il10 inhibits cytokine synthesis by human monocytes

8/14/2019 IL10 Inhibits Cytokine Synthesis by Human Monocytes

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I n t e r l e u k i n 1 0(E L ,.1 0) I n h i b i t s C y t o k i n e S y n t h e s i s b yH u m a n M o n o c y t e s: A n A u t o r e g u l a t o r y R o l e o f I L -1 0P r o d u c e d b y M o n o c y t e sBy Ren 6 de W aal M alefyt, Joh n Abrams,* Bruce Benn ett,Carl G. Figdor,~ and Jan E. de VriesF r o m t h e * D e p a r tm e n t s o f H u m a n I m m u n o lo g y a n d I m m u n o lo g y , D N A X R e s ea r c h I n st it u te ,Pal o A lt o , C al i forn i a 94304; a nd t he t l ~ ' v is i on o f Imm unol ogy , t he N e the r l ands C anc e rIns ti tu t e , 1066 C X Ams t e rdam, The N e t he r lands

S u r n l n l i r yIn t h e p re se n t s tu d y w e d e m o n s t r a t e t h a t h u m a n mo n o c y te s a ct iv a te d b y l ip o p o ly sa cc h ar id e s(LPS) were able to prod uce h ig h leve ls of in te r leu kin 10 ( I1-10) , previous ly design a ted cytokinesynthesis inhib i tor y fac tor (CSIF) , in a dose depe nde nt fashion . I1-1 0 was de tec table 7 h a f te ra c t iv a t io n o f t h e mo n o c y te s a n d m a x ima l l ev e ls o f I1 -1 0 p ro d u c t io n w e re o b se rv e d a ft e r 2 4 - -4 8 h .Th e se k in e t i c s i n d i c a t e d th a t t h e p ro d u c t io n o f Ib l0 b y h u ma n mo n o c y te s w a s r e l a t i v e ly l a t eas com pared to th e pro duc t ion o f IDiot , I1-1 /3 , I1 .-6 , I1-8 , tum or necrosis fac tor o t (TN Fot) , a ndgranu locyte colon y-st im ula t ing fac tor (G-CSF), wh ich w ere a ll secre ted a t h igh leve ls 4-8 h a f te ra c tiv at io n . Th e p ro d u c t io n o f I1 -1 0 b y LPS a c t iv a te d mo n o c y te s w a s , s im i l a r t o t h a t o f IL lo t ,FL-1/3, l 'b6, I1-8, TN Fc~ granulocyte-macrophage colony-stimulating factor (GM -CSF), and G -CSF,in h ib i t e d b y I I -4 . F u r th e rm o re w e d e mo n s t r a t e h e re t h a t I1 -1 0, a d d e d to m o n o c y te s , a ct iv a te db y in t e r f e ro n 3 r LPS , o r c o m b in a t io n s o f LPS a n d IFN -3 ' a t t h e o n se t o f t h e c u l tu re s ,s t ro n g ly i n h ib i t e d th e p ro d u c t io n o f I1 -1 ot , I1 -1 B, I1-6 , I1 -8 , TN FO t , G M-CSF, a n d G -C SF a tthe t ranscr ip t ion a l leve l. Vira l- I1-10 , w hic h has s im i la r b io logica l ac tiv i ties o n h um an ce l ls, a lsoinhib i ted the pro duc t ion of TN FOt and GM -CSF by m onocytes fo l lowing LPS ac tiva tion . Act iva t iono f m o n o c y te s b y LPS in t h e p re sen c e o f n e u t ra l i z in g a n t i - I1 -1 0 m o n o c lo n a l a n t ib o d ie s r e su l te din t h e p ro d u c t io n o f h ig h e r a mo u n t s o f c y to k in e s re la ti v e t o LPS t r e a tm e n t a lo n e, i n d i c a t in gth a t e n d o g e n o u s ly p ro d u c e d I1 -10 in h ib i te d th e p ro d u c t io n o f I1 -1 ot , I 1 - 1 B , I1-6 , I1-8 , TNFot ,GM -CSF, and G-CSF. In addi t io n , I1-10 had auto regu la tory e ffec ts s ince i t s t ron gly inhib i tedI1 -1 0 m R N A sy n th e s is i n LPS ac tiv at ed mo n o c y te s . Fu r th e rm o re , e n d o g e n o u s ly p ro d u c e d I1 -1 0w a s fo u n d to b e r e spo n s ib le fo r t h e r e d u c t io n in c l ass II ma jo r h i s to c o mp a t ib i l i t y c o mp le x (MH C)e x p re ss io n fo l lo w in g a c t iv a t io n o f m o n o c y te s w i th LPS . Ta k en to g e th e r o u r r e su lt s i n d i c at e t h a tI 1 - 1 0 h a s imp o r t a n t r e g u la to ry e f fe c ts o n imm u n o lo g ic a l a n d in f l a m ma to ry r e sp o n se s b e c a u seo f i t s c a p a ci ty t o d o w n .re gu lat e c l a ss I I M H C e x p re ssio n a n d to i n h ib i t t h e p ro d u c t io n o fp ro in f l a mma to ry c y to k in e s b y mo n o c y te s .

