of P'-AA

In vitro '&3#ANCA -gG p!odution in 'MC ultu!e supe!natants 6as measu!ed by 'hadia

-mmunoCA' 2@ analy/e! 7%he!mo :ishe! Sienti+i8 using LiA%M

'&3, and the le$els o+ '&3#ANCA

-gG p!odution 6e!e e5p!essed in !esponse units 7&.8*

+tatistical analysis,

"ata a!e p!esented as median $alues, unless stated othe!6ise* %he nonpa!amet!i Mann#Whitney .

test 6as used to ompa!e data +!om patients 6ith that o+ healthy ont!ols* %he Wilo5on mathed

pai!s test 6as used +o! int!aindi$idual ompa!ison* Co!!elations 6e!e assessed using Spea!manQs !ank

o!!elation oe++iient* %6o#tailed P#$alues less than *@ 6e!e onside!ed statistially signi+iant*


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'esults

Increased percentage of circulating IL-21IL-13-cells in AA-positi$e GPA-patients compared to

AA-negati$e patients and healthy controls

We initially dete!mined the +!e9ueny o+ -L#21 p!oduing C"( %#ells in the pe!iphe!al blood o+ G'A#

patients 7n (28 and HCs 7n 208 a+te! in vitro stimulation* %he pe!entage o+ i!ulating -L#21E%h#

ells 6as signi+iantly highe! in G'A#patients as ompa!ed 6ith the ont!ol g!oup 7:igu!e 18* +

note, %h1 ells may p!odue -L#21 in addition to thei! signatu!e ytokine -L#1* Sine %h1 ells a!e

in!eased in G'A#patients=1, 11>, 6e ne5t e5tended ou! analysis to in$estigate 6hethe! in!eased -L#

21E%h#ells in G'A#patients !esulted +!om an in!ease in %h1 ells* %o this end, -L#1 staining 6as

inluded in the analysis to dete!mine 6hat pe!entage o+ the total -L#21E %h#ells a!e %h1 ells*

.sing this app!oah, 6e assessed the +!e9ueny o+ -L#21E-L#1

#,-L#21

E-L#1

E, and -L#21

#-L#1

E ells

6ithin the C"( %#ells in G'A#patients and HCs* As sho6n in +igu!e 1 7C, " and 8, G'A#patients in

!emission had a signi+iantly highe! pe!entages o+ i!ulating -L#21E-L#1

#, -L#21

E-L#1

E, and -L#21

#-L#

1Eells as ompa!ed 6ith the ont!ol g!oup* Ho6e$e!, the maBo!ity o+ i!ulating C"(

E%#ells that

p!odued -L#21 only, 6e!e distint +!om %h1 ells, that is negati$e +o! -L#1* %o assess the possible

!ole o+ -L#21E-L#1

# %h#ells in ANCA p!odution, 6e ompa!ed thei! pe!entage bet6een patients

that 6e!e ANCA#positi$e 7n 23 --: tite! U1(8 o! ANCA#negati$e 7n 108 at the time o+ inlusion*

Signi+iant in!eases in the +!e9uenies o+ -L#21E-L#1

# %h#ells 6e!e obse!$ed in ANCA#positi$e


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%o !ule out the possibility that the in!eased p!opo!tion o+ -L#21E-L#1

#%h#ells in G'A#patients 6as

the !esult o+ u!!ent t!eatment, the ANCA#positi$e patient g!oup 6as di$ided into t!eated and

unt!eated patients, and the pe!entages o+ -L#21E-L#1

# %h#ells 6e!e ompa!ed* No signi+iant

di++e!enes 6e!e obse!$ed bet6een t!eated and unt!eated patients 7data not sho6n8* We also

ompa!ed the pe!entage o+ -L#21E-L#1

# %h#ells bet6een u!!ently unt!eated ANCA#positi$e

patients 6ith a histo!y o+ gene!ali/ed and loali/ed disease* No di++e!ene 6as +ound bet6een these

patient g!oups 7data not sho6n8*

Increased frequencies of IL-21

IL-13

-

Th-cells correlate positi$ely with Th13 response,

-t has been !epo!ted that -L#21 is a key +ato! !egulating the di++e!entiation o+ naV$e C"(E%#ells into

%h1 ells=32, 33>* -n o!de! to analy/e this !elation, 6e o!!elated pe!entages o+ -L#21E-L#1

