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Failure of lyophilized splenic tissue to produce accelerated rejection of canine renal allografts
FAILURE OF LYOPHILIZED PRODUCE ACCELERATED CANINE RENAL P.
HUTCHIN,
M.D.,*
TRANSPLANTATION of tissues and organs into genetically foreign recipients provokes an immune response which leads to destruction of the transplant. Subsequent grafts of tissue with the same genetic constitution are then rejected more rapidly in a second set reaction. Under certain circumstances, however, pretreatment of the recipient with donor tissue may prevent such a sequence of events. Steinmuller [21] failed to find accelerated rejection of skin grafts in mice pretreated with frozendried splenic or renal tissues. Other investigators found prolongation of allografted ovaries [ 171, skin [ 161, and kidneys [lo] in recipients specifically pretreated with donor antigens. This effect was attributed to “enhancement” although passive transfer experiments were not always successful [ 111. Because of the fundamental importance of the problem as well as possible information which might be obtained on the mechanisms of graft rejection, this study was undertaken to ascertain the effect of pretreatment with lyophilized sp 1enic tissue on subsequent canine renal allograft survival. Part of the study From the Department of Surgery and Division of Immunology, Duke University Medical Center, Durham, N.C. This work was supported in part by U.S. Public Health Service Grants GM 22454 and HD 00668. Submitted for publication Aug. 30, 1966. * Present address: Division of Thoracic Surgery, University of North Carolina, Chapel Hill, N.C.
SPLENIC REJECTION ALLOGRAFTS AND
D.
L.
TISSUE
TO OF
STICKEL,
M.D.
had as its objective to establish the presence or absence of isoantibodies directed against donor antigens. Since immunological enhancement depends for its effect on the presence of humoral antibodies [ 121, their demonstration would favor such a mechanism if prolonged allograft survival was demonstrated. Furthermore, the failure to find accelerated rejection in the presence of humoral antibodies could be taken as evidence against a predominant role of such antibodies in the allograft reaction.
MATERIALS
AND
METHODS
Thirty-six male mongrel dogs were used in this study: 12 dogs served as donors of the spleen and kidneys and 24 dogs served as recipients. The recipients were divided into three groups. Experimental
Group
Eight animals were pretreated with lyophilized splenic tissue, 1 gm. intravenously, 28 and I4 days prior to kidney transplantation, In each instance the spleen and the kidney were from the same donor. Specificity
Control
Group
Four animals were pretreated with lyophilized splenic tissue as in Group 1 but the 357
JOURNAL OF SURGICAL RESEARCH
VOL. 7 NO. 8, AUGUST 1967
kidney was transplanted from an unrelated dog. Baseline Control Group Twelve animals received a kidney allograft from an unrelated dog without prior pretreatment. BLOOD GROUPTYPING
Blood groups of all donor and recipient dogs were determined with specific canine antisera.* The tube method for the determination of blood groups A, C, and D, as described by Altman [l], was used, OPERATIVE
PROCEDURES
Splenectomy Splenectomy was performed on donor dogs four weeks prior to nephrectomy. The spleen was immediately lyophilized (see below) and stored at -20°C. until used. Renal biopsy was always obtained at the time of splenectomy to assure that normal, healthy kidneys were present for subsequent transplantation. Bilateral Nephrectomy plantation
and Kidney
Trans-
After bilateral nephrectomy, recipient dogs received a transplant of a single kidney from the appropriate donor. The kidney was placed in contralateral iliac fossa and vascular anastomoses established with the external iliac vessels of the host. Total renal ischemia time was 17 to 28 minutes. Ureterovesical anastomosis was performed by careful mucosa-tomucosa approximation. Postoperatively the dogs were maintained on ad libitum feedings of commercial dog chow and water. Blood for serological studies was obtained on the third, seventh, and fourteenth postoperative days. Serial blood urea nitrogen determinations were also performed. Complete autopsies were done at the time of death. * Generously supplied by Dr. S. Swisher, University of Rochester School of Medicine, Rochester, N.Y.
