Connective tissue cell mobilization and migration following wounding

ls':rptl Eye Res, (1.970)9, t2-21

Connective Tissue Cell 1Vlobilization and Migration following "Wounding I. Inhibition of ~M[obilization by Chloroq.uine and Inhibition o f ['¢tigration by C o l c h i c i n e * V-IR~:[XI:A '~Vt.ll..',lht~. AND ~ ] . . t r t ! . E X l

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John E. lVecks 3le~nm'bd Laboratory ~ OI)hlhal~olog/t. Department of Ot/~lDalmoloffy. I[niver.s'it!I of Ore,!o~* Aledieal N,:.Do,d, PorHa'.d, OreflOn 9720t. /.7.,~g,A. (Receirat 2J P'ebr~tar?.1 1969, Bo,~'to~) ( 2 o n n e e t i v e tismm cell mobi[iz:~tion a n d m i g r a t i o n in respo~xae to i n j u r y w e r c .studied in o r g a n c u l t u r e s o f w h o l e r a b b i t c o r n e a s . (~ell.~ in J.ht~ c e n t e r o f t h e c o r n e a s w,,.re kilh:d b y fr'eczing, l)tzrir~g {.tin firut 48 h r a f t e r i n i u r y , a zom~ o f cells a i ~ p t ' o x i m a t o l y 401~ ta wide a n d i m m e d i a t e l y a d j a c e n t , t o ti~e Frozen zone- develope~[ m a n m r o u s l a r g e nu(:l~.
I. introduction The i n i t i a t i o l i of" cell m o v e m e n t following i n j u r y i:~ t,h o u g b t to depem~ on two ,1L~t.mc~ c h a n g e s : ([) loss of "e.ont.ae, t. inhit~itiot~", and (2) a ehar~ge lit t h e environ, mell~ (either e x t e r n a l or invenm.1 or boluh) of t h e ce|l.s in. l:he aeighborb_ood of the wo~,~d,, iea.ding to a sin, to of m o b i l i z a t i o n of tire coils (A.bercroml~ie, 1964). C o n c e p t s of the l.al~ure of the eeIlular d ~ m g e s umtarJyir)g eolI i n o b i l i z a t i o ~ are l a r g e l y sper'ula.t.ive. (Vor review see Ahere:rolnbie, 1964.) T h i s s t u d y was untiert~tken to d e t e r m i n e if a period of eel[ mobiIizatior~ d i s t i n g u i s h gtbh:, fz-otll that~ of cell m i g r a t i o n occurs ['o{]owing i n j u r y . A dJsginc'~ period of cell mobilizat, iort has ~log t)een,dem.ollsl;rat, ed. in wot.n...md gissl~es ,~lghougb its ex~stenct:. has beert a.ssumed (Abereromi:de, 1964). q'J~e, cornea is a v a s e u l a r a n d is c o m p o s e d of thre~ d.iserege ce,ll layers---t,h e epighelium, subst>altt, ia. p r o p r i a , a n d l~he e n d o t h e l i u m . T h e cell populagiort of t~lm subsl~an.ti,~ p r o p r i a c o / o connect.lye, tissue fibrocyt~e~s, w i t h t,he remainSnL,e 5'%" coosists of a p p r o x i m a t e l v.. ~,no/ o1" cells eonsisLing p r i m a r i l y of t i s s u e ma.eroph~ges gin,1 oeoasiol~al polymm'phonuelea.r lcukocyt.e-s. T h e substa.nt:ia propri~t is r d a t i v e l y acetlu~,ar, ~he eell:~ c-onsLit.u~ing o n l y abou~ 1/'25gh of t h i s tissue. '_Phe c o r n e a Ls a. p,~rticMa~ly e l e g a n t m o d e l for s t u d y i n g t h e mobillz'~.tio~x a n d migrat~ion of f i b r o b l a s t s i~x orga.tt c,ultttre. T h e cells arc n o r m M t y * "|'his irtvestigat,ion was ~ u p p o r t e d b y ~ 1~1:.t~ r e . a r c h gr~nl, (NI~ 0aTs-~) f r o m t,he ,'gational I n s t i t u t e for Nmirologieat ][)i~eases anti Blh)dneaa, 1_7.8. Public [-lealth Service. t2

C E L L ,MOP, I L I Z A ' I ' I [ 3 X A N D 31I(.;I¢ATI.ON

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{n a q u i e s c e n t .state a n d , a f t e r moL, ilizatim~, mibq'ate in t h e i r o w n l u l t u r a l s u b ~ t . r a t o .... t h e c o n n e c t . i r e t i s s u e m a t r i x o f ti~e c o r n e a . A n o r g u n t.ull;m'e .'system, i~t ~r w i m l l y s y n t h e t i c , m e d i u m , h a s b e e u ~levised for a m d y a h t g the r e s p ( m s e s o f c o n n e c t i v e t.issue fibroc.yte~ t o .~Candar~tize~l i n j u r i e s o f l-h~ r a b b i t ~.o,tt(.o..' ," '"

