Establishment and Characterization o{ a (;ranulocyle-l\IacrophageColony-Stimulating l'actor-Dependent Human \lyeloid Cell Line

By Sadik Oez, H.nne T tre ba.h Fa m!nd Fahso d, nol.id S.h.elz, Chr stoph Bührer,Jens Atzpöd e., and Joach m R. Ka den

A n6w hum.n myeloid cell line has been establishedrecently lrom ihe bone marrow c€ls of a patisnr wtrhchronic myologenous l€!k€mia in bläsr crisis. Th€ äctiv€proliierat on and survivalof rhe c€lls in FPMll640medrumcontaining fetal calf serum are clearly dependenr on rh€pr€ssnc€ of either natural or re6ombinanl humän granulo-cyte macrophage colony-stimularing läctor (rhGM CSF).oespite permanenl culturing in rhcM CSF (100 U/nL), thecells do noi difi6r6ntiar6 and boar the myelomonocyticsurrace markers CD34, CD13, CD36, as well as HLA DR,

l.r lr .l\lircru , s oj.\- ro,, o.o pro\r

'1.rbr .,. ..1e bo-

,,d 9 e .r' t'.i io1

\!.i.u\ ceLL liies hale been rcporrcd from nouse or hum.noriCiis ibr inrcrlcukin-2 llL-:). tunror ne.rosis lactof o(TNFd).r lL 6,igm.ulocyle colonJ stinulalinB facto. (G

CSI),r and gmnuloclrc-na.roph!ge CSF (GM-CSF) "GV-CSF is one of the trellchafucrcrizcd hendtopoler'c

gro*Lh facloß Lhat acts on m)eloid progenitor cells alvärious naLunLi.n sLaSes and induccsproliferation, ditrerenrirrion..nd funclionrl stimularion."' The p..Liferarive ef-fccl ofCNl'CSF is nor absolurelt restricLed ro mreloid cclh,':n.? l. rr' '. ,1. . j ' \. llo, rdc_o.. .'1or. d

celL Liies.' and huhai e.dotheliaL cells' ah. havc bccn

rcportcd 1o .cspond to G\f CSFln rhß rcporr M dcscnbc rhc chamctcristicsofa CM-CSF

deJrendeir n,lclold ccll llne. shich h!5 recentlr been de.,!edftom Lhe bone nrroE cells of a prtient trith chronlcürtelog.rous leukenia (CN{L) in blasr c.isis Althoüghseveful!s|)ects rk stilL under ii!enigation, se rhink rlat thisne\l)_ $ublished cell line, desiemred GM/SO. $illconlrib-uLe Lo undeßr.id Lhe biolo-qic acrion of GNI-CSF on thec.lls Bl usine rheeficct Df G\'I CSFon this cellline. ! 'eryren\iri1e bioassr! has dLrcadr been delelol'ed fo. measurin8rhc C\.1-CSF concenlfurions i. cell culLure supernarrnrs orin human scr. (Ocz S. Bührer C, Atzpodien J, Kalden JR.ninuscripr sübn,irrdl

CASE BEPORT

A 50 rer" old Lmrl. p i.rt tC V.l srs rclci..d ro our crLnic

trom aoorhü bospiraldu. t a bhn crGis ola CVL. Fhich had been

dirai.5ed in Februx.Y 1986

FrDh th. ltl.dt:intt.h. Klint]: llI tn.l tlatjtrl Jüt uhla\aüt t k, L:nt !^ 1 r.t E rl r nge r-Nii rhbetg: dhd M?tlt 2 i nt.h? Hnt 11s.ht 1!

Srbtrttted Jüuatt 2 l9qA;..t?ptttl 1tri1 I 1990

Addrrs rcprhi r.qr?esta Sddik 4... MD M.dizinß.h. XliaikI1l. Unireßitbt Ldaaten Närnb.ry, Ktatk.nhaurt !2 852A

Erldrgen, FRG| tu r,bli.atiar.ais afthit atti.l.rti leii.)!d i" r)dn br tag.

thrt!. . rtn.)t Thi! ani.lt klue th.rafot. he h.r.bt nnrked''rd!.{ßcJncia ,,..r./,,..\i1, It L S a tedt.n li la t.lel) ta