358
PREPARATION AND ADMINISTRATION LYOPHILIZED SPLENIC TISSUE
OF
The removed spleen was perfused with 250 ml. of heparinized saline (1 mg. per 10 ml.) to wash out residual blood. The spleen was then homogenized and lyophilized in 5 to S ml. amounts until dry. The lyophilized tissue was pooled, weighed out in aliquots of one gram and stored at -20°C. until used. Before intravenous injection into dogs, the lyophilized material was dissolved in about 20 ml. of saline, HEMAGGLUTINATION
TESTS
The dextran method of Gorer and Mikulska [7] was employed. Absorbed dog serum was substituted for human serum as the medium for suspending erythrocytes which were obtained either from freshly drawn blood or from blood stored in ACD solution at 4°C. CYTOTOXIC
TESTS
Cytotoxic activity of sera in recipient dogs was determined according to the method of Gorer and O’Gorman [S] or its modification of Amos and Wakefield [3]. Normal dog serum was used as a source of complement [ 131. Test lymphocytes were obtained from the appropriate donor animal when titrating for cytotoxic antibody in the serum of recipient dogs. When necessary, lymphocytes were stored for subsequent testing in TC-199 with 10% dimethylsulfoxide at -20°C. [5]. RESULTS The survival time after bilateral nephrectomy and renal transplantation in the three groups of dogs is recorded in Table 1. The mean survival times in all three groups are remarkably similar. The results of serological investigations are recorded below. INFLUENCE OF DONOR AND RECIPIENT BLOOD GROUPS ON THE SURVIVAL TIME AFTER RENAL TRANSPLANTATION
In agreement with the general prevalence of blood group antigens in dogs most animals
HUTCHIN AND STICKEL: SPLENIC TISSUE AND REJECTION OF RENAL ALLOGRAFTS
Table
KIDNEY TRANSPLANTATIONS I
1. Survival of Dogs With Renal Allografts in Various Groups Individual Survival Time (days)
Mean Survival (days 2 SD.)
Group 1 Experimental
8, 9, 12, 13, 13, 13, 17, 25
13.8 k 5.3
Group 2 Specificity
8, 12, 22
14.0 2 5.9
Recipient
Group
Dogs
control
16, 19, 26
HEMAGGLUTININ RESPONSE TO LYOPHILIZED SPLENIC TISSUE INJECTION AND RENAL ALLOGRAFTS
Figure 1 shows hemagglutinin titers in the three groups of recipient dogs. In agreement with previous reports [l, 221 no naturally occurring hemagglutinins were detected in the sera obtained prior to the injections of lyophilized splenic tissue or kidney grafting. After administration of lyophilized splenic tissue all dogs developed hemagglutinins, and their titers increased after a second injection. After kidney grafting the hemagglutinins showed a precipitous drop by the third postoperative day, followed by a subsequent rise. Hemagglutinin levels present before grafting were not attained, however, except in the A-negative, C-negative dog pretreated and grafted with tissue from an A-positive, C-positive animal (Group 1). In baseline control dogs (Group 3) hemagglutinins were detected in majority
WONEY TRANSPLANTATION SPECIFICITY WNTROL I
7, 8, 9, 9, 11, 13, 15, 15, 13.5 2 5.5
had both antigens A and C [ 11. Only one A-negative, C-negative dog was found. This dog, after pretreatment with lyophilized splenic tissue and a kidney graft from an A-positive, C-positive animal, developed the highest hemagglutinin response observed in this study and interestingly enough had also a very long survival after renal transplantation (25 days). No effect on the hemagglutinin titer or survival time was noted when the donor and recipient dogs differed only with respect to the weak blood group antigen D.
the
GROUP *
control
3
Baseline
10241
of animals
by the
seventh
day
I(IONEY TAAN~PLANTATION
/ I’: ,/ / 64
,/
n2w Fig. dogs lized renal
03
7
/
_.