2. M a t e r i a l s

and Methods

N e w Zeab, n,1 w h i t e r a b b i t s , o|' e i t h e r sex, a n d w e i g h i n g a p l ) r o x h u a t e l y 2 k g were u s e d for all e x p e r i m e n t s . ~ ~ . c o r n e a s were a n e s t h e t i z e d b y t h e l;opical a p p l i c a t i o n of 3 d r o p s o f l},+rs+teaine (benoxi 1;tttQ ;li. 1-mitt i n i e r v a l s a n d the. l ' a } ) b i t s w e r e t h e n g i v e n a b r i e f g e n e t - a [ :, ~e.~t}te ~ir" !~y t.he h~t.ra v e n o u s injeet.io~> of,sodhll n t.hiu m y l a 1 (St~rital • P~trke J)~ vJs a n d Co., {_;otr,dt.. M i c h i g a n , {.,,8.A.), 1 6 - , ) m g / k g . £m~.d,~rd bU~rie.~" were i n f l i c t e d b,v freezh:g ~he ~:m~t.er of e:~¢:h c o r n e a at --,18°0 for 10 see with a "ca.taract.'" ¢:ry('q,r~)be a p i m ~ x h n a . t e i y 2. m m s q u a r e (Li,~d.e C.-3 Cry,~s~trgieal Unit.: U n i o n C'n rbide C o r p o r a t i o n , lAnd.e D i v i s i o n , Cryogenic. l?rodu¢~ D e p a r t m e n t , 2770 ] , e o n i s glv,i., t,os A ngeh..s, Calif., 90058, 1).,.%A..). "['he a n i m a l s , w a k e n e d wit;hit,. 5 - 1 0 rain a f t e r ih,:..aez~,.,r~] a~msthet.i:" was g{ven ;rod we.re t.]~cn killed, b y h ~ t r a v e n o u s air [n~e~t, iol~. T h e vv(:.x were qui<:kly enuc}ea~.ed i n t o I{.inger's sotagicm a n d t h e c o r n e a s c u t ont, w i t h a. I-.2-.,n:,~ margin., r~f sth.~,., ",~ art.ached R,~" Immllit~.. P r i o r to c u l t u r i n g t.he iris and lens wer.e re~,mved a nd di.-.'ca rde.d, .', n d t h e cornea.~ r i n s e d a n d a.ilowed to s o a k for 20 rain in i n c . u b a t i o n ~ m d i a . A m i n i m u m ,.ff 4 c'.orneas w a s t e s t e d for e a c h t,r e a ~ m c n t . T h e mdt'a.re , ; e d i ~ * u s e d w'as .'.1 meal}float-ion of ~hat: u s e d h y D r A a r o ~ J~. tTreeman. (19,;7. per,s,>nnl commuuicv.r, ion) for o r g a n cu!t.ures of c o n n e c t i v e tis,sae. It.s eomposit.ion w,.~:: a s i'~fll<~ws- Eagles" m h ~ i m u n l essm~t-ial m e d . i u m ( E a r ! e ' s base} conl, a i n i n g p h e n o l red ar,,l suppl(:menl.ed w i t h gbtt.omi~:,:, 2 tt~?,i/]; E n r l e ' s ~mu-essential n m i n o acids, 0.I m~f/1; l.mnic.il[/n, tf)t.} un.{ts,:;ntl; st r e . p t o m y o c i n , 1 0 0 p - g / m l ; a ~ d fm~gizone, 0.gSff.g/ml. 3.~:]aoh cm'ae;) w:,.s i n c u l m t e d iu 15 m[ of t h e c o m p l e t e m e d i m n in a 50-ml D e L o u g c u l t u r e flask at. 3.3-5~'C. i~t r~ N e w l_~nmswi('k g y r o t o r y i n ( m h a t . o c - s h a k e r at, m i u i m a l s p e e d (188 r e v / m i a ) , T h e m e d i u m was c t m n g e d d a i l y . T h e p [ I w a s cont~rolled b y g a s s i n g t h e i n c u b a t o r wit.k a (X)2-air mi>:ture r e g u l a t e d by a g a s flown~eeer-mixer. h~ addi*;ion., medi~t co~r.aiaing I 0 °/ 7o £etnl bot.-bm s e r u m (MJerobioiogical Assoe.iates, A l b a n y . Cah£.. U . S . A . ) of c r v s t a | l i n e bovh~e se,r m u a l b u m i n ( S i g m a C h e m i o a l Co.. St, I,,',uis, Me.: U.S.A.), I r a g / m r or 5 mg;,'ml, were t e s t e d , t ' l o w e v e r , since no signifi.eant differerwes were not.eri in t h e r e s u l t s obt, a i n e d w i t h t h e d i f f e r e n t m e d i a , t h e result~s report.ed a re tho::e obt.aip, ed with. t.Ite w h o l l y s y n t h e t i c , p r o t e i ~ - f r e e m e d i u m . F o r ?e~.i,.'.ro.~copic cxa~r-ir~aIion t h e c o r n e a ~ were nu)~lr, t;ecl on a n aWaeou~ pas~e o f g u m t..vag:,c;u, th, froze,t an~l s e c t i o n e d corer, ally t,hrotagh t h e full t h i c k n e s s of t.he c o r n e a . ' t h e .a.ect%ns (.30 I*) were a i r d r i e d , fixed for 10 m e n it~ a b s c h ~ t e met, h a a o l , an<[ .~ta.ined in '~ dih~re (_liems'~ sta.in tbr I0 rain..* A f t e r stah~h~g t.i~ey were r i n s e d i:i d i s t i l l e d w a t e r , d r i e d on a. w a r m i n g p l a t e a t n p p r o x b m , tely 4:5~0, :rinsed itt a e e g o n e and. m o u n t . e d i n P e r m o u n t . T h e ~ptake (g" the vitat dye ~.eu*,.at. red was tier.ermined by" in}:ubat.ing t h e c o r n e a s , imm e d i a t e l y ~oi!owing i n j u r y , for ~ h r in I 0 m l of R i u g e r ' s solut.ion eoai~ainb~g 0 ' 5 m l ot7 t)-t(!.{~ nettgral r e d sgoel<" solugion ( W e i m a r , 1 9 5 9 ) . . F o l l o w i n g ineuba~;ion l:he eornea.s were fixed h~ 1-[olI-'s (1959) n e u g r a l c a | t i t a n - f o r m . e l for 24; hr, r i n s e d in w a t e r , soakewt f o r a few h o u r s ia g u n t a r a b i c surarose (H.olt, 1959} (to mak.e, t h e f o r m a l i n - f i x e d ~CCtaiOns s t i c k t o t h e .~lide.s), s e c t i o n e d wit:h t h e ;freezing m i e r o t . o m e a n d s t a i n e d w i t h O i e m s a as described above. *" rl',he O~e.m..~a stMn was m a d e u p as follows: 50 ml disbil]ed wr~taer, 5 mI o f 1 0 ~ p t ~ o s p k a t ~ buffer ( 0 - t 8 . ~ in anhyd.rous diaba.sio s o d i u m pho~p!,~;te a n d t'}-48S~l h~ m o n o b a s i o p o t a s s i u m l p h o S p l t ~ ) , 1o}{ ~]-4 {Sf..it.f, C ] o u ~ t an(l ]~ran],aJ~, l]~,1.8]; alv~ !-;.3mi O.[ st~ock G i e m ~ a.ol~tion (A!lk~d ChemieM, ..Morristo~vn, N . J . , U.S.A.).