. i990 [) Th?.4hr?ti..h S.rilt | .! Hü,üb|.si0406 a9:)i90i76aJ 00 t 1s3 aaia

bur nor cD3, cD7, CDl0, CD'I1b, CO14, CD2o, or CD42b.The predominanr karyotype, apart rrod tetraploidy inseveral cells,is45,XX, 9, 17, 19, 22,7p ,9q+lder t[9;22]), der {13q1, wirh rhrss addiiional markerchromosomes, f.oh which one was observed in the patienfs l€ukemic cells. On A9lll disesred ONA, Southernblot anälysis with bcr 5 as the probe detected twoadditional hybridizing rsstrictiontr.gm€nts ora.6and 11.0

a | 99O by The American Society ol Hematology,

lh. d,\ sc hrd been dirsno\ed tb.re trin rir no rhe basis ofclinicrl ac !..: olair..s. *hnc blood.cLl (wtsc) coün1 l:3.000($irh 99 blrns.l9i troFlrloqies. I89 .rr-.1..rle\. 9'1 metrnr)el.-.j-r.s, lSL Lrdd, -10r'. .cutopliils, 6a .osinophils. ,1.; brsophiLs, ztl!mphoctrcs)i $r.nelr d.cicrscd xLkaline leukoc!le phosphatse (3

score): as *ell as crlolo-qic aod hnblogic eriminr!io. ol the bone

mano* specrnren oblaried lbrouCh rlir. crei tü..ttrre AllhoLghan HLA tJ-pinghrdbeen penormed, nokart.llte.nxllsn h!dbecnundenake. $ith r.rtccr tolhe Ph .hrono$nc.

InniaLl!, Lhc taticnr had bccn üearöd wirh busLltun onlll untilVly 1937: bur busulfan srs inelleclive, a.d {as disconlhued lndreplaced br nldroxrurea. Th. pali.nfs famill doctor thcn tookoverartendance in June 1987 bul acrinh3d to referhertolhe hosprtalrnDecember 1988 becxüseola blasl incrcascin periphüalblood Twodr'\ l!ter, she 9a! sert l. our clinic

Cn rxrfrinalid.lhd pdtient appeared aculely ill and somnolenr,

"lLh lcrnper.ture! g.eärer lhln J0'C, dJ_spnea, aDd lach!cardix

$BC coünr was 16.1,000 \itb i6? bla$s, l6rt, neutrophils. 5{Irnrphoc]'tes. and raa m.io.!res, xid the pl.telet counr *as 46.000Phenollpeeuminationof rh.teriFheral blood leukocyressavecleareliden.e that rhe blas cells bor. nlcloid a.tilcos su.h !s 5fr CD3(oKTr),1t cD7 {lAr). 2? CDt0 (Bt). l6q. cD?4 (BA r), t6%CDll (\1!7),and20'.CDr3 (M)9) Crrochcdicalnrinrneol bo.cnr.ro* cells sho\red Ihat 25tf oirhc blans *ere po:irivc ior cncnscbut wüc nsgxhve rorperoxidase andPAS

Despn. tf.armcnr sirl ai!ibiotics. the patieot nnaUy died ofanoveit pn.umonia.nd sspsis on the lhhd hospilalda)

MATEN ALS AND METHODS

Bate htr.i.!l!\ \\rrh tle paticifs Fridr infornre{j.onsent.maro* .ells {&c obili.ed br means ol iliac .resr puncture.\l.J.onucl*r cells 1rü. sctrrlrcd by d€nsnr cLedi.nr .c.hfueaiion.n Frcoll lNlcomed '\S, Osl.. N..sar) 1nd culrured in RP\lll6,10ncdiu6 \trlh 10'? retll caliscrum (fCS) and Bro$rb laDtors.Part ol lha cells refe presened br frc.znre in liquid nnro8cn si(h10?dimelhrlsulln\ide (D\lSO) and RPI\'l l640mediuncontarnrie,10t FCS.rid lhese \rc.e üsed Uterlorkrtorypernabsn

Clr,kr,.r Rc.onbnranr huna. G-CSF (rhC-CsF), rhcvCSF, nrlL-1. rhlL.,1, and rhlL-6 {ere Folid.d bJ- L Souza(AI\'!Ceo, Thousand Oaks, C^), \rh-e!s rhlL.la aid rhlL 2 {ereobid,ned lioD Boehri0ser\IanDhcim lFRG)

,.1,rtboltcr Rabbil ami GNI.CSF anliserun ras also a giirr.06 L Souza (AI\'Cen). MonocLondl dntibodies (l{oAbt aeainnCDr4 (HPCA l).CDi (Leu 9). CDl0 (CAl l-A).CDl6 (Lcu I tb),.D.'0. 1 o..l \DP 0 'o".1,. Lg r.d.o..