#%h#ells

6ith pe!entages o+ te!minally di++e!entiated %h1 ells 7-L#21#-L#1

E8 in G'A#patients 7n (28 and HCs

7n 208* -nte!estingly, a signi+iant positi$e o!!elation 6as obse!$ed bet6een -L#21E-L#1

# %h#ells

and -L#21#-L#1

E %h#ells in both G'A#patients and HCs 7! *@D, P *1 and ! *3, P *(,

!espeti$ely8 7:igu!e 2A and 8*

Increased frequencies of 0L-4 56T-cells in peripheral !lood of AA-positi$e GPA-patients


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ANCA#positi$e G'A 7n 18 had a signi+iantly highe! e5p!ession o+ m&NA CL#; than ANCA#negati$e

patients 7n ;8 and HCs 7n 118 7:igu!e 38* -n addition, int!aellula! :ACS#staining +o! CL; 6ithin

i!ulating C"(E % ells on+i!med the in!eased CL#; e5p!ession in ANCA#positi$e G'A#patients

7:igu!e 3A and C8* We ha$e also analy/ed the M:- 7mean +luo!esene intensity8 o+ CL#; e5p!ession

in C"(E%#ells +!om patients and HCs and +ound that the e5p!ession le$el o+ CL#; pe! %h#ell in G'A#

patients 6as simila! to that in HCs 7data not sho6n8* %hus, CL#; e5p!ession is in!eased in G'A#

patients due to in!eased +!e9uenies o+ i!ulating CL#;EC"(

E%#ells*

Sine in !eent studies a ne6 population o+ :o5'3E!egulato!y %#ells has been des!ibed that sha!es

+eatu!es 6ith %:H ell by e5p!essing the t!ans!iption +ato! l#;, 6e also e$aluated 6hethe! the

in!ease in CL#;E%#ells in G'A#patients 6as a !esult o+ an in!ease in :o5'3

ECL#;

E%#ells=3;, 3>*

%his analysis sho6ed that the in!ease in CL#; e5p!ession in G'A patients 6as !est!ited to % :Hells

and although a lo6 pe!entage o+ :o5'3ECL#;

E%#ells 6as +ound 7 *3P8, no di++e!enes in these

ell +!e9uenies 6e!e obse!$ed bet6een G'A#patients and HCs 7data not sho6n8*

Proportions of IL-21-receptor e7pressing 0-cells do not differ !etween GPA-patients and 8

Sine it is 6ell kno6n that -L#21 ats on #ells to suppo!t thei! e5pansion and antibody

p!odution=3D#(>, 6e onduted +u!the! analysis to ompa!e the e5p!ession o+ -L#21& on ells

+!om G'A#patients and HCs* No di++e!enes 6e!e seen in the pe!entages o+ -L#21&E#ells neithe!


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IL-21 induces IgG and AA production !y 0-cells from GPA-patients

%o e5plo!e the inte!play bet6een -L#21 p!oduing %h#ells and #ells in G'A#patients, 6e

in$estigated the e++et o+ -L#21 on -gG antibody#p!odution by #ells +!om G'A#patients* &est!ited

numbe!s o+ patients and ont!ols 6e!e en!olled in this analysis due to insu++iient ell numbe!s*

'MCs +!om G'A#patients 6e!e ultu!ed in vitroin the p!esene o! absene o+ e5ogenous -L#21 +o!

12 days and total -gG 6as measu!ed in supe!natants by L-SA* Sine -L#21 p!omotes #ell

di++e!entiation by syne!gi/ing 6ith A::=12, 13>, 6e 9uestioned 6hethe! the e++et o+ -L#21 on -gG

p!odution ould be augmented by adding A:: to the ultu!e* + note, autologous % ells in ou!

ultu!e system at as a natu!al p!o$ide! o+ C"( ligation +o! #ells, as this ligation is !e9ui!ed +o!

ell ati$ation, isotype s6ithing and memo!y de$elopment* As sho6n in +igu!e @, -L#21 signi+iantly

enhaned the p!odution o+ -gG in vitro in stimulated 'MCs +!om both ANCA#positi$e 7n 8 and

ANCA#negati$e 7n ;8 G'A#patients, 6hile stimulation 6ith A:: alone did not !esult in in!eased -gG

p!odution* %he ombination o+ A:: and -L#21 tended to in!ease -gG p!odution mo!e than -L#21

alone* Ne5t, 6e assessed the e++et o+ -L#21 plus A:: on in vitrop!odution o+ '&3#ANCA* As sho6n

in +igu!e @, spontaneous '&3#ANCA p!odution 6as obse!$ed in ultu!ed 'MCs +!om ANCA#

positi$e patients 7n 1;8 in ompa!ison 6ith ells +!om ANCA#negati$e patients 7n 128* -mpo!tantly,

