1. health and fitness
2. disease

THE AMERICAN JOURNAL
OF PATHOLOGY
VOLUME XLI
NovExii, I962
NuuBER 5
HOMOTRANSPLANTATION OF THE CANINE LIVER
AS AN ORTHOTOPIC VASCULARmIED GRAFT
HISTOLOGIC Al%1 FUNCTIONAL CORRAnONS DURING RESIDENCE IN
THE NEW HOST
RAYMoND A. McBxnw, MD.*; H. BtowwEL Wnmu, MD.;
Louis L SMIT, M.D.; FRANCS D. Mooxz, MD, A"
GUSTAvE J. DAMXKN, M.D.
From the Departments of Pathology and Surgery, Harvard Medical
School and the Peter Bent Brigham Hospital, Boston, Mass.
Previous reports from this laboratory12 have outlined the surgical
technique required to transplant successfully the intact canine liver as
a temporarily life-sustaining, vascularized orthotopic homograft. This
procedure has been carried out on a total of 35 dogs with longest survival to the twelfth postoperative day.
The feasibility of such a procedure in clinical medicine may eventually
become established in some instances of irreversible hepatic damage as
it has for the kidney in selected instances of renal failure. Such heroic
measures are at this time beset with many obstacles other than the basic
one of biologic antigenic incompatibilities which tend to preclude continued viability of tissues exchanged between individuals of the same
or different species. Not the least of these problems is the one posed by
the liver being an unpaired organ, making it necessary to use cadavers
as organ source. Another major problem is posed by virtue of the large
complement of potentially immunologically competent reticuloendotheThis work was supported by grants from the United States Public Health Service
and the National Heart Institute, (H-177I, HTS-5274), and by contracts with the United
States Army Medical Research and Development Command (MLD-2o6I), and the United
States Atomic Energy Commission.
Accepted for publication June 25, I962.
* Research Trainee of the National Heart Institute, NI.H. Present address: Transplantation Biology Laboratories, Queen Vitoria Hospital, East Grinstead, Sussex, England
50S
502
MC BRIDE ET AL.
Vol. 4z, No. 5
lial tissues contained within the liver, making the possibility of graftversus-host reaction a genuine hazard to be feared.
It is the purpose of this report to present the histologic alterations
observed in the transplanted liver at varying time intervals during its
residence in the homologous host and to relate these changes to functional failure. The morphologic features observed are interpreted to be
a result of cellular response on the part of the host to factors (antigens)
present in the homograft. The functional failure which developed is
thought to be related to the loss of hepatic parenchyma incident to infiltration of the graft by host-derived mononuclear cells.
MATERIAL AND METHODS
Mongrel dogs of either sex and of approximately equal size were used. Detailed
description of the operative technique involved has been previously presented1'2 but
will be briefly outlined here. In the experimental group, including those dogs which
underwent whole-organ homotransplantation, there were 35 dogs. In the whole-organ
autotransplant group there were 27 dogs. A third group which underwent what was
essentially a sham operation (complete dissection of the liver with one or more vascular anastomoses) consisted of 13 dogs.
The donor and recipient animals were operated upon simultaneously. The normothermic recipient had a hepatectomy through a midline abdominal incision. The
inferior vena cava was freed to a point 4 inches below the renal veins and, above the
liver, was dissected to the diaphragm. The aorta was mobilized for temporary occlusion above the celiac axis. A shunt was established between the inferior vena cava
and right jugular vein. The portal vein was divided and its blood shunted to the left
jugular vein. The donor animal was prepared in a similar fashion; however, when
the liver was mobilized, the animal was made hypothermic (280 C.) by the application of cold isotonic saline to the peritoneal cavity. The liver was removed and placed
in the recipient animal and the anastomoses established in the following order:
inferior vena cava above the liver; portal vein; inferior vena cava below the liver;
hepatic artery. The anastomoses were all end-to-end, and portal vein perfusion was
accomplished within 30 to 45 minutes after removal of the liver from the donor,
followed shortly by establishment of hepatic artery perfusion. Splenectomy had been
performed early in the series while in later experiments the spleen was left intact.
Cholecystoduodenostomy or cholecystostomy have both been used to re-establish
biliary drainage.
Two control groups were used. (i) Autotranspiants: the same procedure as described for the recipient animal was carried out. The liver was removed from the
animal, cooled in isotonic saline or by perfusion with chilled oxygenated blood, and
was resutured within the same animal. (2) Liver dissections or sham operations:
complete dissection of the liver and establishment of shunts were carried out without
removal of the liver from the animal.
Complete necropsy examinations were performed on all animal at the time of
death. Tissues were fixed in io per cent buffered neutral formalin, embedded in
paraffin, and sections were stained with hematoxylin and eosin. Selected tissues were
fixed in absolute methyl alcohol for the methyl green-pyronine stain.