14
TIMEbAYS) titers of representative 1. Hemagglutinin after intravenous administration of lyophisplenic tissue (Group 1 and Group 2) and transplantation (all groups).
after renal transplantation and their titers rose higher than in the pretreated dogs (Group 1 and Group 2). CYTOTOXIC
TESTS
No cytotoxic activity was detected in any of the tested sera, either in response to injections
of lyophilized
splenic
tissue
or after
kidney transplantation. PATHOLOGICAL
FINDINGS
One dog died from heart worms and another as a result of a surgical failure. These dogs were excluded from the experiment. All other dogs died as a consequence of renal allograft rejection. The vascular anastomoses as well as the ureterovesical anastomosis were patent in all cases. The transplanted kidney was grossly swollen, at times hemorrhagic, and
dark
red in color
on its cut
surface.
The
weights of the rejected kidneys were similar in all three groups of dogs, averaging 186 to 217 gm. 359
JOURNAL
OF
SURGICAL
RESEARCH
VOL.
7
NO.
On microscopic sections marked mononuclear interstitial and perivascular inflammatory response was evident in all kidneys. Focal and frequently generalized interstitial hemorrhages, acute vasculitis with occlusion of the cortical vessels,and medullary ischemia and necrosis were also present. Animals surviving longer did not differ from animals which succumbed early after renal transplantation except for the presence of more chronic changes such as beginning glomerular fibrosis and sclerosis. No difference could be detected in the histological appearance of the kidneys rejected by the pretreated or non-pretreated dogs. OTHER
FINDINGS
The blood urea nitrogen usually rose during the immediate postoperative period, then returned to normal on about the third to fifth postoperative day, remained at this level for several days, and then rose again progressively until death. In a few dogs the rise of blood urea nitrogen was steady and continuous from the time of the kidney grafting, No characteristic differences could be detected between the various groups of animals.
DISCUSSION This study demonstrated that prior exposure to donor lyophilized splenic tissue does not sensitize recipient dogs to subsequent renal allografts. The demonstration of hemagglutinins produced in all dogs in response to lyophilized splenic tissue pretreatment excludes immunological tolerance or paralysis as a cause of the failure of sensitization. Immunological enhancement, on the other hand, could account for some of the findings in this study, despite the fact that prolonged survival of the pretreated animals was not achieved. Administration of killed, as for example, lyophilized, tissue has been shown to result in a high humoral antibody response and minimal cellular immunity [20], especially when given by the intravenous route 14, 6, 141. Under these circumstances, accelerated 360
8,
AUGUST
1967
rejection might not result on second exposure to the antigen, and the presence of isoantibodies directed against donor antigens might actually interfere with the allograft reaction [12, 151. The amount of antibody present relative to the size of the grafted tissue is important in this respect, Prolonged survival of allografted kidneys was not obtained in this study, possibly because of insufficient isoantibody levels attained in the pretreated dogs, resulting in an incomplete antigen blockade of an organ as large as the kidney. This would permit an antigen “leak” to occur into the host’s immunological centers, followed by sensitization of the host and subsequent rejection of the foreign tissue. Alternatively, insufficient time after pretreatment may have been allowed for any existing cellular immunity to subside [6]. The antibodies produced in response to the injections of lyophilized splenic tissue crossreacted with the allografted kidney as demonstrated by the steep fall of hemagglutinins after renal transplantation. Neither the antibodies produced in response to the lyophilized splenic tissue nor the antibodies produced in response to renal transplantation were cytotoxic on repeated tests with the methods available. Previous serological studies after canine renal transplantation likewise failed to demonstrate cytotoxins [9], whereas hemagglutinins were demonstrated [ 191. Dissociation of hemagglutinin and cytotoxic activity after immunization of dogs is not uncommon, and repeated grafts from the same animal may be necessary before cytotoxins can be demonstrated [2, 181. The cross-reaction of antibodies produced in response to lyophilized splenic tissue with kidneys from unrelated dogs (Group 2) may be due to sharing of antigens between individual dogs [l] or due to a certain amount of inbreeding in the population of mongrel dogs available. After kidney grafting the fall in hemagglutinins was not as complete in this group of dogs, however, as in the animals receiving a kidney from the donor of the spleen (Group 1). The subsequent rise in titer was similar in both groups of pretreated dogs but smaller than in the non-pretreated animals
HUTCHIN
AND
STICKEL:
SPLENIC
(Group 3), indicating a partial inhibition of the antibody response after kidney grafting in the pretreated dogs. No correlation could be made between the height of the hemagglutinin titer observed postoperatively and the length of survival of the dogs. Animals which lived longer had higher hemagglutinin titers than those which died early. This finding suggests that the development of hemagglutinins after renal transplantation is not related directly to the development of immunity to the donor tissue and that factors other than the humoral antibody level must be implicated in the allograft rejection.