-L#21 indues a signi+iant enhanement in '&3#ANCA p!odution in 'MCs isolated +!om ANCA#

positi$e patients in ompa!ison 6ith ANCA#negati$e patients* So it is onei$able that auto!eati$e #

ells 6e!e en!ihed in the pe!iphe!al blood o+ ANCA#positi$e patients*


16/34

5iscussion

-n the p!esent study, 6e demonst!ate an in!ease in the pe!entage o+ i!ulating -L#21 p!oduing

%h#ells in G'A#patients* We +ound that ele$ated +!e9uenies o+ -L#21 p!oduing %h#ells 6e!e

!est!ited to ANCA#positi$e G'A patients and that these ells 6e!e distint +!om %h1 ells* We also

on+i!med that -L#21 an enhane the p!odution o+ -gG and ANCA in vitro*

$e! the past +e6 yea!s, %h1 ells ha$e hallenged the lassial %h1* &eently, a

distint %h#ell subset te!med %:Hand ha!ate!i/ed by ele$ated e5p!ession le$els o+ multiple su!+ae

p!oteins and l#; as 6ell as enhaned -L#21 se!etion, has been identi+ied as t!ue helpe! ells +o!

antibody !esponses* We and othe!s ha$e p!e$iously demonst!ated that i!ulating %h1 ells a!e

signi+iantly in!eased in G'A#patients e$en du!ing 9uiesent disease=1, 11>* Ho6e$e!, data a!e

laking to suppo!t a !ole o+ -L#21#p!oduing %h#ells in G'A* Sine %h1 ells also p!odue -L#21, 6e

in$estigated 6hethe! %h1 ells in G'A a!e a sou!e o+ -L#21* St!ikingly, the maBo!ity o+ i!ulating

C"( %#ells that p!odued -L#21 6e!e distint +!om %h1 ells, indiating that othe! %h#ell subsets

suh as %:H ells a!e the sou!e o+ this ytokine* -mpo!tantly, the e5pansion o+ %:H ells in G'A

patients 6as on+i!med by in!eased L; e5p!ession* %o the best o+ ou! kno6ledge, this is the +i!st

!epo!t demonst!ating an in!ease in the +!e9ueny o+ i!ulating -L#21 p!oduing %h#ells in G'A

suggesting that %:H ell#de!i$ed -L#21 may ont!ibute to disease pathogenesis $ia stimulation o+

7auto8antibody p!odution*


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patients* -t seems likely that in!eased %h1 ells in G'A#patients a!e the !esult o+ an enhaned % :H

!esponse, 6hih in tu!n may pa!tiipate in g!anuloma +o!mation and $asula! damage* %he !ole o+ -L#

21 in $asulitis 6as p!e$iously suggested by Chen and o6o!ke!s=(3>* -n thei! study, mie de+iient in

inte!+e!on !egulato!y +ato!#(, a p!otein that inhibits -L#1A p!odution, !apidly de$eloped la!ge#

$essel $asulitis and sho6ed in!eased -L#21 synthesis in addition to in!eased -L#1A p!odution=(3>*

Mo!eo$e!, a !ole o+ -L#21 in !e!uitment o+ %h1 ells to in+lamed tissues has been !epo!ted by

Ca!uso and o6o!ke!s=((> by sho6ing that -L#21 indues gut epithelial ells to se!ete ma!ophage

in+lammato!y p!otein#3X 7M-'#3X8, a hemokine that mediates %h1 ell homing to the skin, Boints,

and muosal tissues* Gi$en that endothelial ells a!e kno6n to p!odue M-'#3X, it is possible that -L#

21 in G'A#patients enhanes the mig!ation and aumulation o+ %h1 ells into the $asula! 6all

!esulting in in+lammation* esides, -L#21 6as sho6n to enhane g!an/yme e5p!ession=(@> and

in!ease pe!+o!in#mediated ytoto5iity by human C"D %#ells=(;> and N) ells=(>* -t is the!e+o!e

onei$able that -L#21 an ont!ibute to $essel inBu!y and disease p!og!ession in G'A#patients* %his is

an a!ea 6o!th o+ +u!the! in$estigation*

-n ont!ast to the p!o#in+lammato!y !ole o+ %:H ells, !eent studies ha$e identi+ied a

distinti$e population o+ %:H ells that displays a !egulato!y +untion and supp!esses the

di++e!entiation o+ GC #ells in +olliles in vivo* %his subset 6as te!med +olliula! !egulato!y % ells