RESULTS
Histologic Features
A major problem in interpreting the histologic alterations of homografted livers was the presence of significant surgical artifact. The enor-
Nov., I962
LIVER HOMOTRANSPLANT
503
mity of the surgical procedure, the exquisite sensitivity of the hepatic
parenchyma to physical trauma and the frequent association of severe
myocardial disease added significant hepatic structural derangement to
those related to host-graft interaction. To some extent, the controls selected provided base lines of structural derangement, above which consistent significant differences might be attributable to factors other than
pure operative manipulation and nonspecific host responses. It was
within this general category of tissue alteration that specific changes
reflecting host-graft interaction were sought.
In attempting to relate morphologic changes to the duration of residence of the graft within the homologous host, it is important to remember that the specinens emined were obtained from animals who
died or were sacrificed at the stated time intervals. The sequence of
histologic alterations suggested might reflect the cause of death and altered physiologic status of the particular animal, rather than representing a true temporal sequence of tissue changes which might be expected
in one particular animal surviving a prolonged period. Sequential representative liver biopsy specinens might have helped solve the logistics
of this problem. However, it was thought that such added trauma would
have jeopardized the survival times that were achieved.
Reimplantation of an animal's own liver failed in several respects to
represent an ideal control situation. The technical aspects of the vascular anastomoses were more difficult because of the shorter lengths
of vessels. Regional hypothermia was not possible in vivo at the time
of vascular occlusion, as this would have resulted simultaneously in a
cooled recipient, which, early in the course of the experiments, was found
to be undesirable. Nonetheless, the clinical course of these animals was
entirely different from those with the homotransplants. Several longterm survivors were obtained, the animals were not clinically as ill, and,
as wil be noted, the histologic pattern was quite different.
Liver Homotransplants
Thirty-five animals underwent the homotransplantation procedure.
Eighteen died during the operative or immediate postoperative period,
8 survived from I to 3 days, 5 from 3 to 6 days, and i each survived
6, 6/2, 82, and I2 days. In those subjects surviving 4 or more days,
the more usual lesions at necropsy consisted of gastric and duodenal
ulceration with hemorrhage, thrombosis of the hepatic artery or vena
cava, peritonitis, infarcts of the liver, atrophy of hepatic parenchyma
and disseminated foci of necrosis in the myocardium.
Gross Features. At the time of necropsy, irrespective of the length of
survival, the most common gross alteration was an increase in size of
the liver and swelling, as evidenced by bulging of the parenchyma above
504
MC BRIDE ET AL.
Vol. 4z, No. 5
the sectioned surface. Alterations in color reflected varying degrees of
vascular congestion. Within the first 3 to 4 days postoperatively the
texture of the liver was resilient and somewhat soft; however, beyond
5 days increased resistance to cutting was usually observed. Infarcts,
when present, were usually small and located over the anterior surface,
particularly along the anterior edge. Their color rapidly changed from
reddish-purple to bright yellow.
The lobular architecture was usually markedly accentuated on the
cut surface with intense reddening and slight depression of central zones.
In longer survivors the lobular pattern was frequently completely obscured. Bile staining of the parenchyma was noted in every instance in
which there was clinical jaundice. Partial or complete occlusion of the
hepatic artery by thrombus at the anastomotic site was an occasional
complication. Complete occlusion of the hepatic artery produced obvious
softening and necrosis of large segments of the liver. The small peripheral infarcts were usually not associated with vascular occlusion of larger
vessels. Thrombosis at the site of venous anastomosis was unusual;
however, it was very common at the site of shunt insertion into the inferior vena cava below the renal veins. The extrahepatic biliary drainage
system, whether cholecystoduodenostomy or cholecystostomy, was usually patent; however, in several of the animals early in the study it served
as the source of ascending suppurative cholangitis.
Microscopic Features. The hepatic lesions observed in the animals
dying within the first 3 postoperative days were essentially similar in
all groups and probably reflected the nonspecific results of surgical
trauma, anoxia, and hepatic outflow obstruction. These alterations consisted of: (a) central and peripheral congestion of the hepatic lobule,
with or without associated hemorrhage into the surrounding parenchyma; (b) central necrosis of hepatic parenchyma, most marked in
areas of hemorrhage; (c) slight neutrophil infiltration, especially in
regions of central necrosis and to a lesser degree within the portal tracts;
(d) generalized interstitial edema with proteinaceous precipitate within
the spaces of Disse and separation of hepatic cell cords; (e) varying
degrees of intracanalicular bile stasis, most marked centrally. The hepatic cells within the peripheral portions of the lobule were usually unaltered. Sinusoids contained red cells, and Kupffer cells were more
prominent than usual. The bile duct epithelium was intact and usually
contained subnuclear vacuoles.