TISSUE
7.
8.
9. 10.
11.
12.
SUMMARY CONCLUSIONS
AND
The survival of dogs after renal transplantation was not affected by prior pretreatment with lyophilized splenic tissue from the donor of the kidney, despite the development of hemagglutinins directed against the donor antigens. The significance of these findings with respect to the allograft reaction and immunological enhancement is discussed.
13.
14.
15.
16.
REFERENCES 1.
Altman, B. Hemagglutinin in skin and kidney homotransplantation in dogs. Transpkznt. 3:326, 1965. 2. Altman, B., and Simonsen, M. Cytotoxic antibody and hemagglutinin in canine homotransplantation. Ann. N.Y. Acad. Sci. 120:28, 1964. 3. Amos, D. B., and Wakefield, J. D. Detection of cytotoxic isoantibodies. Proc. Amer. Ass. Cancer Res. 3:204, 1961. 4. Batchelor, J. R., and Silverman, M. S. Further Studies on Interactions Between Sessile and Humoral Antibodies in Homograft Reactions (Ciba Foundation Symposium on Transplantation). Boston: Little, Brown, 1962. P. 216. 5. Cavins, J. A., Kasakura, S., Thomas, E. D., and Ferrebee, J. W. Recovery of lethally irradiated dogs following infusion of autologous marrow stored at low temperature in dimethylsulfoxide. Blood 20:730, 1962. 6. Chantler, S. M., and Batchelor, J. R. Changes
17.
18.
19.
20.
21.
22.
AND
RE JECTIOK
OF
RENAL
ALLOGRAFTS
in the host response following treatment with lyophilized tissue. Transplant. 2:75, 1964. Gorer, P. A., and Mikulska, Z. B. The antibody response to tumor inoculation: Improved methods of detection. Cancer Res. 14:651, 1954. Gorer, P. A., and O’Gorman, P. The cytotoxic activity of isoantibodies in mice. Transplant. Bull. 3:142, 1956. Govaerts, A. Cellular antibodies in kidney homotransplantation. J. Immunol. 85:516, 1960. Halasz, N. A., Orloff, M. J., and Hirose, F. Increased survival of renal homografts in dogs after injection of graft donor blood. Transplant. 2:453, 1964. Halasz, N. A., Seifert, L. N., and Orloff, M. J. Mechanisms of antigen-induced delay in organ allograft rejection. J. Surg. Res. 6:80, 1966. Immunological enhancement of tuKaliss, N. mor homografts in mice: A review. Cancer Res. 18:992, 1958. Kasakura, S., Thomas, E. D., and Ferrebee, J. W. Leukocytoxic isoantibodies in the clog. Trunsplant. 2:274, 1964. Leskowitz, S., and Waksman, B. H. Studies in immunization: I. The effect of route of injection of bovine serum albumin in Freund adjuvant on production of circulating antibody and delayed hypersensitivity. 1. Immunol. 84:58, 1960. Moller, G. Studies on the mechanism of immunological enhancement of tumor homografts: III. Interaction between humoral isoantibodies and immune lymphoid cells. J. Nut. Cancer Inst. 30: 1205, 1963. Nelson, D. S. Immunological enhancement of skin homografts in guinea pigs. Brit. J. Erp. Path. 43:2, 1962. Parkes, A. S. Enhancement of the survival of interstrain ovarian homografts in rats. Transplant. Bull. 5:45, 1958. Puza, A., Rubinstein, P., Kasakura, S., Vlahovic, S., and Ferrebee, J. W. The production of isoantibodies in the dog by immunization with homologous tissue. Transplant. 