14

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3. Resffas and Discussion M obih~za t io u W h e n the cells in ;u ¢:.etfln'~l zone. of t,h,- eornm~ were kille,,I by .t'reeain~ w i t h tldtc r y o p r o b e at, --.48°C'. t'o[' 1(.)see: t.he cells in gifts frozen zone. gra.ctua,lly disn.I)l)O,av('(]. t')rt t.hc ot:her h a n d , ~ b o r d e r of cells apt)roximat,,fly 400 ~ wide imm0,diat,oly adjncent:. to t;}m frozen-kilh~d zone. began t,o unde, rgo iIrofoul~_d oha.nges in morpllolo,~zi~:al aplm.a,ra.3~.ce including t;]m develo/3nmut~ of ltunterous large uuchm]i a.nd d e e p l y basc>l>hilic cyeopla.sm (P]t~tes t and 2). Wit,his. 24-48 hv a.ft~er injucy these ee.lls became. fibrobh~st.-like i.~t apl~eal'ance. We h a v e de.~igtmt,ed t h i s aeriea of eha.nge~ t,be phn;~ • ~,i' celluh~r mobiIiza,t.ion (,'~ctiw~t,ion.). Similar eh,'mge.s i,t t h e ~ p p e a r a n e e of corneal fii,rc,cvteu following knife, wounds h a v e b e e n describetl in rat, (VVeimar. 1957, 1958; \Veinmr a n d Hara.guchi, 1965, 1966) mid, rabbit) (Bracher, 1967) cor~ma:u "|'hesa morl~}toh.~ica.l c h a n g e s were t b u n d to be aceompa.nied by a. viel~ development, of pre\'ioust}i usdetect, able e n z y m e s (Wei~na.r and ]-Iara.gutdti, 3965, t966) a n d by increased upga.kes ~fi' ~5SO4, [~4C]leucine ~md [3H]m'idine (Bvach('r, 11367), i n d i c a t i v e of incre~l.se(l mu('¢)•p o l y s a e e h a r i d e , t)roteiu., and ribonucteic acid syn.tAtesis. \Ve next, at,t.empt.ed t,o d e t e r m i n e if ~,he cells in this zone of act, iv,%tcd c,?lts, witich d e v e l o p e d be~;ween 24 and 48 hr a f t e r i n j u r y , were (tire,:fly act,ivatexl, by t.hc i n j u r y or if t.he.y were g r a d u a l l y r e s p o n d i n g go changes in t h e i r envi~'onmeato re,sult.ing from eithex t,he p r o d u c t s of ~u~olyzing cel!.s in ~he ,~dj~cent, frozen-killed ".,,,om~or t,he [os.~ (~I7 cortt;ae~ inhibPdion (]3nHougli a n d L a u r e n c e , 1.960). Cornea[ fibrocy~es do ~ot, n o r m a l l y t.~ke up tam v i r a l d y e neut, ra.l red b u g followin.g v~ounding, eoneentra, t.e Iarge quantit, ies of tfl~e (lye itt ~he f o r m of cytopla.smk; vacuoles (Weimar, 1.958, t959). I m m e d i a t e l y following injuxy c o r n e a s were incuba,t(-d for 2 hr in t l i n g e r ' s s o l u t i o n eont, airfi~tg 100 r a g % glucose a.n:.l 1-7 :< 10 -'~ .~[1 neut;ral re~t. ('ells t,hroughott~; t~he eorne,~, except; for t.he, froze~t zone, t o o k up t,he dye i~. t,he tkn'm of small vacuoles but, a Mm'el)ty demaraa.te.d 4¢)0 ix-wide .zov~e o f cells i.m.mediately a d j a c e m to l.he fro~'.:n, zone t o o k u t) e x c e p t i o n M l y large quartt.i~ies o.f t h e d y e i3t t h e form of ext,rerc, ely large vacuoles. T h e reaul.t.s were part~icularty st.riking if one r e m o v e d the d y e w i t h m e t h a n o l aft,er fixathon a.nd ,,sectioJfing. "Phe c y t o p l a s m of such cells w:~.s filled witch h u g e empt, y vacuo].,~s. This v e r y disZincV zone of ceils c o r r e s p o n d e d bot~J.~ in Iocagion. and in w i d t h t,o gke zone of a c t i v a t e d fibrob!asl;s e,h a t a p p e a r e d 24-48 hr a.fter freezing. T h e activat;ed cells ~ea.rest. t,he killed zone h a d dist;irtegly larger ~teul;ra 1 red vae~aotes t h a n 1;he aet, ivat,ed cella t~rtaaest~ fl'om the killed zoom. T h e mechani.~m of t,he u p t a k e of neut, ral red is unknox~nt bug t3he dye appg~rs to be present; in lysosomes (Alfison ,~nd MM]ueci, 1965; Morgan, I,'ernando a ~ d Atousi, 1.966). Beca,use t h e up~MCe of nem;ral red begart i m m e d i a t e l y followi~gg i n j u r y these reault, s were i~:erpreted as indica.gi~g 13hat~t h e i n j u r y a,cted d i r e c t l y eta t h o s e ceils w h i c h were t,h e first~ t,o b e c o m e m o b i l i z e d fo!lo',vi~-g i n j u r y . I a s u p p o ~ of t.he argument: l;ha~; direct, i n j u r y ae*~ivaved the. init.iM cellular mobilizatoion, which ocem-red bet, w r e n 24 a.nd 48 hr a f t e r i u j u r y , we h a v e f o u n d that, it, is possible w i t h a I~rger e r y o p r o b e (the reLiua probe, about. 8 m m il! diamet, er), to aet, iva~e., eornea.1 fibroeyLe8 l;hroughou*s 13he cornea, wit, hout, p r o d u c i n g cell dea~;h. Migrate, on A t eq~proxi~na./:ely -.48.hr ~.fter i n j u r y ~;he p r e v i o u s l y m o b i l i z e d fi.brobtas~s begat~ t~) m i g r a t e iut.o rite frozen-ldlled zone. B y 96 hr a f t e r i n j u r y t h e cells were s p r e a d