Survival beyond 3 days heralded a striking change in the character,
quantity, and distribution of the cellular infiltrate. Mononuclear cells
replaced the neutrophils as the predominant cell type. The first mononuclear cells to make their appearance were located in the loose adventitia surrounding small portal and central veins (Fig. 2 ). In the earliest
Nov., I962
LIVER HOMOTRANSPLANT
505
stages these cells were of the small- and medium-sized lymphocyte
type; however, a minority were larger and more closely resembled
reticulum cells. By the fifth and sixth day the quantity of the infiltrate
had increased to the point of complete replacement of many small portal
tracts, with obliteration of architecture (Fig. 5). In larger portal tracts,
at this time, the infiltrate was never as intense and remained confined
to the perivenous adventitia and the adventitia surrounding the larger
branches of the bile ducts. The infiltrate around the small central and
portal veins increased in quantity to the point of apparent compression
and compromise of the vein lumens. In animals surviving beyond the
fourth day, mature plasma cells were present in the infiltrate. Some of
the cells were of the reticulum-cell type with large nuclei, prominent
nucleoli and scanty, pale basophilic cytoplasm; others were larger than
the mature plasma cells and had intensely hyperchromatic nuclei and
abundant deep basophilic cytoplasm. The latter cells exhibited cytoplasmic pyroninophilia. Kupffer cells lining sinusoids and connective
tissue cells within the portal tracts were prominent. There was no evidence of transition of Kupffer cells to cells present in the infiltrate;
mitotic figures were not seen in either of these cells. Vascular endothelium was unaltered except for slight prominence and occasional vacuolation of that within small arteries.
Central congestion was less intense than that seen in earlier postoperative periods, particularly in regions with the heaviest central infiltrate. Generalized edema persisted. Derangement of the architecture of
the hepatic cell cords in central regions was progressive and was proportional to the quantity of cellular reaction. To a lesser degree, there
was beginning perivenous proliferation of connective tissue elements in
some of the longer survivors.
Bile stas, initially confined to central zones, was present throughout
the lobule beyond the sixth day and was only rarely noted in larger bile
ducts in portal areas.
Evidence of regeneration of hepatic parenchyma (mitosis and polyploidy) was seen in only one animal (X-69) surviving to 8 days.
Sinusoids remained patent; however, they were distorted in the central zones and contained large numbers of mononuclear cells within their
lumens. Lymphatic channels in the walls of hepatic and portal veins
were prominently dilated. This probably resulted from severance of
the vessels at the vascular poles, with consequent disruption of lymphatic
channels. Acute fibrinoid necrosis in the walls of small hepatic arteries
was seen in a few animals surviving to and beyond the fifth day. These
lesions were rare; they were unaccompanied by appreciable cellular
reaction within the vessel wall, and the intima was minimally affected.
The arterial lesions were found within the liver only in the homotrans-
506
MC BRIDE ET AL.
Vol. 4i, No.- 5
plantation group and were not observed in the control autotransplants.
Peripheral infarcts were usually associated with thrombosis of small
portal and central veins and were rimmed by intense neutrophil infiltration.
Progressive degeneration of bile duct epithelium was usually a prominent feature and in every instance was associated with an intense cellular infiltrate within the surrounding tissue.
The final histologic picture seen in the longest survivors was one of
complete disruption of lobular architecture with few surviving hepatic
cells around the periphery of the lobule (Figs. 3 and 4). Distorted sinusoids, filled with mononuclear cells and lined by hypertrophied Kupffer
cells containing phagocytized basophilic debris and hemosiderin, composed most of the hepatic lobule.
Interestingly, the longest survivor (X-58; I 2 days) showed less
histologic derangementthan the animal surviving for 8/2 days (X-69).
The cellular infiltrate was less intense, consisted almost entirely of mature plasma cells, and there was minimal disruption of the cell cord
architecture. Centrilobular proliferation of connective tissue was present
and in several regions connected adjacent central veins.
Whether or not splenectomy had been performed had no observable
effect on the tempo or character of the cellular infiltrate.
Liver Autotransplants
Twenty-seven autotransplantations were performed. Ten survived
24 hours or more and 7 for 4 days or more: 2 for 4 days, 3 for 5 days, i
for io days and i for I4 days. Technically, the autotransplantation
procedure was the most difficult to perform, and the highest incidence
of thrombosis at anastomotic sites was encountered in these animals,
although their postoperative course was far less stormy if thrombosis
did not occur. In those subjects surviving 4 or more days, lesions at
death consisted of thrombosis of the portal vein or hepatic artery, infarcts of the liver, hepatic and subhepatic abscesses and myocardial
necrosis.
Gross Features. The gross anatomic appearance of the livers varied
considerably. It reflected vascular insufficiency, when present, in the
form of multiple discrete infarcts throughout the hepatic parenchyma or
diffuse generalized softening. In addition, those features reflecting vascular congestion, as seen in the homotransplants, were also observed
in this group. Exsanguination through disrupted suture lines was an
occasional complication.