2:722, 1964. Simonsen, M. Biological incompatibility in kidney transplantation in dogs: II. Serological investigations. Acta Path. Microbial. Stand. 32:36, 1953. Snell, G. D., Winn, H. J., Stimpfling, J. H., and Parker, S. J. Depression by antibody of the immune response to homografts and its role in immunological enhancement. J. Exp. Med. 112: 293, 1960. Steinmuller, D. Failure of lyophilized tissues to evoke a secondary response to skin homografts. Transplant. 2:383, 1964. Swisher, S. N., Young, L. E., and Trabold, N. In vitro and in vivo studies of the behavior of canine erythrocyte-isoantibody systems. Ann. N.Y. Acad. Sci. 97:15, 1962.
HUTCHIN,
M.D.,*
TRANSPLANTATION of tissues and organs into genetically foreign recipients provokes an immune response which leads to destruction of the transplant. Subsequent grafts of tissue with the same genetic constitution are then rejected more rapidly in a second set reaction. Under certain circumstances, however, pretreatment of the recipient with donor tissue may prevent such a sequence of events. Steinmuller [21] failed to find accelerated rejection of skin grafts in mice pretreated with frozendried splenic or renal tissues. Other investigators found prolongation of allografted ovaries [ 171, skin [ 161, and kidneys [lo] in recipients specifically pretreated with donor antigens. This effect was attributed to “enhancement” although passive transfer experiments were not always successful [ 111. Because of the fundamental importance of the problem as well as possible information which might be obtained on the mechanisms of graft rejection, this study was undertaken to ascertain the effect of pretreatment with lyophilized sp 1enic tissue on subsequent canine renal allograft survival. Part of the study From the Department of Surgery and Division of Immunology, Duke University Medical Center, Durham, N.C. This work was supported in part by U.S. Public Health Service Grants GM 22454 and HD 00668. Submitted for publication Aug. 30, 1966. * Present address: Division of Thoracic Surgery, University of North Carolina, Chapel Hill, N.C.
SPLENIC REJECTION ALLOGRAFTS AND
D.
L.
TISSUE
TO OF
STICKEL,
M.D.
had as its objective to establish the presence or absence of isoantibodies directed against donor antigens. Since immunological enhancement depends for its effect on the presence of humoral antibodies [ 121, their demonstration would favor such a mechanism if prolonged allograft survival was demonstrated. Furthermore, the failure to find accelerated rejection in the presence of humoral antibodies could be taken as evidence against a predominant role of such antibodies in the allograft reaction.