M urine--IL-10 was recent ly ident i f ied and i t s gen e c lonedbased on i t s cy tokine synthesis inhib i tory (C SIF) 1 ac-t i v i t y (1 , 2) . In m u r in e sy s te ms , I1 -1 0 w a s p ro d u c e d b y th eC D 4 + T h 2 s u b s et a n d i n h ib i t s t h e c y t o k i n e p r o d u c t io n ,p a r t ic u l a r ly IFN -3 ' , b y T h l c lo n e s (1, 2 ). T h e in h ib i t i o n o fc y to k in e p ro d u c t io n b y I1 -1 0 w a s o b se rv e d o n ly w h e n m a c -ro p h a g e s , b u t n o t w h e n B c e ll s w e re u se d a s A P Cs (3 ). Inaddi t ion to i t s CS IF ac t ivi ty , I1-10 was s how n to be p le io tropicand to ac t on d i f fe rent ce l l types, inc luding thymocytes (4) ,

I Abbreviations used in this paper:CSIF, cytokine ynthesis nhibitory actor;G-CSF, granulocytecolony-stimu lating actor; GM-C SF, granulocyte-macrophagecolony-stimulating actor; v-Ib l0, viral-IL-10.

cyto toxic T ce lls (5) , m ast ce l ls (6) , B ce l ls (7) , and m acro-phages (3) .H u m a n I1 -1 0 a lso e x h ib i ts CS IF a c t iv i ty (8 ) . W e h a v ed e mo n s t ra t e d th a t t h e p ro d u c t io n o f IFN - 'y a n d g ran u lo c y te -m a c r o p h a g e c o l o n y - s ti m u l a t in g f ac t or ( G M - C S F ) b y P B M Ca c tiv at ed b y PH A o r a n t i -CD 3 m A b s w a s s tro n g ly i n h ib i t e db y IL l0 a n d th a t t h i s i n h ib i t i o n o c c u r re d a t t h e t r a n sc r ip -t iona l leve l (8) . Bo th h um an a nd rou t ine I1-10 have extensivese q u e n c e h o mo lo g y to a p re v io u s ly u n c h a ra c t e r i z e d o p e nr e a d in g f r a m e i n t h e E p s t e i n B a r r v i r u s g e n o m e , B C R F - 1(2 , 8 ) . Ex p re ss io n o f t h e o p e n r e a d in g f r a me y i e ld e d an a c -t i v e p ro t e in , d e s ig n a t e d v i r a l - IL l0 (v -I1 -1 0 ), w h ic h sh are dmo s t p ro p e rt ie s w i th h u m a n a n d ro u t in e Ib l0 , i n c lu d in g CSIFa c t iv i ty o n mo u se a n d h u ma n T c e l l s (9 ) .

1209 J. Exp. Med. 9 The RockefellerUniversityPress 9 0022-1007/ 91 / 11 / 1209/ 12 $2.00Volume 174 Novem ber1991 1209-1220

Published November 1, 1991


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R e c e n t ly , w e d e s c r ib e d t h a t h u m a n I I~ 10 a n d v - I b l 0 w e r ea b l e t o i n h i b i t a n t i g e n s p e c if i c p r o l i f e r a t i v e T c e l l r e s p o n s e sb y r e d u c i n g t h e a n t i g e n p r e s e n ti n g c a p a c it y o f h u m a n m o n o -c y te s v ia d o w n r e g u l a t i o n o f cl as s II M H C m o l e c u l e s ( 9 a) .H e r e w e d e s c r i b e t h a t h u m a n m o n o c y t e s w e r e a b l e t o p r o -d u c e h i g h l e ve l s o f IL -1 0 f o l l o w i n g a c t i v a t i o n w i t h L P S a n dt h a t t h i s p r o d u c t i o n w a s r e l a ti v e l y l a t e a s c o m p a r e d t o t h a to f o t h e r m o n o k i n e s . I n a d d i t i o n , i t i s r e p o r t e d h e r e t h a t I L- 10s t r o n g l y i n h ib i t e d t h e p r o d u c t i o n o f t h e p r o i n f l a m m a t o r yc y t o k i n e s I L - l o ~ , I L - 1 / 3 , I L - 6 , I I ~ 8 , a n d T N F o t a n d t h e h a e -m a t o p o i e t i c g r o w t h f a ct o rs G M - C S F a n d g r a n u l o c y te c o lo n y -s t i m u l a t i n g f a c t or ( G - C S F ) b y m o n o c y t e s a c t iv a t e d b y L P S ,I F N - ' y , o r L P S an d I F N - y . E n d o g e n o u s l y p r o d u c e d I L- 10h a d n o t o n l y a u t o r e g u l a t o r y e f fe c ts o n I L - lc t , I b l / 3 , I b 6 ,I I ,8 , T N F c t , G M - C S F , a n d G - C S F p r o d u c t i o n b y m o n o c y t e s ,b u t a ls o d o w n r e g u l a t e d i t s o w n p r o d u c t i o n a n d c la ss I I M H Ce x p r e s s i o n o n m o n o c y t e s in a n a u t o r e g u l a t o r y f a s h io n . T h e s er e s u l t s i n d i c a t e t h a t I L - 1 0 h a s i m p o r t a n t r e g u l a t o r y e f f e c t so n i m m u n o l o g i c a l a n d i n f l a m m a t o r y r e s p o n s e s .

