CHRONIC REJECTION is a major cause of late liver

Transcription

1 r' Chronic Livr Allograft Rjction and Oblitrativ Artriopathy: Possibl Pathognic Mchanisms S. Oguma' T. Zrb' B. Bannr' S. Bll2 T.E. Starzl3 and A.J. Dmtris' CHRONC REJECTON is a major caus of lat livr allograft failur. Gnrally accptd pathologic faturs of this condition includ chronic oblitrativ artriopathy (COA) and bil duct loss. Th mchanisms involvd in graft damag ar poorly undrstood. Bil duct loss has bn attributd to dirct ymphocytotoxic attack which sms rasonabl considring th histologic apparanc of rjction. Howvr ischmia du to COA has bn largly ovrlookd as a cofactor in th pathognsis of bil duct loss. t is known that th hpatic artry is th only blood supply to th intra-hpatic biliary tr. n this study w studid th ffct of ischmia. on bil duct loss by studying th rlationship btwn th dgr of COA and intnsity of bil duct loss. Furthrmor in ordr to gt a gratr undrstanding of th pathognsis of COA. w analyzd th linag of clls comprising COA lsion in hart. livr and kidny allografts. Spcial attntion was focusd on dtcting clls with phnotypic charactristics of dndritic cll (DC) linag. DC playa pivotal rol in th initiation of immunological ractions. MATERALS AND METHODS Sourc of Matnals and Slction of Cass Livr allografts.hich faild bcaus of chronic rjction.r rviwd (~ - 28). Th diagnoss of chronic rjction was basd on a charactnstlc chnical cours and confirmd b~ histologic analysis of th rmo'cd allografts. ~ormalli\'rs "'r slctd from autopsy cass n.hich thr \Oias no clinical or pathological vidnc of hvr disas. Tissu sampling for histologic sctions from th faild allografts and control livrs was prformd according to a prdfind protocol. Thr scctlons "r takn from th hilum. two sctons from th lft lob. and thr scllons from th right lob' Th thr scllons takn from th hilum of ach livr.r xamind blindly and mdpndntly by two of th authors for th dgr of narrowing of hpatic artrial branchs and th prcntag of vssls affctd. f gratr than 70'k of th artris dmonstratd mor than 75'1 stnoss. th cas was labld as "svr COA". Convrsly. n "mild COA". gratr than 70'1 of artris sho\oid lss than 35% narro.mg (n - 10) Th intrmdiat cass (n - 10) wr xcludd 10 accntuat any possibl diffrncs btwn th two xtrm groups. v.hich may rsult n rcognition of th rol o( schmia 10 bil duct loss. Gnral patint data is shown in Tabl. Th avrag ag of allograft patints \Oiith mild COA was 31.9 yars compard to 12.6 (or th patints with svr COA (P > 0.05). Th avrag graft survival was much shortr in thos with svr COA (7.7 months) compard to thos with mild COA (33.7 months) (P < OOS). Complt MHC typing of both donor and rcipint was availabl in only 8 of th 18 cass analyzd. No particular trnd \Oias notd in this small sampl. All patints wr maintaind on baslin immunosupprssion of cyclosporin and stroids. Episods of acut cllular rjction wr tratd with a stroid bolus or rcycling doss. f stroids wr unsuccssful. OKT3 (Ortho pharmocuticals. Raritan. NJ) was usd. Histomtrical Study for Artrial Loss Th prsnc or absnc of any artry within th confins of th portal tract and th siz of th only or largst artry in th portal tract was rcordd. This assssmnt was rpatd for all portal tracts in vry sction of th slctd cass (avrag portal tracls/cas). Th siz of th artry was dtrmind by masuring th shortst diamtr of th vssl to th outr :ll.lnt of th mdia using an ocular micromtr. Th prcntag of portal tracts \Oiith artris and th siz distribution of th only or largst artry was calculatd according to a prdfind rang of artrial diamtr « ~ 75 microns). Histomtrical Study for Bil Duct Loss An analysis similar to that don by Nakanuma was also prformd to dtrmin th xtnt of duct loss and siz of vanishd ducts. 