MATERIALS
AND
METHODS
Thirty-six male mongrel dogs were used in this study: 12 dogs served as donors of the spleen and kidneys and 24 dogs served as recipients. The recipients were divided into three groups. Experimental
Group
Eight animals were pretreated with lyophilized splenic tissue, 1 gm. intravenously, 28 and I4 days prior to kidney transplantation, In each instance the spleen and the kidney were from the same donor. Specificity
Control
Group
Four animals were pretreated with lyophilized splenic tissue as in Group 1 but the 357
JOURNAL OF SURGICAL RESEARCH
VOL. 7 NO. 8, AUGUST 1967
kidney was transplanted from an unrelated dog. Baseline Control Group Twelve animals received a kidney allograft from an unrelated dog without prior pretreatment. BLOOD GROUPTYPING
Blood groups of all donor and recipient dogs were determined with specific canine antisera.* The tube method for the determination of blood groups A, C, and D, as described by Altman [l], was used, OPERATIVE
PROCEDURES
Splenectomy Splenectomy was performed on donor dogs four weeks prior to nephrectomy. The spleen was immediately lyophilized (see below) and stored at -20°C. until used. Renal biopsy was always obtained at the time of splenectomy to assure that normal, healthy kidneys were present for subsequent transplantation. Bilateral Nephrectomy plantation
and Kidney
Trans-
After bilateral nephrectomy, recipient dogs received a transplant of a single kidney from the appropriate donor. The kidney was placed in contralateral iliac fossa and vascular anastomoses established with the external iliac vessels of the host. Total renal ischemia time was 17 to 28 minutes. Ureterovesical anastomosis was performed by careful mucosa-tomucosa approximation. Postoperatively the dogs were maintained on ad libitum feedings of commercial dog chow and water. Blood for serological studies was obtained on the third, seventh, and fourteenth postoperative days. Serial blood urea nitrogen determinations were also performed. Complete autopsies were done at the time of death. * Generously supplied by Dr. S. Swisher, University of Rochester School of Medicine, Rochester, N.Y.
358
PREPARATION AND ADMINISTRATION LYOPHILIZED SPLENIC TISSUE
OF
The removed spleen was perfused with 250 ml. of heparinized saline (1 mg. per 10 ml.) to wash out residual blood. The spleen was then homogenized and lyophilized in 5 to S ml. amounts until dry. The lyophilized tissue was pooled, weighed out in aliquots of one gram and stored at -20°C. until used. Before intravenous injection into dogs, the lyophilized material was dissolved in about 20 ml. of saline, HEMAGGLUTINATION
TESTS
The dextran method of Gorer and Mikulska [7] was employed. Absorbed dog serum was substituted for human serum as the medium for suspending erythrocytes which were obtained either from freshly drawn blood or from blood stored in ACD solution at 4°C. CYTOTOXIC
TESTS
Cytotoxic activity of sera in recipient dogs was determined according to the method of Gorer and O’Gorman [S] or its modification of Amos and Wakefield [3]. Normal dog serum was used as a source of complement [ 131. Test lymphocytes were obtained from the appropriate donor animal when titrating for cytotoxic antibody in the serum of recipient dogs. When necessary, lymphocytes were stored for subsequent testing in TC-199 with 10% dimethylsulfoxide at -20°C. [5]. RESULTS The survival time after bilateral nephrectomy and renal transplantation in the three groups of dogs is recorded in Table 1. The mean survival times in all three groups are remarkably similar. The results of serological investigations are recorded below. INFLUENCE OF DONOR AND RECIPIENT BLOOD GROUPS ON THE SURVIVAL TIME AFTER RENAL TRANSPLANTATION
In agreement with the general prevalence of blood group antigens in dogs most animals
HUTCHIN AND STICKEL: SPLENIC TISSUE AND REJECTION OF RENAL ALLOGRAFTS
Table
KIDNEY TRANSPLANTATIONS I
1. Survival of Dogs With Renal Allografts in Various Groups Individual Survival Time (days)
Mean Survival (days 2 SD.)