Microscopk Features. Many histologic alterations were common to
this group and to the homografted experimental group. These included:
Novw., z962
LIVER HOMOTRANSPLANT
507
acute peripheral and centrilobular congestion with or without necrosis;
infiltration by neutrophils into regions of parenchymal damage and occasionally into portal tracts; dilatation of lymphatics in the walls of
central and portal veins; early pericentral and late diffuse intracanalicular bile stasis. A higher incidence of ischemic infarction and ascending
suppurative cholangitis was present in these animals. The single most
striking difference between this group and the homotransplantation
group was the qualitative difference in the cellular infiltrate (Fig. i;
compare with Fig. 2). The predominant cell at all times was the neutrophil. Large reticulum cells and plasma cells, or their precursors, were
not seen. In no animal was there complete replacement of lobular tissue
with mononuclear cells; on the contrary, except for regions of infarction,
the hepatic cell cords were well maintained. Degenerative changes of the
type seen in the homotransplants were not observed in the autotransplants.
An interesting phenomenon observed only in several animals of this
group was the presence of foci of extramedullary hematopoiesis. These
were randomly scattered throughout the lobule and some contained
large megakaryocytes.
Sham Operations on the Liver
Thirteen sham operations were performed. One dog succumbed from
intussusception on the sixth day, another on day i 6 with massive ascites.
The remaining animals were sacrificed after periods of survival ranging
from i8 to 63 days.
Gross Features. Various gross anatomic lesions were observed. These
were clearly related to vascular insufficiency related to thrombosis at
the anastomotic site in the hepatic artery, if the dissection had been
extended to include this procedure. Generalized softening and peripheral
infarcts were observed under such circumstances. In most of the animals
the livers were unaltered grossly.
Microscopic Features. Changes similar to those detailed above, attributable to surgical artifact, anoxia and outflow obstruction, were
present in the early postoperative period. In the longer survivors essentially normal hepatic architecture was found. Dilated lymphatics in
the walls of central and portal veins were frequently observed. Varying
degrees of centrilobular intracanalicular bile stasis were observed in a
few animals who were not clinically jaundiced.
Pathologic Alterations in Other Organ Systems
The vast majority of the lesions present in the experimental group
of animals were classifiable as surgical artifacts and were not attribut-
508
MC BRIDE ET AL.
Vol. 4i, No. 5
able to the temporary residence of a foreign liver within the abdomen.
The experimental and autotransplantation controls shared most extrahepatic lesions, whereas sham-operated animals had fewer and less severe
lesions.
It soon became obvious in the course of the experiment that severe
myocardial lesions were contributing to structural hepatic alterations
and death in all groups (Figs. 6 and 7). These lesions consisted of multiple subendocardial foci of myocardial necrosis without related vascular
lesions. The myocardium of all 4 chambers was usually involved. The
foci of necrosis were more extensive in the right than left ventricular
myocardium, and in some instances were transmural. The affected
myocardial fibers tended to undergo early calcification. In their initial
stages the lesions were present as early as the first postoperative day.
The longer survivors had fewer such lesions. Similar findings have been
observed in this laboratory in dogs subjected to mild hemorrhagic
shock for short periods of time.
Pulmonary congestion, edema, atelectasis and bronchopneumonia
became less of a problem after the operative technique was altered so
as not to include entry into the thoracic cavity. Pulmonary emboli with
infarction were not infrequently associated with thrombosis at the site
of the shunt insertion into the inferior vena cava. Entrapped giant
cells (megakaryocytes) within alveolar capillaries, focal thickening
of alveolar septums and focal interstitial infiltrates of mononuclear
cells were findings not limited to the homotransplantation group. These
lesions were possibly related to prior helminthic infections, so common
in the dog.
The kidneys of the animals with homografts contained nonspecific
lesions which were found with equal frequency in the control groups.
The kidneys were frequently swollen and congested. Peripheral wedgeshaped infarcts in varying stages of organization were common. Many
of the necrotic tubules in the infarcts underwent early calcification.
Areas adjacent to infarcts were infiltrated with neutrophils, lymphocytes and plasma cells, depending on their age. Miliary granulomas,
some of which contained small helminth larvae, were common. Perivascular and periglomerular cellular infiltrates composed of plasma cells
and lymphocytes were observed in the kidneys of the experimental and
control groups of animals; these were found in kidneys without infarction.
Lymph nodes from the porta hepatis and retroperitoneal regions
were xmined. In all groups the nodes were enlarged and soft. Histologic differences between the experimental and control groups were
not always striking. In general, the control lymph nodes had large fol-
Novw, z!92
LIVER HOMOTRANSPLANT
509
licles with prominent, active centers, whereas the experimental nodes
usually had a thinner cortex with smaller follicles and reticulum centers.
In some of the latter group the follicular architecture was obliterated
and the cortex markedly thinned. MIedullary cords, which were more
prominent in the group with homotransplantation, were thickened and
contained moderately increased numbers of lymphocytes and plasma
cells. Sinusoids were dilated and contained many lymphocytes. Hemosiderin pigment was frequently found within the reticuloendothelial
cells lining sinusoids. Typical megakaryocytes were present within the
medullary cords of lymph nodes in some of the animals in the autotransplantation group.
Similarly, marked differences in the spleens of the experimental and
retransplantation control groups were not observed. The majority of
the changes was attributable to acute congestion. A varying degree of
splenomegaly was a constant feature. Well-preserved follicles with
large germinal centers were more common in control animals. Similar
degrees of reticuloendothelial hyperplasia and lymphocytic and plasmocytic content of the pulp cords were present in both groups. Hemosiderin pigment deposition was a more constant feature within the spleens
of the experimental animals.