1 n brif. it is kno\oin that artris and bil ducts of a similar diamtr occur in paralll in th portal tracts of th normal livr. Prcis masurmnt of th diamtr of th only or largst artry in ach portal tract was rcordd as dscribd abov. Tracing an imaginary circl 3 tims th rcordd artrial diamtr from th xtrnal mdia of th vssl circumscribd th ara in which th only or largst accompanying bil duct should b dtctd. Th prsnc or absnc of a bil duct within this ara was thn rcordd for ach portaltrat of vry sction. Th rsult was labld according to th prdfind rang of artnal diamtr for ach livr and xprssd as th rat of bil duct artry (BD-A) paralllism. t must b notd that th rat of paralllism was dtrmind onl~ for thos portal tracts which containd artris. Portallracts ithout artris and bil ducts wr ;t;c1udd from th dtrminations of BD-A paralllism. Study of Chronic Oblitrativ Vasculopathy (COA) Hart (n - 7) kidny (n - 9) and livr (n - 7) allografts which faild from chronic rjction r rviwd. Formalin fixd and paraffin mbddd blocks "'r CU at 4 microns and routinly staind with hmatoxylin and osin. Masson's trichrom and Vrhoff/Van Gison for clastic tissu. Paraffin mbddd sctions wr also subjctd to staining \Oiith various antibods using th avidinbiotin-<omplx (ABC) mthod. From th Dpartmnt of Pathology' and Surgry.' Prsbytrian Univrsity Hospital and Dpartmnt of Epidmiology.' Graduat School of PubliC Halth. Univrsity 01 Pittsburgh. Pittsburgh. Pnnsylvania. USA. Addrss rprint rqusts to A.J. Dmtria. MD. Dpt of Pathol ogy PrsbytTlan UnivT!lty Hospitat. Pittsburgh. Pnnsylvania USA. () t989 by Applton & Lang. nc. 0004' 1345/891$ Transplantation Procding Vo121. No 1 (Fbruary). 1989: pp

2 2204 OGUMA. ZERBE. BANNER ET Al Tabl 1. Dmographic Data and Original Hpatic Disas Grall Original Hapali Osa Survival Ag C.ss (months) Sx PSC CAH HCC SMN PSC AAO OC Mild (n - 10) o o (33.7) (31.9) F:M - 8:2 Svr (n - 8) o o (7.7) ( 12.6) F:M - 2:6 PB - Primary Biliary Cirrhosis. CAH - Chronic Activ Hpatitis (Non A. Non B). HCC - Hpatocllular Carcinoma. SMN - Submassiv Ncrosis. PSC - Primary Sclrosing Cholangitis. AAD - Alpha -Antitrypsin Dficincy. 01 C - Othr Congnital. Antibodis for lysozym. SOO protin and T-ymphocyts (UCHL-l DAKO. Santa Barbara. CAl and for actin (Biognx. San Ramon. CAl wr usd. mmunoauorscnt staining was also prformd on frozn tissu mbddd in OCT compound using a simpl indirct tchniqu with appropriat Auorscinatd scondary antibodis. Primary antibodis includd HLA-DR and Lu 4 (CD3) (Bcton Dickinson. Mountain Viw. CAl. RESULTS Analysis 01 Bil Duct Loss in Chronic livr Rjction Distribution and Si: of Portal Tract Artris. Th prcntag of portal tracts containing artris or artriols wr compard for ach of th 14 subjcts with no OA (controls) th 10 subjcts with mild OA and 8 subjcts with svr OA (Figur ). Ovrall. 90.9'1( of th portal tracts in controls. 61.3'1( of th portal tracts 10 cass with mild OA and 25.3'1( of th portal tracts in cass with svr OA had at last on artry. Th logistic rgrssion modl was usd to dtrmin hthr thr was an association btwn svrity of OA and th prsnc of artris in th portal tract J A statistically significant diffrnc btwn ach pair of groups was found. Figur 2 shows th prcntag of portal tracts in which th largst or only artry was d : 75 microns in diamtr for ach subjct classifid by th dgr of OA. " Partlt Tmts ~~ ~ ~ 21 No COA H t... Mild COA Svr COA Fig 1_ Th prcntag of portaillact. from normal control and fan.d allograft livr. ontalnlnt at la.t on dntlflabl. art.ry hown. Not. th ab.olut to of dntlflabl. portal rtrl n faild.10graft Ovrall th distribution of th siz of th largst or only artry in portal