M a t e r i a l s a n d M e t h o d sIsolation and Cu lture of H uma n M onocytes. H u m a n P B M s w e r eisola ted f rom 500 m l b lood o f normal donors as desc r ibed p rev i -ous ly (10 , 11 ) . Mo nonu c lea r ce l ls were i so lated by d ens i ty cen t r i fu -ga t ion in a b loo d com pon en t separato r , fo l lowed by f rac t iona t ioni n t o l y m p h o c y t e s a n d m o n o c y t e s b y c e n t ri f u g a l e l u t ri a t i o n . T h em o n o c y t e p r e p ar a t i o n w a s > 9 5 % p u r e , a s j u d g e d b y n o n s pe c if ices te rase s ta in ing and con ta ined m ore than 98% v iable ce ll s . M ono -cy tes were cu l tu re d in Ysse l 's me d ium (12) con ta in in g H SA sup-p l e m e n t e d w i t h 1 % p o o l e d h e a t in a c t iv a t e d h u m a n A B + s e ru m .T h i s c u l t u r e m e d i u m w a s e n d o t o x i n f r e e a s d e t e r m i n e d b y t h eL i m u l u s a m e b o c y t e l ys a te a s sa y (< 0 . 2 n g / m l o f e n d o t o x i n ) . T h em o n o c y t e s w e r e c u l t u r e d a t a c o n c e n t r a t i o n o f 4 x l & c e ll s/ m 1in teflon bags (Jansen M NL , St. Nildaas, Belgium), wh ich preventedadhesion o f these ca l ls . Af te r cu l tu re fo r the t imes ind ica ted, mo no-

cy tes w ere co l lec ted and ana lyzed fo r ce l l su r face express ion by in -d i r e ct i m m u n o f l u o r e s ce n c e o r a n a l y z e d f o r l y m p h o k i n e g e n e e x -p r e s s i o n b y N o r t h e r n a n d P C R a n a l y s i s . I n a d d i t i o n , m o n o c y t eculture supernatants w ere collected for determ ination o f Iblc~, IL-1B,IL-6, IL -8 , Ib l0 , TNFot , GM -CSF, and G-CSF pro duc t ion fo l lowingactivation of these ceils . Th e viabili ty o f the ceils after culture alwaysexceeded 95% as de te rmined by t rypan b lue exc lus ion .Reagents. Recom binan t huma n IL-10 and v - I b l 0 were expressedin Escherichia coli as g lu ta th ione-S- t rans fe rase fus ion p ro te ins ,p u r i f i e d , a n d d i g e s t e d w i t h t h r o m b i n t o r e m o v e t h e N - t e r m i n a lfus ion pa r t (13) , re su l t ing in ac t ive hum an and v - IL-10 as desc r ibedprev ious ly (9a ) . Pur i f ied human r lL -4 and r lFN-3 ' were p rov idedby Schering-Plough Research (Bloom fidd, NJ). LPS (E. coli0127:B8)w a s o b t a i n e d f r o m D i f c o L a b o r a t o r i e s ( D e t r o i t , M I ) . T h e n e u -t ra l iz ing an t i - I b l 0 m Ab 19F1 was ra ised aga inst v - IL-10 andef fic ien t ly neu t ra l ized bo th hum an and v i ra l - Ib l0 (S i lve r , J . , andJ . Abrams , manuscr ip t in p repara t ion) .Probes. T h e f o l l o w i n g p r o b e s w e r e u s e d f o r n o r t h e r n a n al y si s:600 bp Sma I f ragm ent (n t 1299 - 1899) o f pCD-hTGF/3 (14), 1200bp Ps t I f ragme nt o f pAL ( /3 -ac t in ) (8 ), 567 bp Ba mH I-Xb a I f rag -me nt (n t 1 - 567) o f pCD-hI I_ ,6 (14), 268 bp H ind I I I f rag me nt(n t 29 - 297) o f SP64-3-10c ( IL -8) (15), 760 bp Bgl I I - H ind I I If ragm ent (n t 159 - 919) o f pCD-SRot-h lL-10 (8 ). Th e fo l low ingo l igonuc leo t ides were used fo r Sou thern ana lys is o f PC R produc ts :I L -l o t: 5 ' - C A T G G G T G C T T A T A A G T C A T C - Y (n t 5 0 0 - 52 1 ) ( 16 ) ;

