I Histopathologic Alterations Associated with the Transplanted i

Homologous Dog ~ iver '

I)cp~zrlrrtt,nts of PnlItoIogy anrl Sttrgrry, Northwestern University Medical School, Chicago 1 1 , Illinois

INTIIODUCTION •

The technical 1)roblerns and clinical course after transidantation of the homologous tlog liver have Iwcn recently described (Moore ct al., 1960; Starzl et al . , 1960, l ( i ( ) l ) . 'l'hc rcljcction pattern was similar to that seen with other vascularized 1io1nogr;tits. Survi\?;il averaged six to ten days but was as long as twenty and a 11:llf tliiys (Starzl ct at., 1961 ). The present study characterizes the histopathologic changes in SO animals in which homotransplantation of the liver was performed.

Aitcr homografting of various organs, pathologic changes have not been generally demonstrable in the host organs. Sinionin et al. (1953) found no consistent altera- tions in the spleen, liver, heart or adrenal glands after renal homotransplantation. In a sttidy of liver homografts, JIoore (Moore et al., 1960) did not mention a systemic rcslx)tise. I n the present study, a marked systemic response in a variety of host or,<nns ivill bc described wliicli, it is tliouglit, was indicative of total reticuloendothe- li:il participation in the rejection of the liver homograft.

'I'lle te~hnique of liver transplantation was previously described (Starzl et al., I ( ) ( ) O ) . The graft \\'as placed within the liver fossa after removal of the recipient dog's livcl-. Sl'lcncctomy was performed.

.-\utol)sics \\.ere performed on all animals and the specimens were fixed in furmalin. IIematosylin and eosin stains were used routinely. Periodic acid-Schiff, hIasson, iron reticulum and oil red 0 or Sudan black B stains were also employed to aid in inter- prctin:: the sequence of events in parenchymal and mesenchymal tissues but data from these stains will not be presented a t this time since they would appear to warrant a heparate report.

RESULTS

.ALTERATIONS RELATED TO SURGICAL ARTEFACTS O F THE TRANSPLANTATION PROCEDURE

\Yell-defined outflow block (Fig. I ) , patterned by constant hepatic vein and \~:~siable distal ' (central) venous congestion with marked perivenous lymphatic

1 .\idctL by Grant A-3176 from the United States Public Health Service, National Institutes of IIc.ilth, Bethesdn, Maryland.

"I'rt-sent address: St. Mary Hospital, 36475 West Five Mile Road, Livonia, Michigan. Jlarkle Scholar. Present address: University of Colorado School of Medicine, Denver,

Colorado.

tli\.ii,ilioll ~~o1111110111!. I * I ~ : I ~ ; I C ~ C ~ ~ ~ X C ' ( I 11111 li\'('l.s oi [11os(' ;11li11);11s s ~ ~ r v i v i n ~ 4 8 ~~~~~~~s nr A loi~on~~clc;rr , - c a l l ;,! 1ct;s. I:oc;~l ;11-c;~?; oi ccilti.;~l \.(*in ci~(lolllcli;~l ~lccl.o.sis ancl ol)sll.~~ction occurretl. ' ' scantily prcsent a t 4 ( 1 licsi,iti:~l c~it lcncr of oulllo\v 1)lock was somctimcs present in long-term survivors.

were ovoirl or irrc-zlll;l Such c11:iugc.s i~rc. I;no\\.i) to be dtie to hepatic ischeinia rather than a specific effect of eccentric deeply basol)' l ~ o n l c ~ t i ~ n ~ l . . : ; ~ l a ~ ~ t n t i o n (St;~rzl c t a / . , 1960).

cytoplasm. The aggre-i 'I'\\u sl'cciilc control proccdurcs were carried out. In the first, cholecystojejunos- 1 ting elongated cells i n

tonly \\-it11 entn-o-cnterostorny was performed after ligation of the common duct in , tion, Similar isolated ce four dogs, since this was the means of provision for internal biliary drainage in the i ti~;irls~~lnnts. ?'he dogs \\.ere sacrif~ced a t 5 , 13, 14, and 15 days. Liver function st~idic>s \\.ere uniiol.n~ly normal. Histologic stlldies of the liver were either normal or rcvc.;~led a minimal periportal polymorphonuclear leukocytic infiltrate always with an undistorbed hepatic architecture. All other orjians were histologically normal.

FIG. 1. Liver from dog surviving 10 hours. Notc portal venous and lymphat ic engorgement characlcristic o i outilow block, X 43.