tracts with at last on artry was significantly (P < 0.001) diffrnt among groups using th chisquar tst of association for indpndnt sampls. Pairwis comparison of th thr groups using th sam statistical tchniqu dmonstratd that th distribution of th siz of th largst or only artry was significantly diffrnt (P < 0.00 ) btwn ach pair. Among 81'1( of th portal tracts of controls. th largst or only artry was lss than 35 microns compard to about 58% of th portal tracts in cass with mild OA and 41 q of th portal tracts in cass with svr ~A. At th othr xtrm. only 47r of th portal tracts in controls containd a singl or largst Jrtry. 75 microns or gratr in diamtr. ranging from 1.9<; to 7.9'1 among th 14 subjcts. Six prcnt of portal tracts in cass with mild OA (rang 3.1'"; to 12.6'1) and 14'"; of portal tracts in cass ith svr OA (rang 3.17 to 30.8r;) ths diffrncs ar also statistically significant (P < Bil Duct-Artrial Paralllism. Th man rat of bil duct-artry (BD-A) paralllism vrsus artrial Sil. classfid by th dgr of OA is shown in Figur 3. Thr S a statistically significant (P < 0.001) trnd toward dcrasing paralllism with dcrasing artrial sil in both th mild and svr ~A. using a tst for linar trnd.' USing th chi- 01 Pilrtli Trlcts : t --'- J sn. ll rrn. 11" s. C» all all all all till C» C» all <35 Microns Microns Microns 75+ MCrons Fig 2. Th prcntag of portal tr.t ccordlng to th.z. of th larg t or only portal artry. r.pr.ntd. Not. th r.latlv. d.r n.mall port. artriol. and rlatlv. ncr n larg.r artrl

3 _as 3' S nr_' m ALLOGRAFT REJECTON AND ARTEROPATHY T.lllls. 100 &D " :.- 20 : -'- l~ ;i'i l~ """!if" ~ ~ 1l1."1ii"'" C <J5 Microns Microns Microns 75+ Microns Fig 3 Th prcntag of portal tract n which a bil duct la dntifid along with th corrlpondlng artry hown Not th.vr. 10 of small bu duct. n th faud allografta. Duct 10 corrsponding to largr artrl. la.n only n th allografts with svr COA. squar tst for indpndnt sampls. statistically significant diffrncs (p < 0.01) for BDA paralllism btwn groups (controls vs. mild COA. controls vs svr COA. and mild COA vs. svr COAl wr found in all siz catgoris. xcpt btwn controls and mild COA for artris gratr than or qual to 75 microns in diamtr t must b rmmbrd that th rat of BO-A paralllism was dtrmind using only thos portal tracts which containd artns. Bil duct loss with associatd artry loss was not spcifically tabulatd. Thrfor. absolut bil duct loss. particularly involving th smallr ducts (<35 microns) is vn gratr than that xprssd as a dcras in th rat of paralllism. Analysis of Chronic Oblilt!fali\ Arlriopath~' Light Microscopic Faturs of COA. \iany histopathologic faturs wr common to th affctd artris of all of th organ allografts. Th vssls most svrly affctd by COA wr thos of mdium siz rang. which wr oftn th first and scond ordr branching vssls of th particular artrial trs. Th intima containd a varying dgr of mononuclar innammation. subndothlial foam clls and apparnt smooth muscl cll prolifration. Plasmacytic. nutrophilic and osinophilic inflammation was lss commonly prsnt xcpt in th cardiac allografts. Th intrnal lastic lamina was oftn disruptd in artris of kidny and hart allografts; in th livr it was mostly intact. Th apparanc of th mdia and priadvntitial spac was similar in all of th organs. Th mdia was infiltratd to various dgrs by macrophags lymphocyts and foam clls. Th inflammatory clls wr locatd in aras of dgnrating smooth muscl clls. Cuffs of priadvntitial inflam' matory clls wr sn around affctd artris. Ths priartrial clls appard to invad into th mdia from without. as if th mdial smooth muscl clls wr th targt of cllular mdiatd immunologic injury. Finally although w did not xamin squntial