Group 1 Experimental
8, 9, 12, 13, 13, 13, 17, 25
13.8 k 5.3
Group 2 Specificity
8, 12, 22
14.0 2 5.9
Recipient
Group
Dogs
control
16, 19, 26
HEMAGGLUTININ RESPONSE TO LYOPHILIZED SPLENIC TISSUE INJECTION AND RENAL ALLOGRAFTS
Figure 1 shows hemagglutinin titers in the three groups of recipient dogs. In agreement with previous reports [l, 221 no naturally occurring hemagglutinins were detected in the sera obtained prior to the injections of lyophilized splenic tissue or kidney grafting. After administration of lyophilized splenic tissue all dogs developed hemagglutinins, and their titers increased after a second injection. After kidney grafting the hemagglutinins showed a precipitous drop by the third postoperative day, followed by a subsequent rise. Hemagglutinin levels present before grafting were not attained, however, except in the A-negative, C-negative dog pretreated and grafted with tissue from an A-positive, C-positive animal (Group 1). In baseline control dogs (Group 3) hemagglutinins were detected in majority
WONEY TRANSPLANTATION SPECIFICITY WNTROL I
7, 8, 9, 9, 11, 13, 15, 15, 13.5 2 5.5
had both antigens A and C [ 11. Only one A-negative, C-negative dog was found. This dog, after pretreatment with lyophilized splenic tissue and a kidney graft from an A-positive, C-positive animal, developed the highest hemagglutinin response observed in this study and interestingly enough had also a very long survival after renal transplantation (25 days). No effect on the hemagglutinin titer or survival time was noted when the donor and recipient dogs differed only with respect to the weak blood group antigen D.
the
GROUP *
control
3
Baseline
10241
of animals
by the
seventh
day
I(IONEY TAAN~PLANTATION
/ I’: ,/ / 64
,/
n2w Fig. dogs lized renal
03
7
/
_.
14
TIMEbAYS) titers of representative 1. Hemagglutinin after intravenous administration of lyophisplenic tissue (Group 1 and Group 2) and transplantation (all groups).
after renal transplantation and their titers rose higher than in the pretreated dogs (Group 1 and Group 2). CYTOTOXIC
TESTS
No cytotoxic activity was detected in any of the tested sera, either in response to injections
of lyophilized
splenic
tissue
or after
kidney transplantation. PATHOLOGICAL
FINDINGS
One dog died from heart worms and another as a result of a surgical failure. These dogs were excluded from the experiment. All other dogs died as a consequence of renal allograft rejection. The vascular anastomoses as well as the ureterovesical anastomosis were patent in all cases. The transplanted kidney was grossly swollen, at times hemorrhagic, and
dark
red in color
on its cut
surface.
The
weights of the rejected kidneys were similar in all three groups of dogs, averaging 186 to 217 gm. 359
JOURNAL
OF
SURGICAL
RESEARCH
VOL.
7
NO.
On microscopic sections marked mononuclear interstitial and perivascular inflammatory response was evident in all kidneys. Focal and frequently generalized interstitial hemorrhages, acute vasculitis with occlusion of the cortical vessels,and medullary ischemia and necrosis were also present. Animals surviving longer did not differ from animals which succumbed early after renal transplantation except for the presence of more chronic changes such as beginning glomerular fibrosis and sclerosis. No difference could be detected in the histological appearance of the kidneys rejected by the pretreated or non-pretreated dogs. OTHER
FINDINGS
The blood urea nitrogen usually rose during the immediate postoperative period, then returned to normal on about the third to fifth postoperative day, remained at this level for several days, and then rose again progressively until death. In a few dogs the rise of blood urea nitrogen was steady and continuous from the time of the kidney grafting, No characteristic differences could be detected between the various groups of animals.