'mouse werc purchased fton lecton-Dicki.son (Mountain Vies,c^) cDl6 (oKM il. cDf b (oKM l). cDl] (oKNt t3), cDl4(oKNl lll and CDr (OKT l) lrom Ofrho-Dirgdo{,c Sr-$enri\eckrrt.dihd. FRG): and CD,1lb (AN jl) fron Dakopatß

srood vo 76 No 3lAusust I i990 pp 5r3 532

6M-CSF DEPENDENT HUMAN MYELOO CELL LINE

Praliletulian aßa\. Afrc. washine tne cells ihree tincs inphosphale-bü|löred saline (rBs), ihe celts { 1 : I01 celh/sell) w*cincubated for 72 hoüa in flat bouon 96-we1l microriter plaFs in ann,l voluhe o1200 /]L RPMI 1640 mediün containi.g lo% FCSand various cltokines. rH-rhlnidine (Aneshan, Brlunschweie,FRC) ras,dded at I aci (r7 kDq) Fer well for 4 houß before theend.rtlecullure Thecelhweretnenhanestedon6bergld$fiheßand deasurcd by liqüid scintillation countin8. Al1 assays wereperlorned in tdplicate and dala are expre$ed as tne nea. counßter ninule ol iH-thynidine ircorporarcd

Im,tunophenat]tihs Cell surlace aniigens sere detec&d bynandard i.direci innünofluorescence .say. ln briel thc cels wereincubated al 4oC lor 30 ninuts üith nouse MoAbs, washed llrcctif,es by cenlrifugltion witn PBS. followed bt .ddins FITC-co.Juglted soat anl1-nouse antibody aor r0 ninutes at 4oc. shinedcclLs sere üen evalüated with ! Uv-nicroscope The cells qereco.sidered positire for ücanligen in question shen tne nuorcscencei.lensny on inlact cells was clearly visible in comparison silh thecells treated only witn FITC-mnjügaLed eoar !iri-mouse airibody

Chronasane analrtß. Krryotyping oaCM/SO c.lh and fyo-pr€served bone narrow cells rvas perforned bt sandard techniqucs,includin8 Ciensa fypsin bdnding. '

Southühblot ahdlttü. High noleculd $eight DNAftoncells*as djgested rnhrg.lll, elecrophoresed on a 0.8% agarosegel, and

blolted onroa nt10. n.nbrane (cenescreen PlüsiNE\. duponrdeNemou$- lad Honbure, FRG). Thc b./-5' prcbe (proridcd by C.R.Barhao, Uln. FRC) was tabeled by .ick-translation and hybridizedaccordinetonandardprotocols.L. -

Staiüi,slor hi.ota Mat-Grtinwald and cytochenicat stainings torPAS, nycloporoxidase, and d-naplryl acerarc esLerase werepertnrmed in our roütine iematoloct laboraroiy

BESULTS

Estdblishment and .ellulü charucte^ti.s. lnitially,bone maros nononuclear celh were cultured in the prcsence of rhG'CSF. .hGM'CSF, and IL-3 (1,000 U/mL cach)$i1h the intertion ofstudyi!81!e ditre.entjarion capacity ofthe blasl cells. Asüsual.1lc cnlture nedium used rvas RPVI1640. supplemefued $ilh L0% FCS. 02 frmol/L L-Slulamine, ard antibiotics. Wlthin ih€ fis1 2 weeks rle.esere no d flerence ro be.erec..d. e .he in pro re% or '. .e

or i! malu.dtion of lle cells in the presence of rhese lhreehemaropoietic gro*lh factoß. However. afte. several pa!sages an active prollfe.alion sas süstailed only by the celhexposed to rhG\,I'CSF. In subsequent srcls, liree slablesubclones sere establlshed bl usire liniling dilution tech-

wfreG

)g

F

&0ä

rGt.w s*#!

"Q

\h-!