I L- 1/ 3: 5 ' - C G A T C A C T G A A C T G C A C G C T C C G G G - 3 ' ( n t 4 4 4 -469) (16) ; Ib6 : 5 'GAGGTATA CCTAGA GTACCTC-Y (n t 510 - 531)(17); IL-8: 5'-TAAAGACATACTCCAAACCTT-3' (n t 200 - 221)(15); IL-10: 5 ' - C A G G T G A A G A A T C C T T T A A T A A G C T C C A A -G A G A A A G G CA T CT A CA A A G C C A T G A G T G A G T T T G A C A T C3 ' (n t 42 9 - 4 98 ) ( 8) ; T N F ~ : 5 - G C ~ G ~ G A G C T G A G A G A T A A C -3 ' ( n t 5 0 0 - 5 2 1) ( 1 8) ; G M - C S F : 5 ' - C C G C ~ G T C T C C T G A A C C T - Y(nt 150 - 168 ) (19); actin: 5 ' - C ~ A A C C C T A A G G C C A A C C G T G -3 ' (n t 250 - 272) (20) ; and G-CSF: 5'-GCCCTGGAAGGGATC-T C C C C C - Y ( n t 4 0 0 - 4 2 1 ) ( 2 1) .m R N A Isolation and Northern Analysis. Tota l ILNA was i so -la ted f rom 20 x 106 mo nocy te s by the guan id in ium th ioeyana te -CsC1 p rocedure (22) . For nor th e rn ana lys is , 10 /~g to ta l tLN A persample was separa ted accord ing to s ize on 1% agarose ge ls con-raining 6.6% formaldehyde, transferred to Nytran nylon membranes(Sch le icher & Schue l l, K eene , NH ) a nd hybr id ized wi th p robes ,labeled to high specific activity (>10 s c p m / / ~ g ) b y t h e h e x a m e rlabeling tec hniqu e (23). Filters were hybridized, washed u nde r str in-gen t con d i t ions , and deve loped as p rev ious ly desc r ibed (24) .Polymerase Cha in Reaction (PCR ) Ana lysis. 1 / x g o f t o t a l t L N Awas reverse transcribed using olig o (dT ) 12-18 as prim er (Bo ehring erMa nnhe im , Ind ianapo lis , I N) and A M V reverse t ransc r ip tase( B o e h r i n g e r M a n n h e i m ) a c c o r d i n g t o t h e p r o c e d u r e o f K r u g a n dBerger (25) in a 20 /z l reac t ion . 2 /x l o f reverse t ransc r ip t (equ iva len tto 100 ng o f to ta l RNA) was used d i rec t ly fo r each ampl i f ica t ionreac t ion . Con d i t ions fo r PC R were as fo l lows : in a 50 /~1 reac t ion ,2 5 n m o l o f e ac h p r im e r , 1 2 5 / ~ M e a c h o f d G T P , d A T P , d C T P a n dd T T P ( P ha rm a c ia , U p p s a l a , S w e d e n ) , 50 m M K C 1 , 10 m M T r is -H C 1 , p H 8 . 3 , 1 . 5 m M M g C 1 2 , 1 m g / m l g e l a t in , 1 0 0 / ~ g / m l n o n -ace ty la ted BSA and 1 U Ven t DN A po lymerase (New England B in -labs, Beverly, MA ). Prim ers used we re as follows: IL-lot sense primer5'-CATCGCCAATGACTCAGAGGAAG-Y (nt 302-325), Iblc ~antisense primer 5'-qroCCAAGCACACCCAGTAGTCTIV_K2TT-Y(n t 770 - 743) (16), IL -1B sense p r imer 5 ' -CC AG CTA CGA AT-C T C G G A C C A C C - 3 ' ( n t 2 3 0 - 2 53 ) , I L - 1/ J a n t i s en s e p r i m e r5'-TTAGGAAGACACAAATIV_~ATGGTGAAGTCAGT-3'(nt 896- 863) (16), 11.-6 sense prim er 5'-ATGAACTCCTTCTCCACA-A G C - 3 ' ( n t 1 - 2 1 ) , I b 6 a n t i s e n s e p r i m e r 5 ' - C T A C A T T T G C C -GA A GA GC C C TC A GGC TGGA C TG-3 ' (nt 810 - 777) (17), Ib8sense p r ime r 5'-ATGACTTCCAAGCTGGCCGTG-3' (nt 1 - 21),I1 -8 an t i sense p r im er 5 ' - T T A T G A A T T C T C A G C C C T C T T C A A -A A A C T T C T C - Y ( n t 3 0 2 - 2 6 9 ) ( 1 5 ), I L -1 0 s e ns e p r i m e r 5 ' -A T G -C C C C A A G C T G A G A A C C A A G A C C C A - Y (nt 323 - 349), IL-10a n t i s e n s e p r i m e r 5 ' - T C T C A A G G G G C T G G G T C A G C T A T C C -CA-3 ' (n t 674 - 648) (8 ) , TNFc~ sense p r imer 5'-AGAGGGA-A G A G T T C C C C A G G G A C - Y (nt 310 - 333), TNFc~ anti sensep r i m e r 5 ' - T G A G T C G G T C A C C C T T C T C C A G - Y ( n t 7 8 2 - 7 6 0 )( 1 8 ) , G M - C S F s e n s e p r i m e r 5 ' - G C A T C T C T G C A C C C G C C C G -C T C G C C - 3 ' ( n t 7 6 - 10 0 ), G M - C S F a n t i s e ns e p r i m e r 5 ' - C C T G C -TTGTACAGCTCCAGGCGGGT-Y (n t 276 - 250) (19) , G-CSFs en se p r im e r 5 ' -G A G T G T G C C A C C T A C A A G C T G T G C C - Y ( n t2 3 3 - 2 5 8 ) , G - C S F a n t i s e n s e p r i m e r 5 ' - C C T G G G T G G G C T G -C A GGGC A GGGGC -3 ' (n t 533 - 508) (21) , /~ -ac t in sense p r im er5 ' - G T G G G G C G C C C C A G G C A C C A - 3 ' ( n t 1 - 2 0 ), B -a c ti n a n tisense p r ime r 5'-GTCCTTAATGTCACGCACGATTTC-3' (n t 548- 530) (20). Reactions w ere incubated in a Perkin-Elmer/Cetus D N AThe rm al cyc le r fo r 20 cyc les (dena tu ra t ion 30 s 94~ annea l ing3 0 s 5 5 ~ e x t e n s io n 6 0 s 7 2 ~ R e a c t i o n s w e r e e x t r a c t e d w i t hCHCI3 and 40 /x l pe r sample was loaded on 1% agarose ge ls inTAE buf fe r. P rodu c ts we re v isua l ized by e th id ium brom ide s ta in ing .S u b s e q u e n tl y , g e ls w e r e d e n a t u r e d i n 0 .5 M N a O H , 1 .5 M N a C 1 ,n e u t r a l iz e d i n 1 0 M a m m o n i u m a c et at e, a n d t r a n sf e r r ed t o N y t r a nn y l o n m e m b r a n e s . M e m b r a n e s w e r e p r e - h y b r id i z e d i n 6 x S S C ,

1 21 0 Interleukin 10 Inhibits Monokine Production



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TIME (h) TIME (h)Fig ure 1. The kinet ics of I I -10, IL-6, TNFot , and GM -CSF product ion b y human m onocytes, act ivated by LPS. Hu ma n monocy tes, i solated by cen-t r ifugal elutr iat ion w ere cul tured in tef lon bags (4 x l f fS/ml) in the absence or presence of LPS (1 #g/m l) and product ion of (A) IL-10, (B) Ib6,(C) T NFoe and (D) GM -CSF w as determined in the cul ture supernatants harvested at the t imes indicated by cytokine specif ic ELISA's.