DISCUSSION This study demonstrated that prior exposure to donor lyophilized splenic tissue does not sensitize recipient dogs to subsequent renal allografts. The demonstration of hemagglutinins produced in all dogs in response to lyophilized splenic tissue pretreatment excludes immunological tolerance or paralysis as a cause of the failure of sensitization. Immunological enhancement, on the other hand, could account for some of the findings in this study, despite the fact that prolonged survival of the pretreated animals was not achieved. Administration of killed, as for example, lyophilized, tissue has been shown to result in a high humoral antibody response and minimal cellular immunity [20], especially when given by the intravenous route 14, 6, 141. Under these circumstances, accelerated 360
8,
AUGUST
1967
rejection might not result on second exposure to the antigen, and the presence of isoantibodies directed against donor antigens might actually interfere with the allograft reaction [12, 151. The amount of antibody present relative to the size of the grafted tissue is important in this respect, Prolonged survival of allografted kidneys was not obtained in this study, possibly because of insufficient isoantibody levels attained in the pretreated dogs, resulting in an incomplete antigen blockade of an organ as large as the kidney. This would permit an antigen “leak” to occur into the host’s immunological centers, followed by sensitization of the host and subsequent rejection of the foreign tissue. Alternatively, insufficient time after pretreatment may have been allowed for any existing cellular immunity to subside [6]. The antibodies produced in response to the injections of lyophilized splenic tissue crossreacted with the allografted kidney as demonstrated by the steep fall of hemagglutinins after renal transplantation. Neither the antibodies produced in response to the lyophilized splenic tissue nor the antibodies produced in response to renal transplantation were cytotoxic on repeated tests with the methods available. Previous serological studies after canine renal transplantation likewise failed to demonstrate cytotoxins [9], whereas hemagglutinins were demonstrated [ 191. Dissociation of hemagglutinin and cytotoxic activity after immunization of dogs is not uncommon, and repeated grafts from the same animal may be necessary before cytotoxins can be demonstrated [2, 181. The cross-reaction of antibodies produced in response to lyophilized splenic tissue with kidneys from unrelated dogs (Group 2) may be due to sharing of antigens between individual dogs [l] or due to a certain amount of inbreeding in the population of mongrel dogs available. After kidney grafting the fall in hemagglutinins was not as complete in this group of dogs, however, as in the animals receiving a kidney from the donor of the spleen (Group 1). The subsequent rise in titer was similar in both groups of pretreated dogs but smaller than in the non-pretreated animals
HUTCHIN
AND
STICKEL:
SPLENIC
(Group 3), indicating a partial inhibition of the antibody response after kidney grafting in the pretreated dogs. No correlation could be made between the height of the hemagglutinin titer observed postoperatively and the length of survival of the dogs. Animals which lived longer had higher hemagglutinin titers than those which died early. This finding suggests that the development of hemagglutinins after renal transplantation is not related directly to the development of immunity to the donor tissue and that factors other than the humoral antibody level must be implicated in the allograft rejection.
TISSUE
7.
8.
9. 10.
11.
12.
SUMMARY CONCLUSIONS
AND
The survival of dogs after renal transplantation was not affected by prior pretreatment with lyophilized splenic tissue from the donor of the kidney, despite the development of hemagglutinins directed against the donor antigens. The significance of these findings with respect to the allograft reaction and immunological enhancement is discussed.
13.
14.
15.
16.
REFERENCES 1.
Altman, B. Hemagglutinin in skin and kidney homotransplantation in dogs. Transpkznt. 3:326, 1965. 2. Altman, B., and Simonsen, M. Cytotoxic antibody and hemagglutinin in canine homotransplantation. Ann. N.Y. Acad. Sci. 120:28, 1964. 3. Amos, D. B., and Wakefield, J. D. Detection of cytotoxic isoantibodies. Proc. Amer. Ass. Cancer Res. 3:204, 1961. 4. Batchelor, J. R., and Silverman, M. S. Further Studies on Interactions Between Sessile and Humoral Antibodies in Homograft Reactions (Ciba Foundation Symposium on Transplantation). Boston: Little, Brown, 1962. P. 216. 5. Cavins, J. A., Kasakura, S., Thomas, E. D., and Ferrebee, J. W. Recovery of lethally irradiated dogs following infusion of autologous marrow stored at low temperature in dimethylsulfoxide. Blood 20:730, 1962. 6. Chantler, S. M., and Batchelor, J. R. Changes
17.
18.
19.
20.
21.
22.
AND
RE JECTIOK
OF
RENAL
ALLOGRAFTS
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