,

tFis 1. Morphol6sy,nd morphotosic difl6r6ntiation or GM/so ool|3r cM/So c6tts growins inm€dium containins loo U /bL rhcM-CSF .tono {A),

dditionat PMA {1oOns/mllror 24 hours {3), Th..d66r6nr cotls w6r€cuiturod on st€rit. slid6s and .n-dri6d beror€staining {May Grünw3ldl.

t

B

q"

-!k"'

E

a

15

G -r a-r-

Fig 2, Prolir.rativ6 r6cponss of GM/SO c6lt3 iorhGM-CSF, rhc-csF, rhll 1a,rhlL 2, rhll-3, rhll-4, or rhll 6€irhor alono ai conc€ntr.iionsb6rw6.n 1 a.d 1,0OO U/mL orin combin.rion wirh 1 U/mLrhGM CSF| mo8lr.n6nt an€r72 houra. Th. n6urrarizing 6ntibodY wls to3t€d in l:4o linaldirurion, and th€ curv€3 f.pf6,

rique. These subclones din'er in their norpholos] andgenerarion time, but thet, still retain their .equkement forexternal supplJ_ of rbGM CSF. The dependencr can be

circumvented if the cells are cultnred by carefully reducingGM-CSF fo. 2 or mo.e wceks. As a conscqü€nce, celh tlathave once becone independent do no1 require GM'CSF fortheir survival and prolile.ation. whereas they süstain to.espold to GM-CSF ivitb increased proliferalion (data nolsho{n). To avoid overly complexda!a. onll thecloncrermedas GM/SO is discused in this reporr.

Although tlc.c is a colsiderable variation in cell size, thetlpical mo.pholosy of GM/SO cells in May-Grü.Naldstainine is a large, rourd, of slighrLl jr.egular nuclcus, threeto six proni.en! nucleoli. ard deep blue cytopLasn wjih aperinu.lear pale zone (Fis lA). There is no s.llnula presentand the cytochemical stainiig tor PAS. m)eloperoxidase.ard a-napht_vl acetate esrerase r.e neeative. Moreorer. 1he

cells grosing in 100 U/mL ibGM'CSF (unless othe.iviseltinulated) rclain rhe; norpholoe] and display no olerrdillerentiation jn liquid cultlre. HoNever. a rapid change

r{

^ ,*{-d-}t- " f{-l f

"-fi-!; tf $-lr i: !i

D 1l-f !-tt- E h-r I s€

1a

1210

t6

13

17

F _r _-l.l

20 21

Fig3. Sr.nrin. ka'yorypo of tin€ cM /SO: 4s, xX, - 9, ,17 ,9q- dor t{S:221 (q3ajq 1 1 l, dor (13q1, otusihr€6 mark6rohromGom€s lM1, M2, and M3)rhat oould not b6 €x!cdv cla$ili6d,

22

GM.CSF DEPENDENT HUMAN MYELOO CELL LNE

rnlo a monocyte-nacrophagc morphologl aid increascdadhe.encc!oplasLicsurla.ecanbcinducedb) theddditionofphorboln!rishrc acerale (P\,1A) (Siema, St Louis. MO) atconcentratio.s bcrween 1.000 ng,lml- and I ne/ml (FiBI B).

Inrmunophenor!I)e anllysis sho\led rhar rhe CM/SO ccUsclea.ly bear surlace dntiecns of mlelononocllic cel15 CD3.1(Sreater llan ?5%), CDlS (g.catü than 967.), and CD36{gieaLer thrn 92:Z), bur iot rhe oller typical granuloc}re,nacrophage anlisens such as CDllb. CDl.1. or CDl6.Whilc HLA DR (greaiü than 98.:i) is slronel) exprcsed onLhe cell\. luphoclric hukers such as CDl, CDr, CDl0.aD'i)

"nd I olb., . i. n.d-l(r ( D-)b . e . d.nl .c..

As shosn 'n

Fig 2. the p.olifer.livc cfccl ol cNI CSF onr'(.( .ll . do'eorFde-."ndr e'' u' r's oo \re-.to be b\' 100 U/i1l-. Hoseve., b) nsing a pol,raalent rabbitanliscrxm a-qainst GN{-CSF at neutralizing titeß. the lrolif-crarion could be conpleteLr abolished in ihe\e assxys Tlisinhibilion rppareitl] is no1 due to anr lo\ic effecl inantise.um. sincc lhe neutralizine elTecr could in ruin be

enrnel! supp.essed bl exces of rhGNI CSF in culturenedium In eddilion. $c simullaneously lested rhc CSF andrhlL-3, as qcll as rhIL-la. rhlL-2. rll]--.l. and rhll-6 to'.le | .n\ Do.' b. r-P. r .1 rL . .e l l.-e .\' rLc g \cexanples demonstratej there {as io deteclable prolileraliveeflict of lhese cytokines a1 conccrtrations betseen I and1,000 ll/nL, either aloie or in combinarion with 1 U/mLrhGl\f-csF.