I n the second, four dogs had autografts of the liver, employing the same technique as \\it11 homografts. These animals were sacrificed after 7 to 15 days. Liver architec- ture \\,as completely preserved with no evidence of infiltrate. Focal myocardial infarcts were present, but there were no other changcs in host organs.

.+\LTER~\TIONS SOT RELATED TO SIJRGICAL ARTEFACTS OF THE TRANSPLANTATION ~ R O C E D U H E

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Generalized Ki~pfer cell enlargement with increased nuclear basophilisni was noted a t 10 hours (Fig. 2 ) . This change was constant throughout all subsequent survival inter\lals. Focal K ~ ~ p f e r cell proliferation did not occur. Endothelial activation was present in all sized vessels as evidenced by increased nuclear basophilism and in- creased size of the endothelial cells a t 18 hours. Endothelial and adventitial prolifera- tion was seen regularly after 5 days. Occasionally, arteriolar hyalin degenera!ion was manifest a t this time (Fig. 3 ) .

FIG. 2 . Liver fror

Parench!.rnal archit1 surgery. Shortly after of hepatic cell loss dc days, undisturbed arct ma1 loss after 5 days cases \\-as virtually (

behaved in a similar n

Despite very esten reticular matris to be

~ l o ~ i ~ ~ ~ \ ~ ~ ~ ~ l ~ ~ ; ~ r ' ~ ' ~ 1 1 ;~l:si'cg;l~t~s \\.l)icl~ \\.crc nl)st.nt (111rii)g 111~ ' 1.11.st 45 11o11rs and s<;lniil>. l~i '~ 'Si ' i I l a t 4 tl;iys \vcrc. very nulncrous ilCler 5 (lays (Fig. 4 ) . Those cclls \\.crc o\r,iti or irrcgril;~rly sll;i[)ild and reseni1)lctl p l a s ~ n a ~ ~ ~ . t c s . They ~)ossessed an prL.cl~itri<- iltycjll!. basol~hilic ovoicl nucleus ; ~ n d \rari;~l~le i~lllou~lts of light b;rso1)hilic c!.t~~l>l;i.~m. The ngg,.rlxgntes were commonly intimately associated with fixed ~>rolifera- tin< clong:~rcd cclls i n perivascular areas or portal tract sulq~ortive tissue. In addi- [ion, similar isolated cells were present difiusely \ ~ i t h i n the hepatic parenchyma.

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- ---- - - J FIG 2 . L~vcr from dog surv~ving 10 hours illustrating Kupfer cell activation, X 26.3.

Parench~mlal architecture was well-preserved during the first 4 or 5 days after surgery. Shortly after the above-described mononuclear infiltration, varying degrees oi hepatic cell loss developed most prominently around the central vein. After 6 (lays, undisturbed architecture was noted in only one dog. The degree of parenchy- mal loss after 5 days was crudely related to the period of survival and in several cases was virtually complete (Figs. 5a and b). Intrahepatic ductal epithelium behaved in a similar manner, being extensively sloughed after the fifth day.

Despite very extensive parenchymal destruction, reticulum stains revealed the reticular lnatris to be intact. Fatty alterations presented no consistent pattern.

Frc:. .i. Artcriolc oT liver showing cndothclial and pcrithclial proliferation with focal hyaline 1 FIG. 4 . Liver: 10 tl;iy +I

dcgcncrnlion, Y, 263. I plasrnacytic.

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FJC. 4. Liver: 10 day survival, X 263. Periportal and parenchymal infiltrate is primarily 1)lasmacyLic.

I)ONI\T.I) X. Dl<OCK AN11 TlIOhlAS Ir:. STARZL

FIG. 5 . Gcncral hcpatic architccturc: (a) 9 day survival, X 42; (b) another dog, 8 day survival, X 42.

FIG. Sb.

I)OS;\l.I) I:. I1ltOC1< r\NI) TIIORII\S 15. STAItCL

FIG. 6. Lymph node from dog surviving 20 days: (a) cortical depletion, X 82; (b) cortical cell typc primarily mononuclear and plasmacytic, X 232.