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l#l~;. I , ()Qf.(fl.R c ~ . l [ t t l r e . ( J ~ [ -~-- ¢JOllf.r~.)( l z l 0 d { l l . " (.'(mq~arcd w i t h 0~:2,1 h r CM. .Mo b i l i z a t itm al~d m i g r a t i o n o1" i~j urt--a(:Li r a t e d c o r n e a l eonneot-ivc t i s s u e ceils i n t o f r o z e n - k i l l e d zo~e +>f c..ornca. Ceils (:ount~;tt a~ 250 X in a t;trip 80 t~ w i d e f r o m nort:aai a r e a bo t.he l n ~ x i m u m dist, a n c e o f ee|I mi£;'ralion i n t o t h e f r o z e n ave.Co (~:ee :l'ab~c [ t b r s u m m a ~ : y o f statisLica[ analys~s.) A v e r a g e n u m b e r o f cells per s(l I,. squa['~, in a o r m a l z ~ n e , m~tside z o n e o f mohitiz~tt.h>n w a s 5.39_-4:.0,2(3 (s.~).).

.lctivaNo~ ar~.d, mig',',.~lio~ of eo;'~wa~ connect,ive tiss,ue celL~ (~t various zbne 7)c,,'iods

after i~t',ur~J i ( h N : l n ctflt,ure, c o n t r o l m e d i u m )

":l'ime alter injury (hv)

Zone of ~obiliza.ti(m no. c f n e t , i v a t e d ceils in 80-.~, w i d e sl.rip]"

Zont~ o f m [ g r a t l o n no. o f act-ivatcd cells in SO.~ wide str~p?

II:'btat no. o f aet, i v a t e d colts in ,~0-~ ,,,lois ~trip~"

24

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LoveI o f s i g n i t i c a n e e 0-050 ( c o m p a r e d w i t h 24 br). :~"M o b i l i z e d a n d m i g r a M n g cells counLed a t 2 5 0 × in s t r i p 80 ~ wMe f r o m n o r m a l m-ca tm t h e m a x i m u m d i s t a n c e o f cell m i g r a L i o n i n t o t h e froT,cr~ a r e a ( - t - s t a n d a r d de~,iation). StatisfAeM ~tnab'ses c a r r i e d out- o n a. d i g i t a I e O m l m t e r ~vit.h a n a l y s i s o f v a r i a n c e , m u l t i p l e c o m p a r i s o n Lest a f t e r N e u m a n . K e u l s 0,Viner.

1062). Le-,'el o f s i g n i f i c a n c e O.00i ( c o m p a r e d w i t ~ 2-~ llr)

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MAt.:.L,I,:NE

Pi4i, I,,~dAN

sp;~rscly t h r o u g h o u t , t h e woumk.'.d a, rca. T h e g r a d u a l ~nigrat, lon o1' tlbroi4ast, s it~t.o t.ht' frozcn-kil]('(I zon(; is sh,,,vn i,~ Fig. ~. Migrat.ion ]w.r~- is ++leti~etl as a c t u a l n~ovc~ncnt: ()f c t+.lJ..+ i~t.() t.l~(.' (:,'i~{ina.l f'r(~zen-killed z o n e . T h e (-(.lls were. counbt.tl (250 × ) wil:h bh(; n.i(I of -m o c u l a r e y e p i e c e w h i c h w a s (iivith:~/ inL() s q u a r e s ,~0 × N0/z e a c h . A st)vi i) ol" cells g 0 / 4 in w i d t h w a s c-;oune(:(l t't.(:,~ t.he d i s t a l c~Lge o f bht; a(;tivat:c(t ¢;(:11 /.one to t,lm m a x i m u t ~ clist;}tn(:c o f cell ~tigra.r.ion it+. (.hat, st,rip. '.t'Ite cell cmmr+.s t, lm..~ i n c i n d c d a.|l a c t i v a t . e d ceils i~t a.ny sbri}~, i n c l m | i n g t+',:.tls w]~ic.h wer'~+" ilOt~ ycl~ Jnigral;..ng. M i g , . a l j n g cells were; c o n s i d e r e r l t;o b e thc}.s(3 (;hat. hml ~,ntere.d t,[}c, ft'ozen-killed zone. ".Pl~e. n u m b e r of a(.-i~ivat.ed and ~novi~g cells iacrca.sed f r o m 1(3.7 per st, rip a,t, 2.1 l.~r a~'te,r k~jury to 34.3 per s t r i p at, 96 hr ~zl't,er injury (3:~d)]e .[). '.l'he mm~l)(;r of ecl],~ i~ iF,, +~ctivat.ed z o n e (,I00/x) r e m a i n e d reL, ttivcl+v co+tst+utlu ( T a M e [): a l l ; h o u g h c e l l s w,zre (_[.i~[l+ finu+dly migr~t.ing £rom this zone.