C),tagereti. antl DNA andlr:sts To find ou1 xhellc.GN1/SO celis possess thc typical characrerislicr of .) CMLceLl, eg. PI' chrohosome or ,.F r/ rearangefreil. seanxl)zed the karyorype with a C-bandin-s rech.ique andcramined the Dr.\A hybridization will a ,..-5 probe onSouthern blot\. Tle srcmline karyoL\pe rls determined as

45, XX. 9. 17. 19, 22. 7p . 9q- (der t[9i22]tq34:ql ll), de. ( l.lq). and tlree additiondl ndrke. ch.omo-somcs (Fis 3). Since v!rious numerical chronosona I anona-lies $ereobserved in the bonenanox bl.sl cells, ar leasr oneoftnesc markeß (Ml), toCether sith the 9q+ chromosome,seenr\ to bccharacLe.isticforlhepalienfs leukemictransformaüor because it was also presenr in all neraphases of thebone marol cells freshl! frozen during rhe btasl crisis. t!thc couße of establishneir. an incrcasing raLe oirelraploidkaryotypes Na\ found in sübsequen1.nal!ses Soulhern blotanaltsl! oft!/11dieested DNA wirh bcr 5' as h)bridizationprobesho{ed tso AglII reslriction fraemenrs ofE.6 and I1.0kilobasepair (kbp) size, girineclcarcvidencctbr aicaffance'ment ltpical fo. CNfL (Fig 4. lane

^). ÄddirionalL), tlc

sisnalof tle 8.6 kbp fraghent. ifcompared 10 1le 5.0 kbp, js

m.n) tlme\ st.ongcr, and in rlis case indicares a. anplifica'tion proces. The occurence oftwo aberrani fragnents $asobse.ved rn scyüal blouing a.d h)bridiz.tion cxperinenrslthus,.n artifact due to p.fiial dieesrions is ercluded.

D SCUSSION

Thecelllines Lhat eere cstabiished in.ecenl dccades havebe.one an c$ential pa.1 ol our conre porar) biomcdicat.csearch Their conlribulions ro d;coveri!8 laflous iew

DcBA

t *

11.O

8.67.2

5.5

5.Oqse

Fig 4, Sourhsrn hybridizaiion of DNA from cM/so .s tin€u3ins bcr 5 pfob*. Each rans conlainod 5 ps ol ,g/ digoslod DNAfrom GM/so cs s (a), K562 c€ tino (a), bon€ marow csrrs ofanorh€r pati€ni wirh cML lcl,.nd p.riphsrat btood mononuctdafcorls or a h€a thy p€13on lDl.

diaenoslic and therapeuric a-qenrs can actxallt be exempliäed $i1h 11. developmelt of Mo^bs,,i plriltcarion olc)tokines,tr! bioasays, :.r'and so fo.ll. ]n Lhis con!e:r, theIictor deFndent cell lines, bcing an ideal sub.jecr ior srudy-'-8,'. fh\'oog. p e .n.c or 'he a or que..io....undoubtedh oi paniculai inleresr.

I. this rcpon we descrjbc a nes lunan myeloid cell tineGltl/SO. of xhictr sxrvival and p.oliferarion in vnro ßp.inarily dependent on the presence oi hGM-CSF. eillernatural or reconbinanl.

^part f.om exchding p.obable

eiTecls of rhl]- 1a. rhIL-2, rhIL-3. rhIL.1. .hIL,6, andrhG-CSF on C\1/SO b) lesljng thcm direclly, *e provid€devide.ce bl using a neurralizing anribody rhar rhe laclor.espoisible fof the detecred proliierarion is reslricred 10

G\'I-CSF. ln contast to 1le reporled synereistic efecl olrhc-CSF.rr IL-6,11xnd IL-II on leukefric blasrs or no.fralCD3,1- ceLlr. respectivel). {e tailed 10 observe such }lyncrgisnonCM/SOcells.rl\{ CSFand lL-5travenor!e1

In addition. $e conlirned rhrough inmunophenotrping

and dilit.cntiatio. induclion lbat GM/SO cells posessexclusivelJ myelogcnous, bu1!o lymphoid, features. This isindeed the najo. differenc€ in conparison with the othcr celllines reporled to be dependent on the prescnce of externalGM CSF.r6 Another charrclerislic to be noled is rhe addi-lio.al second sienal \irh D./ 5' probe in Soulhern blotanalysis ofDNA f.om GM/SO cells. This nnding is actuallycoisistent wilh lhc obsematio! made bl Bartran e1 allTLE colld ,l.o dere. . n "dd,u 1-l 1 \eL { bc -tnemen. inblast cells ofa parient with CNIL. and the) suggest that one

OEZ ET AL

ofthe 1{o Philad to be secondarily rearanged a1lhc 5 b./

In summar), rhe data presenlcd herein suggest that rleGV/SO ccllline possesses d functionally iltacheceptor forbG\,I CSF as weLl as some characreiislics ol its nornalcounterpa.l. Apart lrom the use for bioassay, ir js likeh tharprospecrile studies on lhis cell line nar conrrjburc ro thend!r'. d ! orrLe o\\' olog ccoNequeiceo he,1.erd.-

iioi betwccn GM-CSFand n],elogenous cclls.