1 % S D S , 1 0 x D e n h a r d t ' s s o l u t i o n ( 0 . 2 % F i c o l l , 0 . 2 % p o l y v i n y l -p y r r o l id o n e , 0 . 2 % B S A , p e n t a x f r a c ti o n V ) a n d 2 0 0 # g / m l E. col it R N A ( B o e h r in g e r , M a n n h e i m , F R G ) fo r 4 h a t 5 5 ~ O l i g o n u -c l eo t ide p r obes ( 200 ng) , spec i f i c f o r a sequence in t e r na l t o t hep r i m e r s u s e d i n t h e a m p l i f i c a t i o n , w e r e l a b e l e d a t t h e 5 ' e n d b yT 4 p o l y n u c l e o t i d e k i n a s e ( N e w E n g l a n d B i o l a b s) a n d q r -3 Z p-A T P( A m e r s h a m , A r l i n g t o n H e i g h t s , I L ) a s d e s c r ib e d ( 26 ) . P r o b e s w e r es e p a r a te d f r o m n o n i n c o r p o r a t e d n u c l e o ti d e s b y p a s s a g e o v e r a N i c kco lum n ( Pharmac ia ) and added to the hybr id i za t ion mix . Fo l lowing

hyb r id i za t ion f o r 12 h a t 55~ f il te r s wer e wa shed in 0 . 1 x SSC( 1 x S S C :1 5 0 m M N a C 1 , 1 5 m M N a - c i t ra t e p H = 7.0) , 1 % S D Sa t r o o m t e m p e r a tu r e a n d e x p o se d t o K o d a k X A K - 5 f il m s fo r 1 - 2 h .Lymphokine Determinat ions. T h e p r o d u c t i o n o f l L - lc r a n d T N F c ~b y m o n o c y t e s w a s m e a s u r e d b y l y m p h o k i n e s p e c i f i c E L I S ~ s o b -t a i n e d f r o m E n d o g e n ( B o s t o n , M A ) . T h e l o w e r d e t e c t i o n l i m i to f t hese E L I SA' s wer e 50 pg / m l and 10 pg /m l r espec t ive ly . P r o duc-t i o n o f I b 1 3 w a s d e t e r m i n e d b y l y m p h o k i n e s pe ci fic E L I S A o b -t a ined f r om Ci s t r on ( P ine Br ook , NJ) . T he sens i t i v i ty o f t h i s E L I SA1211 de W aal Malefyt et al .



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was 20 pg/m l, lb 6 levels were determined by lymphokine specificELISA purchased from Ge nzym e (Boston, M A). Th e sensit ivityof this assay was 0.313 ng/ml. Ib8 and G -CSF specific ELISA'swere obtained from R &D Systems (Minneapolis, MN ) and usedto quantitate Ib 8 and G-C SF production. Th e sensitivity of theseELISA's was 4.7 p g/m l an d 7.2 pg /ml respectively.GM-CSF produc-tion w as determined by lymphokine specific ELISA as describedpreviously (27). The sensitivity of this ELISA was 50 pg /m l. IL-IOproduction was determined by a specificELISA n which the anti-IL-10 mA b JES 9D7 w as used as a coating antibody and the a nti-IL-10mA b JES3-12G8 as a tracer antibody (Silver, J., and J. A brams,manuscript in preparation). Th e sensitivity of this ELISA was 50pg/ml .Immunofluorescence An alys is. Ce lls (10 ) were incubated in Vbottom microtiter plates (Flow Laboratories, McLea n, VA) with10/~1 of purified mAb (1 mg /ml) for 30 rain at 4~ After twowashes with PBS containing 0.02 m M sodium azide and 1% BSA(Sigma Chemical Co., St. Louis, MO ), the cells were incubatedwith 1/40 dilution of FITC labeled F(ab')2 fragments of goatanti-mouse antibody (Tago, Inc., Burlingame , CA) fo r 30 min at4~ Afte r three additional washes, the labeled cell samples wereanalyzed by flow m icrofluometry on a FACScan (Becton Dickinsonand Co., Sunnyvale, CA). T he a nti-M HC class II mAbs PdVS.2( H L A - D R / D P / D Q ) (28) , Q5/13 H LA -DR /DP (29) , and L243(HL A-D R) (30) were described previously.

Resu l t sI L I O I s Produced by Hu man M onocy t e s. W e h a v e s h o w n

p rev io u s ly t h a t I1 -1 0 was p ro d u ced b y ac t i v a t ed h u man Tcel l c lones , act ivated per ipheral b lood T and B cel l s , EBVt ran s fo rmed B ce l l l i nes (8 ) an d mo n o cy t es (Ab rams , J ., H .Yssel, and M. G . Ronc aro lo , manuscr ip t in p reparat ion) . He rew e f u r t h e r c h a ra c te r iz e I b l 0 p r o d u c t i o n b y h u m a n m o n o -cy t es . H ig h ly -p u r i f i ed h u man mo n o cy t es , i so l a t ed b y cen -t r i fugal elu t r iat ion , p roduced I1 .10 fo l lowing act ivat ion byLP S . In ad d i t i o n , i t i s sh o wn th a t t h ese h u man mo n o cy t eswere ab l e t o p ro d u ce h ig h l ev el s o f I1 .6, TN F c~ , an d G M -CS F (Fig. 1 ). K in e t i cs o f cy to k in e p ro d u c t i o n b y LP S ac t i -v a t ed mo n o cy t es i n d ica t ed t h a t I1 .1 0 p ro d u c t i o n b y m o n o -cy tes was relat ively late . I t w as f i rs t detected in supe rnatan tsh arv es t ed a t 7 .5 h , b u t max imal p ro d u c t i o n was o b se rv ed2 0 -4 8 h a f t e r ac t i v a t i o n . In co n t r as t , TNF o l an d I1 -6 were

f -v0

- - I

1 2 -

1 0

_m1 1 0

i0 0

me=

N

L P S ( n g / m l )Figu re 2. The production of ILl0 by human monocytes n responseto increasing concentrations of LPS . Hum an monocytes (4 x 106/ml)were culture d in teflon bags w ith increasing concentrations of LPS fo r24 h and the production of Ib10 was determined by ELISA.