biopsis. thr appard to b a tmporal progrssion of th changs. Early lsions wr 2205 rich in inflammation and accompanid by smooth muscl cll prolifration. Thraftr. th inflammation gradually subsidd and th intima bcam sclrotic. Phnotypic Analysis by mmunohistology Various admixturs of macrophags (lysozym +) T-ymphocyts (UCHL - J +). prolifrating smooth muscl clls (actin +) and SOO protin positiv clls wr sn in th thicknd intima of involvd vssls. Th variation. to som xtnt. was dpndnt on th "ag" of th lsion and organ. A dtaild dscription of th findings prsnt in ach of th allograft organs is prsntd lswhr. On closr xamination. two typs of intimal S 00 + clls wr sn. Th first was round to oval with an oval. clf td or angulatd nuclus. Ths clls wr uncommon. Whn prsnt. thy wr locatd immdiatly subjacnt to th ndothlium. Th scond typ of Sioo + clls was dndritic shapd and usually found in contact with narby T-ymphocyts and/or plasma clls; occasionally macrophags (Figur 4). Th Sioo + dndritic clls wr usually locatd dpr in th intima. nar th intrnal lastic lamina. Likwis. activatd and prolifrating smooth muscl clls wr sn in two locations. immdiatly subjacnt to hyprtrophid ndothlial clls and dp intima. nar th disruptd lastic lamina. Macrophags. T-ymphocyts and rar S 00 + dndritic clls wr also sn in th mdia. Th cuffs of inflammatory clls in th priadvntitial spac consistd prdominantly of macrophags; T-ymphocyts wr lss common. S 00 + dndritic clls and/or trminal nrv twigs wr th minority group. but wr also asily rcognizd in th priadvntitia. Srial frozn sctions cut at 4 microns conscutivly ~~ ".. ~. ~ ~.. t ~- & tt ;; ~~ ~ i.- r: - ~ Fig". S 100 protin polltlv dndritic hapd cll n th thicknd and nft.mld ntlm. n br.nch of.n allogr.tt coronary artry )( 400.

4 - - " -" : - -. ~:'.. ' OGUMA. ZERBE. BANNER ET AL staind for SOO protin and HLA-DR wr x.amind. Round H LA-DR positiv clls wr sn in th intima. Othr HLA-DR positiv clls wr spindl shapd. Howvr. th morphologic rsolution in th frozn sction immunofluorscnt stains wr not of sufficint quality to sparat smooth muscl clls from dndritic clls. Nvrthlss. th longatd HLA-DR positiv clls wr in th sam gnral location as th S 00 + ons. DSCUSSON Analysis of th data rvals that quantifiabl bil duct and small artrial loss ar faturs of chronic livr allograft rjction. Th dgr of damag to both artris and bil ducts incrass as th siz of ach dcrass. Additionally. th svrity and siz of artrial loss and vanishd ducts is dirctly proportional to th dgr of COA. Bil duct loss was rcognizd undr two circumstancs. On occurrd whn an artry without a corrsponding bil duct was found. Th othr was rcognizd whn thr was loss of both th artry and th bil duct. Thrfor. th rsults of this study offr indirct. but supportiv vidnc that two mchanisms may b rsponsibl for bil duct loss: dirct immunologic attack and ischmic injury. Th absnc of a bil duct whn an artry is found is supporting vidnc for a dirct immunologic attack on th biliary pithlium. Othr vidnc also suggsts that a primary immunologic insult to th biliary pithlium occurs. Th histologic apparanc of acut cllular rjction is charactrizd by lymphocytic invasion and vidnc of dgnrativ changs in th biliary pithlium.' Although biliary nflammaton is lss svr in chronic rjction. carful light and lctron microscopic xaminations of livrs undrgoing chronic rjction rvals lymphocyts in clos contact with dgnrating bil duct clls. lo Th loss of ducts tn conjunction with artrial loss is indirct but suggstiv Vidnc that ischmic injury contributs to bil duct loss. W also found that th svrity of duct loss and th siz of th "vanishd" ducts is dirctly proportional to th dgr of COA. Sinc th biliary tr in humans