I Gillis S, Fern l'l, Ou W. Snitn KA T celi gtu*th factor:Pannetcs ofFrodüction a.d quanlitative nicrcassay lor activitt. J

lnnunol120:2007,19?82. EsperikT, Nissen-Maycr J: Ä nignlt scnsnivc c.llline, WEHI

16.1clonc ll lor ncasurinC cltoloxic iactor/tunor ne{osis factorironhunanmonocytes. JlmnunolMelhods95:99, 1986

3. Shißizu S, Yoshioka R, Hnose Y, Sugai S, Tachibana J,

Konda S: Eslablishnent of two inte.leuki. 6 (B ccll slrnulaloNlactor 2/interldo. t2)-dependent human bone naro* deiveddlel.ma ccll lincs. I ExF Mcd 169 139,1989

t Weinstein Y, Ihle JN, Lavü S, Reddy El: Truncation of thec-nyb gene by a retovnal i.regration in aninterleukin 3-dependenlnvoloid lcukcnir colllinc. Proc Natl Acdd Sci USA 83 5010, 1986

5. Lanee B, valtieri M, Santoli D, Caracciolo D, Vavilio F.CcmFcncin I, Cri$n C, Enlnuel B, Fina. J. \o*cllP, Rovera GCrowth laclor requirene.ß olchildhood acntc leukenia: Eslablnb-me.t ol CV-CSF lepe.dent cell lines. Blood l0:19:, 1987

6. Valrieri M. Santoli D, Crracciolo D, Kreid.r BL. AltnannSw, Tseardy DJ, Gcnpcrlcin I. Mavilio F, LrnCe B. Rovera CEstablishment and characrerizalion ofan undifrerenliated nunan Tloukcnir coll linc shich röqulres cV CSF lor groRlh, J Innu.olD8 4042. r98l

7 Metcali Dr The sranulocyle'nacropha8c colonyitinulatingiactos. Science 229:16, 1985

8 Sietr CA: Henalopoietic grortn facloß JClinlnvest?9:1549,t987

9. Cason JC, Wcisbad RH, (rulnan SE. Clark SC, Ee$ichR\I, Wong CC, $a8ner H Puriied hunan sranulocrle-macro-pha8o colony{rinulatine tuctor: Dnecl action on neulrolhils. Sci

10. Siof CA. Emeßon SG, Donahue RE, Naüan DC. $ansEA, wong GC. Clark SCi Hünan reconbin.nr eranülocrterd, ot\de' coo. ) ri '. r i13 ,-ro- \ _ül l -8" t" . oI Fcti. s.ience 230:l l?1,1985

11. Weisbart RH, Kqan L. colde D\\,, Cason JC: HunanGM-CSF prines ncüüophlh lor enhanced oxidalive netabolisn inrciponsc ro rhe najor pnlsioloeical cnen.attmctanls. Blood 69 18,

1987

12. Orab$ein KH, Urdal DL. Tushinski RJ, Ivlochizuki DY,Price VL, Canrell MA, Cillis S, Conlon PJ: Induction of macro-phage luooricidal activity by granulocyr€-m$opnage colonysimulatins factor Scicnce 212:506, 1986

13 Berdel wE. DanhauselRiedl S, Steinnauser G, winton EFi

Vaious human nenatoFoietic gro*lh lactos (interlcukin-1, cl\lCSF, C-CSF) sinulate clonal grosLh of nonhenatopoierc tunor

14. Aussolino f, Wane JV, D€lilipti P, Tunini !, Sanovio !,Edgcll CJS, Aelietla I{. Arese P, Mantovxni A Granulocr-tc- andgranulocrte-nacropnaSo colonj"{tinulating aa.to( induce hudanendorhelial celh to migrate and proliferate. Natüre ll7 4?1, 1989

l5 ScabrighrM ^

rlpid ba.dine techniqüe loriuhdn chrofro-sones Lancell:971. l9ll

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