p r o d u c e d r a p i d ly u p o n a c t iv a t e d a n d r e a c h e d m a x i m a l l e v el sof product ion at 3 .5 and 7 .5 h fo l lowing act ivat ion respec-tively (Fig. 1). How ever, GM-CSF produ ct ion was a lso f irstd e t ec t ed 7 .5 h a f t e r ac t i v a ti o n o f mo n o c y t es b y LP S , b u t i nth is case maximal p roduct ion levels were reached at 20 h .Do se r esp o n se s tu d i es i n d i ca ted t h a t ac t i v a t i o n o fmo n o c y t esby LPS at 10 ng /ml al ready resu l ted in s ign i f ican t levels o f11 .10 prod uct io n , w hereas the m axim al I1 .10 syn thesis wasobserved at LPS concent rat ions o f 1 /zg /ml (F ig . 2 ) .IL, O Inhibi ts Cytokine Production by Hum an Monocytes . IL-10has been shown to inh ib i t IFN-3" and GM -CSF produ c t ionby act ivated PBM C (8) . To determ ine the effects of I1-10 onthe pro duct ion of cytokines by monocytes, highly-purif iedmo n o cy t es were ac t i v a ted fo r 2 4 h b y LP S in t h e ab sen ce o rpresence of I1 .10 . In a dd i t ion , m onocy tes we re act ivated wi th

Table 1. Effects of Exo genous IL-IO, Endogenous IL-IO , and IL-4 on C ytokine Production by H um an M onocytesIL-l t~ IL -I~ IL-6 IL-8 IL-10 TN F c r GM-CSF G-CSF

ng/mlMedium 37~ 0 0 0 150 0 0 0 0LPS 1.2 44.8 261.7 479 30.6 21.2 0.6 90LPS + IL-4 0 9.7 135.7 418 11.9 5.1 0 17.5LPS + IL-IO 0 13.6 78 434 N D 2.6 0 21.1LPS + alL - 10 2.7 50.6 323 672 ND 47.6 5.5 110

Hum an m onocytes, solated by centrifugalelutriation were cultured in teflonbags at a concentrationof 4 x 106 ceUs/ml and activated by LPS (1/zg/ml) in the absence and presenceof IL-10 (100 U/m l), IL-4 (100 U/m l) or anti-IL-10 mAb 19 FI (10 #g/m l) for 24 h and production of cytokineswas d etermined in the supernatants by cytokine specific ELISA 's. ND : not done.12 12 Inter leuk in 0 Inhibits Monokine Production



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A 3 B 2 0

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I ' ~ ' " l " I " I " I " I " I '' 0 0 0 , r - 0 0 0 , r -

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F i g u r e 3 . T h e e f fe c ts o f I b l 0 o n t h e p ro d u c t i o n o f c y t o k in e s b y m o n o -c y t es a ct iv a t ed b y IF N - % L P S o r L PS a n d I F N - % H u m a n m o n o c y t e s ( 4x 1 0 6 / m l ) w e r e c u l t u r e d w i t h I F N - 3 , ( 1 00 U / m l ) , 1 , 1 0 , 1 0 0 , o r 1 , 0 0 0

n g / m l L P S a n d c o m b i n a t i o n s o f L P S (1 , 1 0, 1 0 0 , o r 1 , 0 0 0 n g / m l ) a n dI F N - ' y ( 1 0 0 U / m l ) e i t h e r i n t h e a b s e n c e (E ] ) o r p r e s e n c e ( [ ] ) o f I b l O ( 1 00U / m l ) f o r 2 4 h a n d p r o d u c t i o n o f ( A ) I b l o ~ , ( B ) I L -1 /~ , ( C ) I L -6 , ( D ) T N F o 4a n d ( E ) G M - C S F w a s d e t e r m i n e d b y c y t o k i n e s p e ci fi c E L I S A ' s i n t h e s u -p e r n a t a n t s .