Myeloid hyperplasia of the bone marrow was present to some degree
in most of the animals. Perivascular accumulations of lymphocytes and
plasma cells were nonspecific changes found in both groups.
Acute gastroduodenal ulceration with hemorrhage was the cause of
death of several animals in both the experimental and control groups.
Acute arteritis, characterized by fibrinoid necrosis in the walls of
small muscular arteries, with minimal involvement of the intima and
sparse lymphocytic response in the adventitia, was seen occasionally in
various organs in the experimental group and retransplantation controls.
The organs involved were the intestine, gallbladder, heart and pancreas.
Involvement of the liver, though rare, was observed only in the experimental group.
DIscuSSION
Previous studies of the renal homograft in man and renal and splenic
homografts in the dog have demonstrated the prominent feature of
mononuclear infiltration of the graft, the intensity of which tended to
parallel functional decline and eventual "rejection" of the graft by the
host. Most experimental evidence supports the concept that graft rejection, whether of skin or of a vascularized organ transplant, is mediated
by cells elaborated by the immune-centers of the host in response to tissue antigens present in the graft but lacking in the host. The mecha-
5IO
MC BRIDE ET AL.
Vol. 4i, No.- 5
nism(s) by which such "sensitized" cells mediate the sequence of events
which precede death and functional failure of a graft is unknown. The
apparent necessity for the "sensitized" cells to come into close contact
with structures within a graft in order for rejection to proceed implicates
either adverse mechanical factors incident to the presence of such cells,
e.g., altered blood flow, or antibody-like factors produced by and effective only in the immediate vicinity of the infiltrating cells.
In a recent publication from this laboratory,2 the clinical course and
chemical values in the animals discussed in the preceding pages have
been outlined in detail. It was demonstrated, by the parameters chosen,
that the liver homograft was functional and temporarily life-sustaining.
The cause of death in most instances was attributable to identifiable
factors other than liver disease or failure. Animals with homografts surviving the immediate postoperative period showed evidence of liver function up to 24 hours prior to death. Death was preceded in these instances by rapid rises in bilirubin and alkaline phosphatase values, and a
lowering of the prothrombin level. In many ways the picture looked "obstructive" and might well have been associated with an infiltrate in the
portal areas rather than primary liver cell necrosis. The autotransplantation control animals, besides having a distinctly better postoperative
course, did not demonstrate these chemical abnormalities consistently.
The morphologic counterparts of these clinical differences between
the experimental and control groups of animl were quite evident in
the qualitatively and quantitatively different histologic hepatic lesions
within the two groups of animals surviving beyond the third postoperative day. The constant feature of extensive loss of hepatic parenchyma
with replacement of large portions of the lobule by mononuclear cells was
the readily apparent feature peculiar to the experimental group which
corresponded to the clinical differences observed. The constant relation
between the loss of hepatic parenchyma and mononuclear cell infiltration was striking enough to suggest a cause and effect relationship. Such a
relationship was also suggested by the degenerative changes in bile duct
epithelium, limited to the homotransplantation group. In each instance
an intense cellular reaction was present in the periductal adventitia. The
question of whether or not the parenchymal cells were destroyed by
cytotoxic factors (antibody) released by the infiltrating cells or by
altered intrahepatic circulatory dynamics incident to obstruction and
distortion of sinusoids by the massive infiltrate, is of basic importance.
No decisive answer is forthcoming from this or previous studies. From
the observation that comparable or even more intense cellular infiltrate
into the human liver in leukemia is not associated with such extensive
parenchymal necrosis, added emphasis is given to the possibility of
Now., z92
LIVER HOMOTRANSPLANT
5II
elaboration of cytotons by the infiltrating cells in the homotransplanted
livers. The paucity of cellular infiltrate in the longest survivor in our
experimental series may account for the survival time obtained on the
basis of less parenchymal damage incident to fewer cells. A similar observation was recorded by Starzl, Kaupp, Brock and Linman 3 in their
recent publication.
Other factors unrelated to the cellular infiltrate, yet which certainly
contributed to hepatic failure, were nonspecific, as they were also present in the control series. The most prominent among these were: (a) intense centrilobular congestion, possibly related to extensive myocardial
damage and hepatic outflow obstruction; (b) generalized edema and
multiple parenchymal infarcts, possibly related to hypervolemia and
subsequent plasma dispersal hemoconcentration. The latter was frequently a complication in the early postoperative period in both groups
of animals.
Arteritis of the type found in the experimental and control animals
has also been described in kidney homografts in man4 and the dog.5'6
Another type of arterial lesion consisting of subendothelial, medial and
perivascular infiltration with mononuclear cells (sometimes noted in
huiman and canine renal homografts) was not observed in the liver
homografts. Possibly this was because of the relatively short period of
observation. The early cellular infiltration of the veins in the liver
homograft resembled the pattern seen in the rejection of renal homografts in man and the dog.7
The cause of the myocardial lesions remains obscure. These were
found with equal frequency in the experimental and control animals.