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. 190 I)ONI\LI) W. nKocli I\NI) THOMAS I:. STAIUL

phagic rcticlllllm cells, sc atlvcntilial prolift I.!r~rplc ~rodcs : l'rogrcssive changes occurrcd in the enlnrged mediastinal, ccrvical f

:ind n~cscntcric lymph nodes. As early as the fifth day definite cortical thinning was ~ W S C ' I I ~ with decrcascd nuillbers of follicles. The follicles were discrete and composed

By the eighth day, m \\rere complete1 y absent ( p s e d of densely packed p r c v i ~ u ~ l y abundant lyn within sinusoids and lyr cells occasionally actively Kidney: Within 4 da

rnacytoid cells were seen principally cortical. At 1:

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lial and adventitial proli during the first 6 to 7 da At this time definite a r centuation of nuclei in and periadrenal suppa proliferation with periva:

Lung: After 4 days, nine cells in thickness, I

with a hyperchromatic : bizarre giant cells were areas of hyaline thicken

i\lononuclear cell infil supportive tissue and a1

Bone marrow: P0st-1 animals surviving more

FIG. 7. Kidney: (a) plasmacytic aggregation, X 97; (b) tubular basement membrane activa- 1

tion, X 97.

prim:lrily of s1naI1 round tlecply basophilic lyniphocytcs and occasional reticulum cdls. The adjacent cortical area anrl n~edullary cords revealed a loose pattern of

1

plasmacytes and larger similar but irregular plasmacytoid cells. I n addition, significant numbers of small round lymphocytes and a small number of nonphagocyti- zing reticulum cells were present. The congested sinusoids contained many macro-

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l \ l l ;~,~ic rt.riz11lt1111 ct,lIs. solllc of \vliich contained heniositlerin. Definite endothelial ;irl t l t)r ;1tf\-c11 t i t ial l,rolilcrntioo was present.

liy thc c i ~ h t h day, recession of follicles had ~)rogresscd, and occasionally these \\.t,l.e cnml~l~tc ly absent (Fig. 6a). The thin cortes ant1 medullary cords were com- ;,owcl oi tlt~rlst-ly paclit'tl plasnli~cytes arid larger plnsm;icyloid cells (Pig. 61,). The Irc\.iously ;~l)urltlant lynlld~ocytes were greatly tlccrcascrl in numbcr. The cells \\.itlli~l silll~soi(ls and lymphatic channels were almost esclusively large reticulum cells occ;~sionally actively phagocytic and often with PAS positive cytoplasm,

Kitltrc~t: \iTithin 4 days small focal aggregates of plasmacytes and large plas- macytoid cells were seen in the periglomerular and perivascular areas. These were principally cortical. At this time occasional vessel wall edema was noted. Endothe-

FIG. S. Perircnal plasmacytic and rnononuclcar infiltration, X 97.

lial and adventitial proliferation occurred. The cellular aggregates developed 'slowly tlurin: the first 6 to 7 days after which cellular reaction was quite marked (Fig. 7a). A t t h i h time definite areas of thickening of tubular basement membrane and ac- c.cntuation oi nuclei in this area were noted (Fig, 7b). Concomitantly, perirenal ~ i i d periadrenal supportive tissue demonstrated capillary engorgement and/or proliferation with perivascular aggregation of plasmacytes and lymphocytes (Fig. 8 ) .

Lznrg: After 4 days, alveolar wall proliferation, diffusely or as nodules three to nine cells in thickness, occurred. These cells were ovoid or spindle-form frequently with a hyperchromatic nucleus. Mitoses were infrequently present. Multinucleated !)izarre xiant cells were irregularly formed in the alveolar wall (Fig. 9). Occasional ; i r c n ~ of hyaline thickening of the alveolar wall were present.

llononuclear cell infiltrate, predominantly plasmacytic, involved the peribronchial supportive tissue and alveolar walls.

Boilc i i l u i r o a : Post-mortem bone marrow studies were performed routinely on animals surviving more than 4 days. All revealed increased plasmacytes (Fig. 10).

I)ON,\LI) l i . DROCI< A N D THOhfAS E. STAHZL

FIG. 9 . Lung. Focal alveolar wall proliferation, mononuclear cell infiltrate and giant cell iormation, X 225. I

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ccli FIG. 10. Bone marrow. Note increased plasmacytes, X 392.

~&~ 1 > 0 8 A I . I > W, nl<OCK A N D TlIOMAS r . STAH7.1,

FIG. 11 . Small intestine. Mucosal slough and mononuclear infiltration, X 42.

(;o.~lminlcsli~rnl I m c

~;ln;~l l ir~lrstinr n.oc , tion o f ~)lasmacytrq T h e ~~lasniacyt ic inlillr I I ). l ' he s u l ) m ~ ~ c o i ~ \ with areas of intrritil ulcers were present i n perforation had occurr

Other organs arrd arteries and veins, ;I

revealed small myocnr and similar to those survivors the infarcts lowed by minimal inli

T h e occurrence of donor organ is uniic c.? al., 19h0) , kidney liver (Moore et al., infiltrate is seen in fr this cellular acc~rmul; functional detcriorati early a s the fiith da j

T h e origin of the homograft has been (1953), working wit donor and not in the t h e host. Hume el I

ever, that kidneys hc o f infiltrate. The 1 thought by Hurne ct origin and not indic, (1960) showed tha prompt appearance c host.