t'J+ff+'.c'~of inhibitor, T o d e t e r n g n c , it" tA~e h~cr~u~sing m n n b e r s o f m o b i l i z e d a ~ d znigral,ing c c.lls w e r e avi.-.i~z b y cell divisiont or r e p r e s e n t e d a co,~tAnuing acbiwtgio~t o(" I+'reviously q u i e s c e n t eells. c o l c h i e i n e (! x 10 "'-5 5~/1) w a s a t l d e d ~o t h e c u l t u r e s at, +;he f o l l o w i n g i n t e r v M s : 0.-24 b.r,

F

Z o n e of

mobiCizoliofl

6 Zone

. . . . . . . .

,5 0

oe

-

ol

Migr~tio,-~

4

o~..,..<..~..

4

O.3 ¢.~

3 .Lt ,; 2 <-x

--

%

:

~"

O-4a

_

O

"%

4B-Ta

80

2"40

hr

CMI--

hr c0~ch,-,n~

.300

%

, %. 0 - 4 8

he CM

q"'~o.),

560

Distence

~.

"N,.

720

O - 7~-. ~ hr CM

O-OO1

880

10410

(1'

K i n . 2. O r g a n c.ulturu. C31 ~-: c o n t r o l m(~di~u~l. C o i ~ h i c i ~ 0 == co'-~t n'ol na~dim~t-i-coici:iei,~.e {I x i O - ; ~ / l ). :~ C o n ~ p a r e : l wit& e{1,her 0 - 2 4 h r C M o r 0 ~ 4 8 h c CM, t.~Jen 48.-72 h r c o i v h i c i n e , i n h i b i t i o n o f cell m i g r n t . i o n b y col,-llieiJm a d d e d follc..wing to.It m o b i l i z u l . i o n , St;a~i~tic~al m~als'si~ (~e~+, f o o t . n o t o t o . T a b l e [). T h e r e is n o sigmificat:l, difrcrene,:~ i~ f.ho tol:M m ~ m b e r o f cells f o u n d iJa t.he " z o n e uP m o b i l i ' - . a t . i ( m " f o r a n y o f 1,he t.reat-ment.s. T h e r e is a l s o t)o sign.itietu~<: d i t t ' e r c n e e ~n f~he tot.ai nullll~t.r (,f c e l l s {r, tl:: +," ' z o n e o f m i g r a t i o n : ' I)eiavee.t t.hc t r e a t m e n ts 11--t8 h r e o n ~ r o ! m e d i a rind 0-.48 h r e(.'.nWol medh~ I:ollowcd i)y :24 h r o ? colehici;~e. T h e t o t a | n u m b e r o f cel:~s in f.tle, " z o n e o f m i g r t t t i o n " f
t'l",Lt. MtII~II,IZ..~'I+ION

AND

MI(;I-'A'I'Ii~N

17

2-1--i8 hr, -t,'.¢--72 he. ;tin! 7"2-9t; hr after i n j u r y . No ,Jtitobic fignre.s :yore, e v e r seen ,.ithm" i tt l,ltc colchicim>treatc
Zor~e Of rnoblh2ol~or~

6

?,-4,. ~..J CO

Zorl, e

o~' r n ~ g ~ t i u , ~

4

"5 p I

l ,l [-

~s

1

-0

{ ~

O - 72

I 0

72- ~ 6

L ._.t

610

at

240

Fie:. 3. ( ) r g ; t n c u l t u r e .

h:

< 000t

CM

,~u c o l c h i c i n e - - " t

.t

400

560

(:.tl :.-- cu~ttt',ol m e d i u m .

Colchivino

-.-,..._

e---._ ~ - - .

7~_.0

~

-* ~3BO

_j

+ It040

1200

~: uontr~}l mcdJm~.-,~-colchiei,oa I1 X l O -z ,M]]~}.

* (:omla:tred w i t h Li-'/:?. tar f.'3l, ti~(~z~ "72-- < 0"001 ] for ~he cre~mm~t. 0-06 h r toni ~,>im e d i ~ than for tbe trea~men~ 0-72 hr eontrol m e d i a foil.roved by 24 hr of eoR:h{eine..

.-'~.vecagS{~. t+ttttrtl)et + o f '-~:lls p e r S 0 ~ wide. s t r i p ht " z o n o o f m o b i l i z a t i o n ' * ; 0--96 b r I.;M: 17-'~l::k;bt:~l); 0 - 7 2 h r CM, O~en 72-9[; }n- eo|,.'hicine: .15-OO.-~::I-SS. ,a v:-rvge, nt+mbt:r o f curls pt+r ,~O-ff w i d e str+p hi "zoP.e o f : n i g r a t i o t F ' : 0 - 9 8 Jar CBt/: :t~3-5tI-1-:],87 ; 0-Vg h r C31, t | : u n 7 2 - 9 6 } t r e<}tchicine: ;->-71:~:0-58.