It is difficult to implicate possible etiologic factors at this time because
of the numerous procedures which can produce myocardial necrosis in
experimental animals, as recently outlined by Selye.8
That the cells infiltrating a homograft originate in the host has gained
support from studies of renal homografts in man9 and skin and renal
homografts in the rabbit and dog."0 Simonsen, Buemann, Gammeltoft,
Jensen and J0rgensen 11 and Dempster,5 studying the histologic pattern
of renal homograft rejection in the dog, first introduced the concept that
grafted tissues may be capable of reacting agan st the foreign antigens
present in the host's tissues. Such a reaction is evident in transplantation
of the spleen2'12 where early enlargement of the lymphoid follicle appears
to be associated with an early "graft-versus-host" reaction. Such a reaction, occurring in an immunologically competent organ such as spleen,
may or may not find a counterpart in tissues that are not active in the
production either of immune bodies or lymphocytes. A graft-versus-host
reaction would result from the local production of immunologically
512
MC BRIDE ET AL.
Vol.,4z, No. 5
competent cells within the graft, which upon their transport to the host's
tissues, would be capable of producing adverse effects. Indeed, the capability of immunologically competent cells, or their stem-cell precursors,
within grafted tissues to produce deleterious effects on the host is the
generally accepted mechanism of the runting syndrome in tolerant
mice 13 and of homologous disease in x-irradiated chimeric mice.'4 The
large complement of reticuloendothelial cells within the liver, in the form
of Kupffer cells lining sinusoids, provides a potentially similar situation
in which evidences of a graft-versus-host reaction must be sought. Histologic evidence of such a reaction would, in its simplest form, consist of:
(a) demonstration of the local production of cell clones from reticuloendothelial cells within the liver; and (b) the finding of cells thought
to be immunologically competent within host tissues where they would
not normally be found. The former consideration warrants an examination of the possibility that the mononuclear cells found in the homotransplanted liver were entirely or in part produced de novo from stemcell precursors in the liver in response to host antigens (leukocytic)
present in the perfused blood.
The competence of Kupffer cells to produce clones of hematopoietic
cells has representation in human pathologic conditions (erythroblastosis
fetalis) and in the hematopoietic foci found in several of the autotransplantation controls. In the latter situation the stimulus for hematopoiesis
must be sought in factors other than response to foreign antigens. Welldefined foci of hematopoiesis were not observed in the liver homotransplants. The distribution of the cellular infiltrate within the homografts
differed markedly from the focal character of hematopoiesis. In the
homografts, the earliest foci of cellular aggregates were within the loose
adventitia of small central and portal veins (reminiscent of perivenous
inflammation in tuberculin sensitivity). Hematopoiesis was intrasinusoidal. Marked parenchymal necrosis accompanied the infiltrate
within the homografts; whereas there was little to suggest a similar reaction to foci of hematopoiesis. A similar infiltrate with predilection for
vessels has been described in the homografted kidney in man'-5 and the
dog,5'6"' an organ in which the reticuloendothelial system has little representation as compared to the liver. For these reasons we concluded that
the cells within the homograft were host-derived.
If, indeed, many or all of the cells observed in the liver homografts
were produced within the liver, they would presumably possess a complement of individual specific antigens similar to those of the Kupffer and
parenchymal cells and hence would be subject to similar consequences of
an immune reaction directed against them. Such a mechanism may account for the absence of hematopoiesis in the homograft. Conversely, if
Nov7, zI962
LIVER HOMOTRANSPLANT
5I13
Kupffer cells do possess features of stem cells which would permit them
to elaborate clones of immunologically competent cells, there is as yet
no experimental evidence which would deny them the prerogative of
becoming tolerant to host antigens. Such a tolerant state could arise as a
result of selective influences in the host's response favoring mutant noncompetent clones or from the fact that such clones would have been
exposed to host antigens during their entire period of ontogenesis.16 This
last proposition makes the assumption that the adult canine liver contains cells sufficiently immature to actively acquire tolerance in the classic sense.
Some of the large mononuclear cells of the infiltrate within the liver
homografts bore a striking resemblance to the "large lymphoid cells"
described by Scothorne 1 in the regional lymph nodes of rabbits bearing
skin homografts. Scothorne postulated that these cells were producers
of antibody and were intimately involved in the rejection of skin homografts. He demonstrated the cells within the regional lymph nodes as
early as the third day after homografting; however, they were fewer in
number at day io, when the skin homografts were in an advanced stage
of destruction. Scothorne stated that at this time "the cortex (of the
regional nodes) gives the impression of being depopulated of cells." Presumably, these large mononuclear cells left the lymph nodes via the efferent lymphatics and reached the graft by way of the bloodstream.