1 2 1 3 d e W a a l M a l e f y t e t a L



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LPS for 24 h in the presence of IL-4 (100 U/ml ) or neutralizinganti -Ibl 0 mAb 19F1, which was raised against v- Ib l0 butefficiently neutraliz ed bot h hu ma n I1:10 and v-IL-10 (Silver,J., andJ. Abrams, manuscript in preparation) (The latter resultsare discussed below). Cyt okin e producti on was determinedin the s upernata nts of these cultures, harvested 24 h after ac-tivation, by cytokine specific ELISA's. As sho wn in shownin Table 1, monocytes which were incubated in m ediu m aloneat 37~ did not pro duc e I1:1c~, 11:1/3, I1:6, Ib l0 , TNFcrGM-CSF, and G-CSF. U nde r these conditions, o nly significantlevels of I1:8 were synthesized. Activation o f monocy tes wi thLPS (1/~g/ml) resulted in p rodu ction of high levels of I1:1ol,Ib113, I1.-6, Ib 8, II:10, TN Fot, GM-CSF, and G-CSF. In-terestingly, IL-10 inhib ited the pro duc tio n o f I1:1o~, I1:1/3,I1:6, I1:8, TNFc~, GM-CSF, and G-CSF to various extents(Table 1). The stron gest in hib itory effects of I1.-10 were o b-served on the produ ction o f I1:1c~, TNFo l, GM-CSF, andG-CSF, which were blocked by 80-100%. The inhibition ofI1:1/3 and I1:6 p roduc tion was less pronounce d, whereas thesynthesis of IL-8 was only slightly affected by I1:10.ILI OAlso Inhibits Cytokine Production of Monocytes Activatedby IFN-% IL-10also inhibited cytokine production by mono -cytes activated by IFN -% or co mbina tions of IFN-3, and LPS.In Fig. 3, it is shown that IFN-3, at optim al concentrationsof 100 U/m l generally was a less potent indu cer of cytokinesecretion than was LPS at opt imal concentrations of I/~g /ml .Furthermore, it is demonstrated that the effects of combina-tions of IFN-3/and LPS on cytokine production by mono -cytes generally were additive. T he strong est inhibi tory effectsof Ib l0 were observed on IL-la, TNFo~ and GM-CSF produc-tion. TNFc~ and GM-CSF secretion was suppressed by morethan 90%, even following activation of the monocytes byoptimal LPS and IFN -y concentrations. Alth oug h consider-able inhibitory effects on I1:1/3 and IL-6 secretion were ob-served at optimal stimulation conditions, their inhibition wasmore prono unced when the monocytes were stimulated bysuboptimal concentrations o f LPS, either in the absence orpresence of optimal concentrations of IFN-%Viral 11_,10 nhibits Cytokine Production by Monocytes. ViralI1:10 and human I1:10 have similar effects on human cells(9). In Fig. 4, it is shown that both I1:10 and v-I1:10 in-hibited TNFc~ and GM-CSF production by monocytes in asimilar fashion. I1:10 and v-I1:10, added at concentrationsof 100 U/ ml , had significant inh ibito ry effects on TNFo~ andGM-CSF production by monocytes following activation byLPS (1/zg /ml) . Thes e inhib itory effects of IL-10 and v-IL-10on TNF-o~ and GM-CSF secretion were reversed when incu-bations were carried ou t in the presence o f mAb 19F1 (Fig.4), d emon strating the specificity of the inhibitory effects ofv-I1:10. In fact, activation of mono cytes by LPS in the pres-ence of I1:10 or v-IL-10 and th e neutralizing anti-I1:10 mA bresulted even in enhanced prod uctio n of TNFc~ and GM-CSF,indicating that endogen ously produced Ib l0 suppressed theproduction of these cytokines.Endogenously Produced ILIO Inhibits Cytokine Production byMonocytes. The inhibitory effects of endogenou sly producedIL-10 on cytokin e producti on by mon ocytes was further evalu-

Fig ure 4. The effects of human Ibl0 and viral-I bl0 on the productionof T NF ~ and GM -CSF by LPS activated monocytes. Hum an monocytes(4 x 106/ml) were activated by LPS (1/zg/ ml) and cultured with hu manII.-10 (100 U/ ml) o r viral-IIr in the absence or in the presence of neu-tralizing anti-IL-10 mAb 19F1 (10/~g/ml) for 24 h and prod uction of (.4)TNF tx and (B) GM-CSF was determined by cytokine specific ELISA's.

ated by quan tifyin g cytokin e levels prod uced by LPS activatedmonocy tes in th e presence of neutralizing an ti-I1:lO mAb.In Table 1 it is shown that LPS plus anti-I1:10 treatmentof mon ocytes resulted in high er levels of cytokin e produc-tion as compared to activation by LPS alone, indicating thatendogenously produced I1:10 in addition to its inhibitoryeffects on TNFcz a nd GM-CSF prod uction blocked the produ c-tio n o f IL-lo~, I1:1/3, IL-6, IL8, and G-CSF. Th e m ost sig-nificant inhibitory effects were found on the production ofIbla, GM-CSF, and TNF~ whereas the inhibitory effectson IL-1/3, I1:6, IL-8, and G-CSF expression were consider-able, but less pronoun ced. Taken together these results indi-cate that b oth exogenous I1-10 and endog enously producedI1:10 inhibit the p rodu ction o f I1:1c~, I1:1B, IL-6, 1I:8, T N F ~GM-CSF, and G-CSF by LPS activated monocytes.11.4 Inhibits ILIO Production by Activated Monocytes. Wedemonstrated previously that I b4 inhibited production ofIL-1/3, IL-6, and TNFc~ by LPS activated mon ocyt es (31). Todetermine whet her IL-4 also inhibited l-b10 productio n, mo no-cytes were activated by LPS for 24 h and Ibl0 secretion wasmeasured. A s sho wn in Table 1, II.-4 stron gly inhi bited I1:10produ ction by LPS activated mono cytes. Furthermore, Ib 4,

1214 Interleukin 10 Inhibits Monokine Production



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Fig ure 6. The effects of I1:10 on the expression of cytokine specificmKN A levels by human monocytes activated by LPS. mKN A was iso-lated from human monocytes (4 x lO~/ml), activated by LPS (1 #g/m l)in the absence or presence of 11:10 (100 U/ml) for 7 h and expression ofI1:6, I1:8, Ibl 0, TGF-/3, and B actin was determined by northern analyses.