Sin~:o tamre h~s boe*t some cri~ic[sm (Lac~s, 1965) o:f l~b,e use o f eolci-i~;i,m t~t ~ oortcent.rat.iot~ o f I x 10 -'~ >~/J, l~he ent;Jrc e x p e r i m e n t , wa, s }:epe.~,te,d wit;h a colohic[ne ~on-

cenbr,q.tion of t x ] 0 -~ _~[/1, .-I'2xactly t.he s a m e rcs~.lt, s we.re obbai~ed" Co]ehieiua d i d not} i n h i b i t c d l mobilizatim~ lint- it- t,o t a R y i a h i b i t e d cell m o v e m e n g . & h e reqult~s arc s h o w ~ in P l a t e s 3 m~d 4 a n d .Figs 2 a n d 3. Miszttrski (19,t9) f o u n d r h a t eotr:lficin.e (1 "< 10 -a t.o 1 × 10 -~ .~]t) added, to resLing s t a g e m o u s e Iibr~_~blas~s h~ haugirtg d r o p c u l t u r e s c~m~ed f h e ('.ells t~ rof, radt; ~heir prouessc~3. O~her nom~:il;otic effeot~ of colchieine hcrve bee+z reporl:ed: c o I c h i c i n e de.pre.~sed p ! m g o c y t o s i a o f u r i c acid pm'bicle,s, b y polymorpho_tmetear leu2kooy~es

V.[Rt':INI-\

tS

~Ar.E[51AI >, A N D

MAJI¢.LI'~NE 1 , ' E L L M A N

(Seegmi]ler, Howell a n d M.'d:~wista, 1962); i u h i h i t e d (t-5 × 10 -~ ~/i) c h e r o o t s × i s by i ) o l y m o r p h o n u c l c a r ]eukocy~es (Cane,-, ]965); d i s r u p t c . [ (1 × 10 ..-5 to i × l0 -s ~t/]) th,.~ s t r i a t i o n s ( G o d m a n , 1955) a n d m y o t u b u l e ~ (B~schoff aud Hol~zer. 1968) of muscle cells in ~i.~uue cult, ure; and is l)clicved. I:.o atl%c't the sol-gel s t a t e of tim e yCot)lam~ ( M a l a w i s t a , 1,965). T h e period of cellular m o b i l i z a t i o n ( a c t i v a t i o n ) hasted a b o u t 48 hr I)cforc m i g r a t i o n be.ga.. B r a c h c r (i967) t;a,:~s h o w n i,~ :tutort~diographic studies ~hat, su(:b a c t i v a t e d ceils ~tt t,hc w o u n d edge in, the c o r n e a i n c o r p o r a t e increased q u a n t i t i e s of [al-l]urid.iue, i n d i c a t i v e of increased ] l N A s y n t h e s i s . ]in addibion, all migrat:mg ('elI~ possessed n u m e r o u s large nucleoli whcrc~.~ ClU[es0el~t (:,or.~.ea.1 fibroeytc~ (Lid. nob have nuelcoJi (l(:tect, alAe w i t h t i m s t a i m n g m e t h o d s used in these st:udins. Our eflbrts to u.~c a c t i n o m y e i n 1) to dctermhm i[" ]~NA syntims~s w a ' s :~ prcrequisite. t:o ce,ll movcntent, h a v e so f~r been tmsuccessfu] because of t.oxicit.y of t h e act.inon).yci~t D. \Ve t h e r e f o r e chose e h l o r o q u i n e (liphozl)hatc, w h i c h inhibi.t,s DN':k--directed I{NA polynm:rnsc {(,'.otm~t a n d Y i d d i n g , .I.965; O~Brimt. O!enick ao,d H~d~.,~.. 1966). Ctdoroquine, Mso i;dfibits D N i p o l y m e r a s e (Cohen :rod Yielding, 1965; O'iBrier~ et al,, ],966), b u t ])~rA syn1",hcsis does nol~ seem ~o be i~,volv'cd here since no c.ell divi,sions were. ever f o u n 4 e v e n in. the presence of coiehicinc. Therefore, t h e use of c h l o r o q u i n e t o in,Mbi~; DNA-(iirectect R N A s y n t h e s i s a p p e a r e d to be valid. A m o r e serious ct.ifl'k',utty is Vim t';tct; tha~ chtoroquine, stabilizes lyso.,~omM n~end)ra:les (Weissr~',ann, 1964). As imtiented. J

' " Zone

--~

o f mobHizolion

I 6

-

0

°%,. "%. "%Q

i

Zone

of migrotion

-!

.5 -

o

\

/%/ I

O-I~h~Cm I

....

o

80

',, \

*'-

,

~X

~\

t

__1

,

2qO

400

560

D}sto~lce Fie,. 4. Orga.t; o u J b u r e . C ~ { == oont.rol m e d , h m l .