However, in Scothorne's experiments, the cells were not found within
the graft at any time. In our experience with the homografted liver, the
cortex of the regional lymph nodes was thinner than corresponding nodes
of the autotransplants. Our failure to find "large lymphoid cells" within
the regional nodes may be the result of the more rapid mobilization of
these cells from the nodes in response to the more massive antigenic
stimulus of a liver homograft as compared to skin. We believe that the
large mononuclear cells reached the liver via the bloodstream and there
became part of the cellular response of the host.
We have spoken of the immunologic competence of the hepatic
reticuloendothelial cells as "potential," intending to survey the existing
confusion concerning this point. Stavitsky 18 demonstrated that homologous adult liver was incapable of producing circulating antibodies under
conditions which allowed for their production by homologous spleen,
marrow and lymphoid cells of the rabbit. Siskind 19 was unable to demonstrate competence of liver tissue to produce the runting syndrome in
immature nice. However, there are conflicting reports on the capability
of fetal liver to protect lethally x-irradiated mice and to produce delayed
death via the homologous-disease syndrome.20 Simonsen 6 has shown
that in certain experimental situations, fetal mouse liver cell grafts could
5I4
MC BRIDE ET AL.
Vol. 4, No. 5
attack the host. The fetal liver is, of course, normally an active hematopoietic center. Nevertheless, it is of interest that the hepatic reticulum
cells can produce immunologically competent cells even in the fetal stage.
Examination of tissues from subjects in the experimental group revealed no specific lesions which might be interpreted as evidence of
a graft-versus-host reaction. Focal accumulations of mononuclear cells
within various tissues and organs were found with equal frequency in
both the experimental and control groups of animals. The presence of
such cellular accumulations in animals who died during operation or in
the early postoperative period argues against the possibility of their
representing a graft-versus-host reaction. Splenomegaly, which is a
valid indicator of a graft-versus-host reaction in mice 1620 must be considered in these experiments a response to altered circulatory dynamics.
Starzl and co-workers' observations 3 differ from our own insofar as
they found lesions in various host organs designated by them as specific
for the homotransplantation group of animals. They attributed such
extrahepatic lesions to a "general host reticulo-endothelial response to
the antigenic stimulus of the homograft." Lesions of this type were noted
in our control groups and were, therefore, not interpreted as host-versusgraft reactions.
SUMMARY
The homotransplanted canine liver is temporarily a life-sustaining and
functional organ. Death was usually preceded by functional failure which
correlated with the loss of hepatic parenchyma incident to infiltration by
mononuclear cells derived from the host.
The microscopic patterns of homografts and autografts was similar
up to the third postoperative day, after which the homograft was progressively infiltrated with mononuclear cells.
No specific lesions interpretable as representative of a graft-versushost reaction were found. Such lesions were sought because of the large
complement of potentially immunologically competent reticuloendothelial cells contained in the liver.
REFERENCES
I. MOORE, F. D.; Shmr, L. L.; BURNAP, T. K.; DALLENBACH, F. D.; D~imw,
G. J.; GRUBER, U. F.; SHOEMAKER, W. C.; STEENBuRG, R. W.; BALL, M. R.,
and BELKO, J. S. One-stage homotransplan-tation of the liver following total
hepatectomy in dogs. Transpl. Bul., 1959, 6, 103-IO7.
2. MOORE, F. D.; WHELTER, H. B.; DEmiSSANOS, H. V.; SMITH, L. L.; BALANKURA, O.; ABEL, K; GREENBERG, J. B., and DAmxw, G. J. Experimental
whole-organ trlantation of the liver and of the spleen. Ann. Surg., i96o,
I52, 374-387.
3. STARZL, T. E.; KAuPP, H. A-, JR.; BRocK, D. R., and LiNEw, J. W. Studies on
Nov., 1962
LIVER HOMOTRANSPLANT
5I5
the rejection of the transplanted homologous dog liver. Surg., Gynec. & Obst.,
i96i, II2, 135-I44.
4. DAMxEN, G. J. Serum Sickness and Related States. In: Cellular and Humoral
Aspects of the Hypersensitive State. LAwRvNcE, H. S. (ed.). Paul B. Hoeber,
Inc., New York, 1959, P. 58I.
5. DEMPSTER, W. J. Kidney homotransplantation. Brit. J. Surg., I952-I953, 40,
447-465.
6. DAmMIN, G. J. Problems of Homotransplantation. Proceedings of the Seventh
Annual Conference on the Nephrotic Syndrome, I955. National Nephrosis
Foundation, New York, I956, pp. Io8-II5.
7. DAmmh, G. J. The kidney as a homograft and its host. Univ. Alichigan M.
Bull., i960, 26, 278-283.
8. SELYE, HE The Chemical Prevention of Cardiac Necroses. Ronald Press Co.,
New York, I958, 235 PP.
9. MuRRAY, J. E.; MERRILL, J. P.; DAMmE G. J.; DF.ALY, J. B., JR.; WALTER,
C. W.; BROOKE, M. S., and WILoSN, R. E. Study on transplantation immunity after total body irradiation; clinical and experimental investigation.
Surgery, i960, 48, 272-284.