7%:&8, 6hih e5p!ess the !egulato!y t!ans!iption +ato! FoxP3 in addition to thei! spei+i lineage

t!ans!iption +ato! CL#;=3;, 3>* As i!ulating FoxP3E%#ells a!e in!eased in G'A#patients=>, it is

onei$able that the obse!$ed in!ease in %:Hells in patients is due to an in!ease in %:&ells that o#

e5p!ess FoxP3 and CL;* We ha$e in$estigated this possibility but +ound that the in!ease in


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at di!etly on #ells th!ough the -L#21* %he e5p!ession o+ -L#21& on #ells

+!om ANCA#positi$e and ANCA#negati$e G'A#patients 6as ompa!able, 6hih suggests that both

patient populations ha$e the same ability to !espond to -L#21* Ho6e$e!, in vitrostimulation 6ith -L#

21 enhaned the p!odution o+ ANCA in ell ultu!es +!om ANCA#positi$e patients only, although

enhaned total -gG#p!odution 6as obse!$ed in both patient g!oups* So it is onei$able that

auto!eati$e #ells 6e!e en!ihed in the pe!iphe!al blood o+ ANCA#positi$e patients* %his might be

linially !ele$ant as 6ell sine ANCA positi$e patients a!e at in!eased !isk +o! disease !elapse=@,

@1>*

-n this study, patients 6e!e e$aluated +o! the dist!ibution o+ %:H ells du!ing !emission* We ha$e

p!e$iously sho6n that ati$ated %#ells a!e p!esent at the time o+ linially 9uiesent disease=0, 1>*

:u!the!mo!e, du!ing ati$e disease e++eto! %#ells appea! to mig!ate to6a!ds in+lamed tissue=@2>*

%he!e+o!e, in o!de! to study dysbalane o+ %#ells in G'A#patients using pe!iphe!al blood samples, 6e

hoose to selet patients 6ithout o! 6ith lo6 dosages o+ immunosupp!essi$e mediation and at the

time o+ linially 9uiesent disease*

onclusions

-n onlusion, the data p!esented he!e demonst!ate a p!ominent in!ease o+ i!ulating %:Hells in

ANCA#positi$e G'A#patients* %he key ytokine o+ these %:H ells, that is -L#21, ont!ibutes to the


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A!!re$iations

ANCA anti#neut!ophil ytoplasmi autoantibodies A(DD Ale5a :luo! (DD A'C Allophyoyanin

A:: #ell ati$ating +ato! IAS i!mingham Iasulitis Ati$ity So!e Ca#- alium ionopho!e

L-SA en/yme#linked immunoso!bent assay :CS +etal al+ se!um :-%C +luo!esein

isothioyanate%:H +olliula! helpe! %#ells G'A g!anulomatosis 6ith polyangiitis HC healthy

ont!ol -g immunoglobulin --: indi!et immuno+luo!esene -L#21 inte!leukin#21 -L#21&

inte!leukin#21 !eepto! ' pe!iphe!al blood 'MC pe!iphe!al blood mononulea! ells 'S

phosphate#bu++e!ed saline ' 'hyoe!yth!in 'e!C' pe!idin hlo!ophyll p!otein 'MA pho!bol

my!istate aetate '&3 p!oteinase 3*

ompeting interests

%he autho!s dela!e that they ha$e no ompeting inte!ests*

Authors9 contri!utions

All autho!s ont!ibuted to the design, a9uisition o+ data, analysis and inte!p!etation o+ data* WHA

ont!ibuted to onept and design, pe!+o!med the statistial analysis, and had +ull aess to all o+ the

data in the study and takes !esponsibility +o! the integ!ity o+ the data and the au!ay o+ the data


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Ac/nowledgments

We a!e g!ate+ul to the patients and healthy dono!s +o! thei! o#ope!ation and pa!tiipation in this

study* %he !esea!h leading to these !esults has !eei$ed +unding +!om the u!opean .nion Se$enth

:!ame6o!k '!og!amme 7:'