rcivs its blood supply xclusivly from th hpatic artrial systm..12 COA and priphral artrial loss compromis blood flow to th dpndnt ducts. Two mchanisms may b rsponsibl for small artrial loss in th portal tracts. Small portal artris may b subjct to ischmic ncrosis bcaus of mor proximal vssl occlusion. Th othr possibility is that small portal artris may b damagd by dirct lymphocytic attack. Rgardlss of th mchanism. th rsult is th sam: diminishd blood flow to dpndnt bil ducts. ) Th rsults of this study also offr som insight into th puzzling qustion of whthr primary biliary cirrhosis rcurs in livr allografts. Nakanuma also discovrd that thr was a dcrasd rat of BD-A paralllism for artris and microns in PBC livr compard to controls.2 n contrast. w found that th loss of bil ducts in th livr allografts was mor rstrictd. Only thos ducts with corr- sponding artris lss than 75 microns in diamtr wr missing. A dcrasd rat of BD-A paralllism for largr artris was sn only whn thr was coxistant svr COA. Howvr. all of th patints transplantd for PBC in this study fll into th group with mild COA. Thrfor. non of th patints with PBC prior to transplantation who wr includd in this study dmonstratd loss of largr ducts as shown by Nakanuma. Furthrmor. largr xcrtory bil duct damag similar to that sn in sclrosing cholangitis is obsrvd in livr allografts with COA but not in PBC. COA is not only a problm in livr. but all othr solid organ aliografts. 16 W thrfor invstigatd th phnotypic charactristics of clls involvd in COA lsions. Th artris affctd by COA dmonstratd a markdly thicknd intima. which containd activatd smooth muscl clls. macrophags. T-ymphocyts and S 00 protin positiv dndritic shapd clls (DCs). Thy wr prsnt whn ongoing inaammatory dstruction was occurring and bcam lss frqunt as th inaammation subsidd and th organ assumd a "burnd out" apparanc. Th dtction of dndritic clls in th inaammd intima might b significant for undrstanding th dvlopmnt of COA. bcaus thy ar fficint accssory clls. promoting T-ymphocyt dpndnt immun ractions.).11 nformation on th rol of dndritic clls in th immun cascad is still limitd. sinc thy hav only rcntly bn studid to any xtnt. DCs ar bon marrow dril'd and constitut up to 0.5'( of priphral blood mononuclar clls Thy ar xtrmly fficint accssory clls. normally xprss high dnsity surfac HLA-DR antigns and ar potnt stimulators of mixd lymphocyt ractions; tims gratr than macrophags in ths rspcts.:::) DCs may also xprss complmnt rcptors. but ar unabl to "procss" particulat antigns bcaus of th rlativ lack of ndoplasmic and lysolomal activity..:4 Thrfor. DCs may associat with typical macrophags whos phagocytic and ysozomal activitis compnsat for ths rlativ dficincis. Onc an ffctor cascad is dirctd against som antigns. rlas of lymphoklos and local production of luko trins in th inaammd intima may induc smooth muscl cll prolifration and collagn dposition which might b ultimatly rsponsibl for luminal narrowing. 1l Whn stimulatd. smooth muscl clls xprss class antigns which may b a potntial targt for humoral and/or cllular immunological ractions.: A vry similar scnario may occur during th dvlopmnt of athrosclrosis 10 th gnral population. Finally. ths studis dmonstrat that oblitrativ artriopathy is a major problm in livr transplantation. similar to kidny and hart allografts.' ll Thrfor. sinc arly graft and patint survival has incrasd with advancs in th fild of transplantation. attntion must b focusd on lucidating th pathognsis of "graft athrosclrosis." A bttr undrstanding of this problm will prmit th trial of novl. and prhaps ffctiv thrapy for this prsntly untratabl condition.

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