IO. PORTER, K. A., and CALNE, R Y. Origin of the infiltrating cells in skin and
kidney homografts. Transpl. Bull., i960, 26, 458-464.
II. SIMONSEN, M.; BUEMANN, J.; GAmMELTOFT, A.; JENSEN, F., and JoRGENSEN, K.
Biological incompatibility in kidney transplantation in dogs. I. Experimental
and morphological investigation. Acta path. micr. scandinav., 1953, 32, I-35.
I2. WHEELER, H. B.; BALANKURA, 0.; PENDOWER, J. E.; GREENBERG, J. B.; DAMMIN, G. J., and MOORE, F. D. The homograft response to whole-organ transplantation of the canine spleen. J. Surg. Res., i962, 2, II4.
13. BILLINGHIAM, R. E. Studies on the reaction of injected homologous lymphoid
tissue cells against the host. Ann. New York Acad. Sc., i958, 73, 782-788.
I4. TRENTIN, J. J. Tolerance and homologous disease in irradiated mice protected
with homologous bone marrow. Ann. New York Acad. Sc., 1958, 73, 799-8io.
I. HisE, D. M.; MERRILL, J. P.; MILLER, B. F., and THORN, G. W. Experiences
with renal homotransplantations in the human; report of 9 cases. J. Cln.
Invest., I955, 34, 327-382.
i6. SimONsEN, M. On the acquisition of tolerance by adult cells. Ann. New York
Acad. Sc., I960, 87, 382-390.
I7. SCOTHORNE, R. J. Studies on the response of the regional lymph node to skin
homografts. Ann. New York Acad. Sc., 1957, 64, I028-iO39.
I8. STAVITSKY, A. B. Antibody synthesis by homotransplanted cells and tissues. I.
Study of factors which influence the process in the rabbit. J. Immunol., I957,
79, i87-I99.
i9. SIsKiND, G. Personal communication.
20. SIMONSEN, M., and JENSEN, E. The Graft-versus-Host Assay in Transplantation Chimaeras. In: Biological Problems of Grafting. ALBERT, F., and MEDAWAR, P. B. (eds.). Blackwell Scientific Publications, Oxford, I959, P. 2I4.
[Ilustrations foUow]
5I6
MC BRIDE ET AL.
Vol. 4i, No. 5
LEGENDS FOR FIGURES
Photomicrographs were prepared from sections stained with hematoxylin and eosn
FIG. I. Dog X-64. Liver autotranspant. This subject survived imtil the tenth postoperative day. The centrilobular vein is normal, as is the cellularity in the
sinusoids. Compare with Figure 2. X 500.
FIG. 2. Dog X-i8. Liver homotransplant on the fifth post-operative day. Mononudear cells obscure the wall of the central vein and occur in abudance in the
sinusoids around the vein. Hepatic parenchymal cells in the infiltrated area have
been reduced in number, and those remaining are altered. X 360.
FIG. 3. Dog X-69. Liver homotrausplant on the eighth postoperative day. The findings here characterized most of the portal areas in the liver homotranslants, in
tht the portal area is prominent because of mononudear cell infiltration. There
is degeneration of bile duct epithelium, and the portal vein is invaded, with subendothelial cellular infiltration (arrow). X 300.
NovT.,
I
5I7
LNIER HOMOTRANSPLANT
62
i't:
"C iw.
.
-.
13
.
zil
I
.....
.e-
-
V
I,
:dwo-V
^W
v
I
ip.i
t4
k."&
eq
0
2
-w-
-4rappl, VW
0.-
WI
..
r-
41 ...
-
-0
0
S.
A
I!r
40 zo
r
.1
.
a
1
5I8
MC BRIDE ET AL.
VOI. 41, No. 5
FIG. 4. Dog X-69. This illustrates another portal area from the liver shown in Figure
3. There is more advanced degeneration of bile duct epithelium and cellular debris
in the bile duct (arrow). No hepatic cells are recognizable. X 300.
FIG. 5. Dog X-i8. A prominent portal area is observed in the homograft illustrated
in Figure 2. The mononuclear cell infiltration is well established by post-transplant daY 5. The cells are found in the wall of the portal vein, as well as surrounding it and other structures in the portal area. The arrow indicates a hepatic
arterial branch with focal necrosis and fibrinoid. X 240.
FIG. 6. Dog X-35. Heart. In this subject with a liver homograft the clinical course
was good for the first 4 days. Jaundice and weakness appeared, and the dog succumbed 3 days later. Focal necrosis of the myocardium with calcification. as
shown here, represented a widely disseminated lesion. Similar lesions were noted
also in dogs with autotransplants (see Fig. 7) and sham operations. Only in the
relatively longer survivals. however, was calcium deposition noted in addition
to necrosis. X 250.
FIG. 7. Dog X- I. Heart. The operation here was autotransplantation with survival
of the subject for 24 hours. The myocardial necrosis with acute interstitial inflammatorv reaction characterized much of the mvocardium. X 200.
Nov.. I952
LIVER HOMOTRANSPLANT
5I9
4
6
7