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'eference list

1* :aui AS, Haynes :, )at/ ', Wol++ SM *egener:s granulomatosis% prospecti$e clinical

and therapeutic e7perience with ;< patients for 21 years*Ann Intern Med10D3, =;;#

D@*

2* $an de! Woude :Y, &asmussen N, Lobatto S, Wiik A, 'e!min H, $an s LA, $an de! GM, $an

de! Hem G), %he %H Autoanti!odies against neutrophils and monocytes% tool for

diagnosis and mar/er of disease acti$ity in *egener:s granulomatosis* Lancet 10D@,

1(2@#(20*

3* Niles YL, MCluskey &%, Ahmad M:, A!naout MA *egener:s granulomatosis autoantigen

is a no$el neutrophil serine proteinase* Blood10D0, 361DDD#1D03*

(* !ou6e! , %e!$ae!t YW, Ho!st G, Huitema MG, $an de! GM, Limbu!g 'C, )allenbe!g CG

Predominance of IgG1 and IgG6 su!classes of anti-neutrophil cytoplasmic

autoanti!odies (AA in patients with *egener:s granulomatosis and clinically

related disorders* Clin Exp Immunol1001, ;30#3D;*

@* )omosi A, Lamp!eht ', Cse!nok , Muelle! A, Holl#.l!ih ), Seit/e! ., Moosig :,

Shnabel A, G!oss WL Peripheral !lood and granuloma 56(52;(- T cells are a

ma#or source of interferon-gamma and tumor necrosis factor-alpha in *egener:s

granulomatosis*Am J Pathol22, 14>11#12(*

;* Lamp!eht ', Moosig :, Cse!nok , Seit/e! ., Shnabel A, Muelle! A, G!oss WL 52;

negati$e T cells are enriched in granulomatous lesions of the respiratory tract in

*egener:s granulomatosis* horax21, ;(#3*


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ele$ated in patients with AA-associated $asculitis* $ephrol %ial ran!plant 21,

2


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22* "ol++ S, Abdulahad WH, West!a Y, "oo!nbos#$an de! M, Limbu!g 'C, )allenbe!g CG, iBl

M Increase in IL-21 producing T-cells in patients with systemic lupus erythematosus*

Arthriti! Re! her211, 1&1@*

23* Simpson N, Gatenby 'A, Wilson A, Malik S, :ulhe! "A, %angye SG, Manku H, Iyse %Y,

&onado! G, Huttley GA, Goodno6 CC, Iinuesa CG, Cook MC 7pansion of circulating T

cells resem!ling follicular helper T cells is a fi7ed phenotype that identifies a su!set of

se$ere systemic lupus erythematosus*Arthriti! Rheum21, 4223(#2((*

2(* Ma Y, hu C, Ma , %ian Y, aidoo S, Mao C, Wu W, Chen Y, %ong Y, Zang M, Yiao , Ru H,Lu L, Wang S Increased frequency of circulating follicular helper T cells in patients with

rheumatoid arthritis* Clin %ev Immunol212, 2>12D2(D*

2@* Yennette YC, :alk &Y, And!assy ), aon 'A, Chu!g Y, G!oss WL, Hagen C, Ho++man GS,

Hunde! GG, )allenbe!g CG, * omenclature of systemic $asculitides, Proposal of an

international consensus conference*Arthriti! Rheum100(, 31D#102*

2;* Lea$itt &Z, :aui AS, loh "A, Mihel A, Hunde! GG, A!end W', Calab!ese LH, :!ies Y:,

Lie Y%, Light+oot &W, Y!*, * The American ollege of 'heumatology 1==> criteria for the

classification of *egener:s granulomatosis*Arthriti! Rheum100, 111#11*

2* Lu9mani &A, aon 'A, Moots &Y, Yanssen A, 'all A, me!y ', Sa$age C, Adu "

0irmingham Basculitis Acti$ity +core (0BA+ in systemic necroti@ing $asculitis* )JM

100(, ;3;1#;D*

2D* %e!$ae!t YW, Mulde! L, Stegeman C, lema Y, Huitema M, %he H, )allenbe!g C

"ccurrence of autoanti!odies to human leucocyte elastase in *egener:s

granulomatosis and other inflammatory disorders*Ann Rheum %i!1003,


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33* Nu!ie$a &, Zang R, Ma!tine/ G, hang Z, 'anopoulos A", Ma L, Shluns ), %ian [,