Contemporary eq strengthened our be1 primarily of Iiost ori; 24 hours before tr mononuclear infiltra were untreated.

I f the cellular infi of the similar infilt nodes, bone marrol scribed with most I

( ; , I . C I I . ~ J ~ / I ~ I , . V ~ ~ I I ~ I / tri1t.t: C'o~igvstive (-II;IIIX(>S with ;lssoci;~te(I I I I I I C O S ~ I s l o u ~ h o i the .ril:~ll i~i(c~.sti~ic \\.;IS ;I constant li~~cling reg:irdless ol si~rvival time. Mucosal inliltra- [ion oi 1)l;1sni;1cytes ; ~ n d less numbers of lymphocytes was present in all animals. 'I'lic ~'l;~sniacytic infiltrate increased to a maximum a t the fourth to fifth day (Fig. I 1 ) . 'l'hc. sul)~nucos;~l and muscularis of the entire tract was markedly congested \\.it11 arcas oi interstitial edema. Single or multiple discrete punched out duodenal t:lcers \vcrc. prcscnt in many animals surviving more than 4 days; and in some dogs I)crior;ition had occurred.

0tlri.r orgurrs arrrf tissrdcs: Adrenal gland parenchyma, skeletal muscle, large arteries and veins, and skin revealed no significant alterations. Numerous dogs revealed small nlyocardial infarctions which were ofterl limited to the right ventricle

t and sinlilar to those provoked in control animals. Initially in the 10- to 24-hour survivors the infarcts were characterized by focal necrosis. This typically was fol- lowed by minimal infiltrate with early organization evidenced in the longer survivors.

DISCUSSION

The occurrence of mononuclear, lymphocytic and plasmacytic infiltrates in the tlonor organ is uniformly cited in early phases of whole organ [spleen (Moore r t trl.. 1960), kidney (Dempster, 1953; Hunie et al., 1960; Simonsen e t al., 1953), liver (Moore ct al., 1960; Starzl et ad., 1961)l homotransplantation. A similar inliltrate is seen in freegrafts of skin (&ledawar, 1944). After the third to fifth day, this cel1ul;ir accumulation becomes accelerated. Various degrees of parenchymal and iunctional deterioration occur. Histologic evidence of rejection is well established as e;lrly as the fifth day.

Tlie origin of the mononuclear, lymphocytic and plasmacytic accumulation in the Iiomocraft has been of considerable interest. Simonsen et al. (1950) and Dempster ( 1953). \vorl<ing with renal transplants, suggested that these cells originated in the donor and not in the host and represented an effort of the kidney transplant to reject the host. Hume ct al. (1960) and JIannick et al. (1959) recently showed, how- ever. that kidneys homotransplanted for long periods to irradiated hosts were devoid of infiltrate. The lack of donor organ infiltrate under these circumstances was thought by H u n ~ e et al. (1960) to be strong evidence that the infiltrate is of host oriein and not indicative of a graft versus host reaction. Conversely, Hume et al. (1960) showed that massive irradiation of the donor kidney did not present the prompt appearance of a mononuclear infiltrate after homotransplantation to a normal host.

Contemporary esperience in this laboratory (Starzl et al., 1962a) has also itrengthened our belief that the cellular infiltrate in the rejecting liver homograft is primarily of host origin. Donor livers were subjected to 1000-1200 r irradiation 18 to 24 hours before transplantation to normal hosts. The tempo and character of mononuclear infiltration in these grafts was no different than when the donor tissues \\,ere untreated.

If the cellular infiltrate in the liver graft is of host origin, what is the explanation of the similar infiltrates to be found in a variety of host tissues, including lymph nodes, bone marrow, lung, and kidney? These host changes have not been de- scribed with most homografts, and have only been seen after whole organ hepatic

( Si.iri.1 1.t (11.. 1 Q O 1 ) or nil11 til)lc org;rn (Sti~rzl ct t r l . , 19621)) lio~notr;t~ls~,lant;~tio~i. i'o~i~icicr;~l)lc cvidcncc intlicatcs that these ch;lnges are part of a host response to the m;~ssivi, ;Intigenic stimulus of a bulky graft. The cellular aggregation is limited to thosc tiss~~c,s know11 to have a reticuloe~idothelial potential. The involved tissues (lo not 1111tlrrgo ir~nctional deterioration as wor~ltl be exlwctrtl in a graft versus host rc;tction. I;in;~lly. imnlunolo~ic paritlysis of the tlonor liver by irradiation with 1 0 0 0 to 1 2 0 0 r docs not ;~l)olisli the host response (Starzl et al. , 1962a).