720

(.p.}

CtQ ~ - c o n t r o l m e d i u m q - e , hlocoquine d i p h o ~ p h a t e

(l x 10-* ~#l),

I n h i b i t i o n ot' cell mobilization b y e h l o r o q u i a e a d d e d a t 18 hr after injury. Stat.is~iegl analy.~is {see {O :l:ab}o .~). (['here is n o siguificaht difference i n him t~)ta~ t m m b e r o f ceils in the " z o n e of mobilizai;ion" or it~ t,hc " z o n e o f m i g r a t i o n " b~t:we~rx the t r e a t m e n t s 0 - 2 4 h r c o ~ r o I n~edia a n d 0 - 1 8 hr control m e d i a followcd, b y ehlorotluit~ f r o m 1 8 - ¢ 8 h r . . I . h e r .' is. eLsignifieaut irtereo-~.e in t|m tot.el n u m b e r o f ¢all~ in b o t h t,ho ~'zo,m o f m o b i l i z a t i o n " (1><0-01.) a n d tAm."zone o f m i g r a t i o n " ( P ~ 0 . 0 2 5 ) with bhe t.rcat, mcut, 0 - 4 3 h r coatrol nmdia. Averag6 n~btr o~" cells poe 80*it wide ~_trip in "'zone o f m o b i l i z a t i o n " : 0-'2.% hr OM: ] 6 . 5 0 q - 2 , 4 8 ; 0 - - 4 8 h r C~.t: 20.33-j:3-92; 0 - 1 8 h r ( : M , t h e n 1 8 ~ 4 8 h r C Q : 12'67~_2"~¢3. A v e r a g e r~umber of o~l}s i?~r 80-/~ wide a~rip in " z o n e ofzuJ&watior~": 0 - 2 4 hc CM: 0.0O-.~0.10; 0 - 4 8 hr CM: 3-25.:j=2-~7; 0-18 hr O~l, t l ~ c n 18--15 ha* C Q : 0 . 8 3 . - u 0 - 6 8 . footnote

r.

i

(.?ELI, M(.)BII,IZA:I'ION

AND

19

MI(.'II{ATION

above, alterabion:~ in t h e ]ysosonm] memt)r,mes mr~y be of signific~tnce i~ processe,s of cclluh, r mobilization. Experimen.f,s with. ehlovoquinc were the.refore carried out as follows: T h e b t ~ m f i u g s of R N A synt, heMs, as indicated by t h e a p p e a r a n c e 0£ v~.ry small nucleoli in ceils iu the nmbilizing zone e,~n be detected by t8 hr ~fi.:er injury, Since ib is clear fr(~m the sbudi~ above w i t h neuCra,t r(;(l tha~ t,l,e lysosonms were MOored i]nme(lia~ely following injury, Z o n e of r n o b i l i z o t i o n

I

5

Zone o f

micjro~ion

.

.

.

.

.

I

.

eL.

--G

"~Q.

0-72

hr CM

p-e0,00t ~

-

"-.. a

I ,

Q

48- 2

"el........

.

o-~8

h:

CM

,,~ 4 e h ~ c ~ ~

80

co"

P
\ ~*"" " " ~

K

::;.,.._ - ' ~

1 240

-'N . . . . . o .....

I 400

",,"~,

f

"" " 0 . . . . . .

560 Oislonce

~

.

.3

. . . . . . . -0,.

7~0 (/.)

""~"-0} 880

"Q--

a

,A

--~

i040

1200

i,'lca. 5. OrR:~n euh.urc. C.M == c o n t r o l m e d i u m . C Q =~ c o n t r o l m e d i u m - ~ - e h l o r o q n i n e diphosphai~¢ {1 >: 10 -~ ~,11 ), * C o I l l p a r c d wil.h 0--18 hi" CM, tlxt;n 18~18 ]'.r CQ, t h e n 4 8 - 7 2 h r CM. lnhibit, ioa~ o f coil m o b i l i z a t i o n b y c h l o r o q u i n e a d d e d d m ' i n g pm.iod o f ceiI mobiliztd;ion {18--48 Lr a f t e r i n j : , r y ) ~ n d la~q¢ o f i n h i b i t i o n o f celt migrat.ion b y c b l m ' o q u i n e a d d e d a f t e r lmriod o f celt ~nobilization ( a d d e d .18 h r following i n j u r y ) . 81::~bisCic~l a n a l y s i s (see f o o b n o t e t o T z , t~le'.[). Tht.~rt~is no signifieani; differcm'.r~ h~ t h e Lotn] mmahc.r o f cells in P~il.hor t},e " z o , e o f m o b i l i z a t i o n " o r in t h e " ' z o n e o£ m i g r a t i o n " b e t w e e n t h e t r e a l m o n t e 0 - 7 2 h r c o n t r o l m e d i u m a n d 0 - 4 8 hv c m , t r o l m e d i u m followed b y 4 8 - 7 2 h r o f o h l o r o q u l n e . E a c h o f vbese t.rcatment,s ha.s s i g n i f i c t m t l y g,'ea.ter n u m b e t ~ 0£ cells in b o t h t h e " ' g o n e o f m o b i l i z a t i o n " a n d blte ~'zone o f m i g r a t i o n " w h e n c o m p a r e d w i t h t h e t,'e,z~ment 0 - t 8 h r ez)ntrol m e d i u m , 1.8-48 h r e h ] o r o q u i n e , t h e n 4 8 - 7 2 l,r in c o : t i r o l m e d i u m {f ~ -<0-001). A v e r a g e n u m b o c o f c~tls p e r ~g0-/, w i d e s t r i p i~~. " z o n e o£ mobitiz,tt,[o~"- 0-.72 h r 0 M : tg.2t~.+_O't8; 0 - 4 8 h r C~'[, t h e n 4 8 - 7 2 b r OQ: 19-58 .--~2-33; 0 - [ 8 h r 0.~, t h e n [ 8 ~ 8 h r CQ. th~n 4 5 - 7 2 h r (i"5I: 8'17.-_k ] "67.