Wato6ih SS, Yetten AM, "ong C ssential autocrine regulation !y IL-21 in the

generation of inflammatory T cells* $ature2, 66;(D#(D3*

3(* Zu ", &ao S, %sai LM, Lee S), He Z, Sutli++e L, S!i$asta$a M, Linte!man M, heng L,

Simpson N, llya!d Y-, 'a!ish -A, Ma CS, Li [Y, 'a!ish C&, Makay C&, Iinuesa CG The

transcriptional repressor 0cl-4 directs T follicular helper cell lineage commitment*

Immunit"20, 1(@#(;D*

3@* Nu!ie$a &-, Chung Z, Ma!tine/ GY, Zang R, %anaka S, Matske$ith %", Wang ZH, "ong C0cl4 mediates the de$elopment of T follicular helper cells* 'cience 20, 2


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((* Ca!uso &, :ina ", 'eluso -, Stol+i C, :antini MC, Gioia I, Cap!ioli :, "el Iehio G, 'aolu/i

A, Madonald %%, 'allone :, Monteleone G A functional role for interleu/in-21 in

promoting the synthesis of the T-cell chemoattractantD &IP-alphaD !y gut epithelialcells* &a!troenterolog"2, 121;;#1@*

(@* Liu Z, Zang , Ma Y, Wang H, Huang :, hang Y, Chen H, Wu C Interleu/in-21 induces the

differentiation of human Tc22 cells $ia phosphorylation of signal transducers and

acti$ators of transcription* Immunolog"211, 12@(#@(D*

(;* be!t C Interleu/in 21 up-regulates perforin-mediated cytoto7ic acti$ity of humanintra-epithelial lymphocytes* Immunolog"20, 1232;#21@*

(* Liu , Zang L, Cui Z, Wang R, Guo C, Huang , )an [, Liu , Liu Z Il-21 enhances ) cell

acti$ation and cytolytic acti$ity and induces Th13 cell differentiation in inflammatory

!owel disease* In,lamm Bo.el %i!20, 1


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Ta!le

Ta!le 1%

linical and la!oratory characteristics of GPA-patients at the time of !lood

sampling,

No* o+ patients (2

No* male


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.igure legends

.igure 1% &ultiparameter flow cytometric detection of IL-21 and IL-13 in circulating 56T-cells in

GPA-patients and 8s,

Whole blood +!om G'A#patients and HCs 6as stimulated 6ith 'MA


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.igure % 7pression of transcription factors 0L4 and .o7P,

(A &ep!esentati$e :ACS plots o+ CL; $e!sus :o5'3 e5p!ession in i!ulating C"( E %#ells +!om an

ANCA#negati$e# 7!ight plot8 and an ANCA#positi$e# 7middle plot8 G'A#patient, and an age# and se5#

mathed HC 7le+t plot8* Ialues in eah gate !ep!esent the pe!entages o+ positi$e ells* (0 &elati$e

m&NA e5p!ession o+ CL; in leukoytes +!om ANCA#positi$e# 7n 18 and ANCA#negati$e# 7n ;8 G'A#

patients, and age# and se5#mathed HCs 7n 118 6as analy/ed by !eal#time &%#'C& no!mali/ed to the

housekeeping gene GA'"H*

( 'e!entage o+ CL;E:o5'3

#ells in i!ulating C"(

E%#ells 6as dete!mined in ANCA#positi$e# 7n

118 and ANCA#negati$e# 7n 18 G'A#patients, and age# and se5#mathed HCs 7n 18* a!s !ep!esent

the mean $alues FS"* P#$alues 6e!e alulated using the nonpa!amet!i Mann#Whitney .#test* ] P

*@ ]] P *@*

.igure 6% omparison of IL-21' e7pressing 0-cells from GPA-patients and 8s,

(A&ep!esentati$e :ACS plots o+ -L#21& e5p!ession on C"10E

#ells +!om an ANCA#negati$e# 7!ight

plot8 and an ANCA#positi$e# 7middle plot8 G'A#patient, and an age# and se5#mathed HC 7le+t plot8*

Ialues in eah gate !ep!esent the pe!entages o+ -L#21&E#ells* (0 %he pe!entage o+ -L#21&

E#ells

6as dete!mined in pe!iphe!al blood +!om ANCA#positi$e# 7n 138 and ANCA#negati$e# 7n 1(8 G'A#

patients, and age# and se5#mathed HCs 7n 108* a!s !ep!esent the mean $alues FS"* P#$alues 6e!e


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.igure


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Figure 2


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incremento de il-21 pam y gw

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