Figu re 5. The effectsof exogenous IL-10, endogenously produced Ib l0and I1:4 on the expression of cytokine specificmRN A levels by humanmonocytes activated by LPS. Human monocytes (4 x 106/ml) were cul-tured in medium at 4~ and 37~ or activated by LPS (1 mg/ml) in theabsence or presence of II:4 (100 U/ml), I1:10 (100 U/ml) and neutralizinganti-IL-10 mAb 19F1 (10 #g/m l) and mK NA was isolated after 24 h. Ex-pression of/~-actin , I1:1o~, I1:1B, I1:6, I1:8, TNF-o~, GM-CSF and G-CSFwas determined on reverse transcribed mRN A by P CR analyses withcytokine specific primers followed by Southern blotting of the reactionproducts with internal probes, cDNA obtained from a CD 4 + T cell clonewas included as control for expression of TN Fol and GM-CSF.

in addit ion to i ts inhibi tory ef fects on IL-I~, Ib6, and TNFc rsecret ion, ef fic iently blocked the p roduction of GM-C SF andG-CSF. However, as observed for IL-10, the production ofIL-8 was o nly slightly affected by IL-4. CoUectively these dataindicate that IL-4 and Ibl0 have comparable inhibitory effectsof cytokine production by act ivated monocytes .

Inhibition of Monokine Production Occurs at the Transc@tionalLevel. To determ ine at whi ch level Ib1 0 inhibited the produ c-t ion o f cytokines by monocytes , comp arat ive PC K analyseswere per formed on KN A isolated f rom monocytes , act ivatedby LPS in the absence or presence of IL-10, IL-4 or the neu-tral iz ing anti- IL-10 mAb for 24 h. The cytokine measure-ments of this exper iment are shown in Table 1. mK N A iso-lated from these samples was reverse transcribed into cDNAand subseq uently amplified with cytok ine specific primers.A relatively small numb er of cycles was used for amplificationto ensure that the amount of amplified DN A was propor-t ional to the cycle num ber and correlated with the amou ntof specific m R N A in the original sample. I t is show n in Fig.5 that under these condit ions equivalent amounts of /~-act inspecific cD NA were amplified. Monocytes incubated at 4~in medium alone for 24 h expressed very low levels of IL-8mR NA . Incubation of these cells a t 37~ resulted in an in-

1215 de Waal Malefyt et al.



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Figure 7. The effectsof exogenous bl0, en-dogenouslyproduced I.-10and Ib4 on the ex-pression of IL-IOand TGFI3 specific mRNAlevels by human monocytesactivatedby LPS.Human monocytes(4 x 106/ml) were cul-tured in mediumat 4~ and 37~ or activatedby LPS (1 m g/m l) in the absence or presenceof lb 4 (100 U/ml), II-10 (100 U/ml) and neu-tralizing anti-Ib l0 mAb 19F1 (10/~g/ml) andmR NA was isolatedafter 24 h. Expression of(/1) Ibl0 , TGFBand 13-actin was determinedby nor thern analysesand (B) expression of IL-10 was determ ined on reverse transcribedmRN A by PCR analysiswith cytokine pecificprimers followedby Southern blotting of thereaction products w ith internal probes.

creased expression of IL-8 m RN A, but did not induce ex-pression of IL-lc~, IL-113, IL-6, IL-10, T N F ~ GM-CSF, orG-CSF m RN A. LPS activation resulted in a strong expres-sion o f II.-lo~, IL-113, IL-6, IL-8, and G-C SF m R N A , whereasTNFc~ and GM-CSF m R NA were moderately induced. Fur-thermore, it is show n in Fig. 5 that IL-lc~, I1.-6, T N F ~ GM -CSF, and G-CSF expression was strongly inhibited by IL-10and II.-4 at the m R N A level (Fig. 5), whereas IL-13 and I I :8expression were only slightly affected by IL-10. Activationof monoc ytes by LPS in the presence of the anti-IL-10 mA bresulted in a moderate enhancement in expression of IL-lc~,IL-113, IL-6, IL-8, and G-CS F m R N A and a stro ng increasein TNFo~ and GM-CS F m R N A synthesis. The levels of ex-pression of cytokine specif ic mR N A and their m odulat ionby exogenous and endoge nous Ib lO or IL-4 correlated wellwi th secretion of the corresponding proteins as shown in Table1 and indicat ed tha t IL-10 and IL-4 inhib ited cy tokin e expres-sion by LPS activated monoc ytes at the transcriptional level.IL-IO Regulates 11_,10 m R N A but not TGF/3 mR N A Ex-pression in Activated Monocytes. Having demonstrated that

human monocytes produced IL-10 relatively late followingactivation by LPS, we determine d wh ethe r II.-10 could affectendogenous IL-10 m R NA synthesis. H um an monocytes wereactivated by LPS in th e presence or absence o f IL-10 for 7 hand mR N A expression was analyzed by northern blot ting.In Fig. 6 it is show n that IL-10 m R N A was detected 7 hafter activation by LPS and that IL-10 had no or on ly min-imal inh ibitor y effects on IL-IOm R N A expression at this timepoint. In contrast, the expression of IL-6 and IL-8 m R N Awas s trongly inhibited by IL-10. However, in anothe r seriesof experiments where m onocytes were activated for 24 h byLPS, Ib l0 strongly reduced the expression of IIr ml~ NAas shown by northern analysis in Fig. 7. Furthermore, it isshown in Fig. 7 that activation of monocytes with LPS inthe presence of a neutra lizing anti-IL-10 mAb resulted in anupregula tion of I1.-10 m R N A expression at 24 h. These resultswere confirmed by PC R analysis wi th Ib l0 specific primerssince the RNA used in this latter experiment was also usedfor the PC R analyses shown in Fig. 5. It is shown in Fig. 7B that the quantitative differences observed in II~10 mRNA

1216 Interleukin10 Inhibits Monokine Production



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