'I'hc dcmonstrntion of gcneralizetl host reticulocntlothelial participation in the rcjcition oi I;~rgc homografts provitles a link between the rejection process ant1 ccrt;~in other hyl,ersensitivity phenomena. For example, Bjornboe and Gormsen (194.3) have shown that prolonged ant1 intense stimulation with bacterial and ~xotcin antigens evokes a gener;tlized reticuloendothelial response similar to that 5cc.n wit11 liver liomografts, ant1 these observations have been extended by Kojima (1950) to include lipid antigens. In their experiments, Bjornboe and Gormsen ( 1913) showed :t correlation between host reticuloendothelial activity and increases in g;1111111;1 glol>ulin, and concluded that the plasma cell aggregates were the chief 5oul-cc of this protein fr;tction. Working with liver transplants, Kukral et al. (1961) have >llo\vn a similar rise in galnnia globulin during the rejection phase.

SUMMARY

-HomotmnzpIantcd li\.ers in dogs developed mononuclear, Iymphocytic and plasmacytic infiltra- tion and hcpatic cell degeneration roughly paralleling survival time.

Estcnsivc histologic alterations of host rcticulocndothelial structures occurred. Proliferation and infiltration of mononuclear cells, principally plasmacytes, were noted in lung, kidney, perirenal supportive tissue, bone marrow, and lymph nodes. Lymph nodes, in addition, were characterized by cortical and iollicular depletion. These changes were considered to reprrsent extensive host reticulocndothclial mobilization coincident to liver homotransplant rejection. The relation between these alterations and those found in other hypersensitivity states is discussed.

REFERENCES

B ~ ( ~ K S I ~ O I : . >I., and G o ~ x t s e s , H. (1943). Experimental studies on the role of plasma cells as antibody producers. Acla Patltol. hlicrobiol. Scand. 20, 649-692.

DEXII>STER, W. J . (1933) . Kidney homotransplantation. Brit. J. S~rrg. 40, 447-465. '

H v a i ~ . I>. ST., J a c ~ s o s , B. T., ZUKOSKI, C. F., LEE, H. M., KAUFFMAN, H. M., and EGDAHL, R . H. (1960). The homotransplantation of kidneys and of fetal liver and spleen after total body irradiation. A n t i . S~rrg. 152, 334.373.

Iiojrar.\. &I. (1960). Morphologic changes accompanying RES stimulation. Ann. N.Y. Acad. Sri. 8 8 ( 1 ) , 196-202.

KI;KR.\L. J . C., LITTLEJOIIN, M. H., BUTZ, G. W., JR., and SIARZL, T . E. (1961). Biochemical studies oi the homotransplanted canine liver. Surg. Forzrnl 12, 112-113.

~I . \SSICK, J . A,, LOCIITE, H. L., JR., ASHLEY, C. A,, THOMAS, E. D., and FERREBEE, J. W. (1959).

.\ lunctioninp kidney homotransplant in the dog. Surgery 46. 821-828. MEnAwaR, P. B. (1944). Behavior and fate of skin autografts and skin homografts in rabbits.

J . ; I t ~ a f . 78, . 176-199. A M o o ~ ~ . . F. TI., ~VIIEELER, H. B., DEMISSIANOS, H. V., SMITH, L. L., BELANKURA, O., ABEL, L. K.,

C;RI.ESBER(:, J . B., and DAMMIN, G. J . (1960). Experimental whole organ transplantation of

thc liver and of the spleen. Ann. Strrg. 162, 374-387. S r x r o s s ~ s , hl. , BUEM,\SS, J., GAMMELTOFT, A., JENSEN, F., and JORGENSON, K. (1953). Biolopi-

cal incompati1)ility in kidney transplantation in dogs. I. Experimental and morpholo~ic investiga- tions. Acla Pathol. hlicrobiol. Scand. 32, 1-35.

post~q,f.rati\.e rolv of STARZI., 'r. E , KAI:I 'P ,

rejectirin of thr tranc STARXI., T. E.. H I - T Z , C

effect of host and pr STARZI., T . E., K A U P P .

Homotransplantation