A v e r , t g c .r,u m b e r o f cells l)cr SO-# wide $,trjp ]11 "'zor, o 0~[ migr~tbion" : 0--~2"hr C~[: 10-75-{:2-20; 0--{8 h r C~1, t.hel: 4 8 - 7 2 he C Q : 9.27-.1-.3-68; 0--IS h r CM, ~hefl 18--48 h r CO, t h e n 4 8 - 7 2 lit" C~ir: 1-84--~: 1.(34.

the inj~lred cornea~ were irmub~ged for 18 hx" M cmltrot m e d i u m ~.m(l 6kort chcmged go m e d i u m t i n , r a i n i n g c h l o r o q m n e (1 × 10 -4 ~/1) for t h e period 18~t8 hr after i n j u r y . A few calls ,~howed some degree of aet,iv~tion aud migeixt.cd b u t cell mobilization was efl'eclfivct.y inhibited (Fig. 4 a n d PL~td5), Those few cetJs which did. m i g r a t e m a y h~ve syn~heMze.d ertough :({N-& prior to tahe e.ddition of okloroquine to "'escape'" its inhibit o r y effects. There was no reeovery when come&s t h a t had been ixtcubaVe.d in cl-doroquine for bhe-period 18-48 h r af*er i n j u r y were dtangext to chloroqnine-free m e d i u m for art a d d i t i o n a l i n c u b a t i o n of 24 hr or 48 hr. As before, ,~. few 0c118 h a d become ac~ivate.d and reign:sled bu~ furLhem mobilizatiort was c o n t p I e ~ l y suppressed (Fig. 5).

2(*

VtII(IINI:A

WEIM, AR. AN.D MAI/LEN[;;

FE.LLMAN

On the o t h e r hand, if."cell mobilizatriort wa ~ allowed to occur for 48 hr or 79 br before chloroquine was added, the ce.1]s cont.inuexl, to mobilize and t,o migrat;e ag ra, l;es a.nd in qua.ntit, ies c o m p a r a b l e to t.he control tissues (Figs 5 a n d 6). 3 I a n y of t~hcse migraeing ceils wore dis~oxted in a.ppearcmee wi~,h v~muolaeed c y t o p l a s m ,~nd distorted rmeleoli (complete Pla~ses 4 a,nd 6). These results show clearly ~ha~ eh[oroquirte inhibited tJ~e inil~ial cell mobiiizagion bu~ did not, iuhibil; cell migra.tion. The degree, of hydrat:ion of the corneas with ~he different t,reabmen~s a p p e a r e d t.o be similar, judged by t-he numbe.: of microgome ,~ections cub. Therefore, di{t'crence~. iz~ celt migrations o b t a i a e d wit.It 6he v a N o u s t,rea.~ment,s would a.ppear to be iztdependeng of cor.ne:d hydr'~l;ioa.

l

Zor~e. o. f . m obil;za;ion .

l

6

)

5 0

ZOne Of mi<:Jt.~tJOn

I

r

.~_

k oL

80

~ 4,9

4 O0

56.0

720 Distance

88 0

~,040

1200

(/z}

E~g. 6. O r g a n c u l t u r e . CSI =: c u l t u r e m e d i u m . C Q - ~ c o n t r o l m c d i u m q - o h t o r o q n h m d i p h o s p h a t ~ ( 1 + 1 0 - ' ,WI). * C o r o p a r c c l wil;h 0--72 h r C5t, t.ben 7 2 - 9 6 h r CQ. L a c k o f i n h i b i t i o n o f cell m i g r a . t i o u b y e h l o r o q u l n e ~L(tded ,~ftcr p e r i o d o f cell m o b i l i z a t - i m l ( ~ d d e d 72 b r foHowiug i n j u r y ) . Sl~.t:.is~ical a n M y s i s (see f o o t n o l c to T ~ b l e 1). T h e m is u o sig~fitlean+~ d;_q'erenee b e t w e e u t h e Lwo tree,tremors in e i t h e r t h e t o t M n u m b e r o f cells £otmd Pt t h e " z o n e o f mobHizatioD." o r ha t.he tot,~l n m n b e r o f c e l l s f o u n d iu t h e " z o n e o f migr~tiol~ ~'. :-kveeage n u m b e r o f cells p e r 80-/z w~d.e st,r i p iu " ' z o a e o [ m o b i l i z a t i o n " : I)-.-gfih r ()3[: i7,71 _~3-09; 0 - 7 2 h r C~I, t.hea 72--96 l i t C Q : I,-3.62:t:2.07, A . v e r a g e n u m b e r o f celts p e r S0-p. w i d e s t r i p in " z o n e o f mig,u~tion'" ." 0 - 9 g h r CSt : t 6 - 5 6 - - 3 . 8 7 ; I)-72 h r CM, t h e n 7 ~ - 9 6 h r C Q : 1;~,.97_-1:3.42.

The results i n d i c a t e t h a t t h e rl'lov£,lllO..ll~of Cf.)r]'l.f~al. COt/I~ect)~V(~ 1~,i$Sl]~ /ibroblasts into a cell-free zone tbllowing i n j u r y m a y be clearly divided into at least ~wo s|;eps: (1) a stage of cellular mobiliza~ior, which m.uy require I~._NA syl~thesis mid which is hzhibitcd by cllloroquine b u t hog b y colc hici,te, ,~nd (2) u stage of mJgra.tion, of t h e a l r e a d y mobilized cells. Migration is i a h i b i t e d by colchieine (1 x 10 -v ~/I) bu~ not by chIoroquine. ~.Ve ~re cm~tilmiag these s~udics on the mcchaai,sm o.f cell mobiliza~iot~ in ~esponse to i n j u r y w i t h analyses of I{NA polymerasc, RN-zK profiles ~z~d qu~Dtitative biocheznical aualyses of t h e c h a n g i n g e n z y m e p a t t e r n s d u r i n g mobilization.

Gf.;[,i, M O B I L [ Z A T t O N

AND M L G R A T I O N

2t

l i e [;:N R t~;N(,' 17.:

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