- Email: [email protected]
Immunologic Aspects of Renal Transplantation
Symposium on Cardiac and Renal Surgery
Immunologic Aspects of Renal Transplantation
WilliamE. Braun, M.D.
TERMINOLOGY The most common type of transplant performed is that between two genetically disparate members of the same species - an allogeneic graft (also called an allograft or homograft).72 A graft performed between genetically identical individuals is an isogeneic graft (also called a syngeneic graft or an isograft). If grafting is done by transferring tissue from one area to another on the same individual, this is still called an autograft. When grafting crosses species lines (e.g., pig to dog, or chimpanzee to man), this is a xenogeneic graft (also called a xenograft or heterograft) . When unspecified, the direction of the immunologic assault is from recipient (host) toward donor (graft), giving the host-versus-graft reaction. 51 It occurs because the donor graft contains antigens which are lacking in the recipient and to which the recipient responds. On the other hand, if the antigen deficit is solely in the opposite direction - that is, if the donor lacks antigens contained in the recipient-the graft itself can successfully mount a reaction against the foreign antigens of a susceptible recipient, giving rise to a graft-versus-host (GVH) reaction. 70 When a graft is placed in a recipient who has not been previously sensitized to the graft's foreign antigens, the resulting rejection is called a first-set rejection. The duration of graft survival may vary according to a number of factors, including the type of graft,27, 80 and donorrecipient characteristics such as species, strain, and sex. 51 When the recipient has already been sensitized to the same foreign donor antigens which the graft contains, the duration of graft survival is considerably shorter and rejection is called a second-set rejectionY
From the Division of Research, The Cleveland Clinic Foundation This work is supported by Contract PH-43-68-1298, Public Health Service, United States Department of Health, Education, and Welfare.
Surgical Clinics of North America- Vol. 51, No.5, October 1971
1161
1162
WILLIAM
E.
BRAUN
MECHANISMS The rejection of a foreign graft may be separated conceptually into two major areas: sensitization of the host and destruction of the donor graft. Sensitization, or the afferent limb of the immune response, begins with the recognition and processing of foreign antigens by the host. Donor antigens may leave the graft in renal venous blood,55 to produce sensitization in the lymph nodes, or host cells may be sensitized to donor antigens as they pass through the graft.77 Of fundamental importance is precisely where in the graft the antigen resides. The two prime sites are vascular endothelium and passenger leukocytes. Recent studies on this point will be summarized later. It has been shown that exclusion of lymphatic drainage from the kidney does not prevent the development of rejection, indicating that the vascular link is a sufficient route of sensitization for that organ. 36 For skin rejection to occur, however, the lymphatics must be reestablished. 2 Sensitization proceeds with the enlistment of regional lymphoid centers in a coordinated immunologic response of some magnitude following the entrapment of circulating antigen or the arrival of sensitized lymphocytes. Mitchison's demonstration that the passive transfer of regional lymph node cells from sensitized animals could confer an active immunity ("adoptive immunity") on immunologically naive animals 52 has clearly implicated lymph nodes in allograft rejection. Donor graft destruction (efferent or effector limb of the immune response) may be mediated by a cellular (see p. 1163) or antibody response (p. 1166), or both. Other mediators of tissue injury such as complement, polymorphonuclear leukocytes, the coagulation system, and certain vasoactive substances, are closely associated with these primary mechanisms (p. 1168). When a graft survives despite histoincompatibility one must consider that other mechanisms - such as enhancement or tolerance - may be responsible for sustaining it. Enhancement is an antibody-dependent phenomenon which occurs under circumstances that might typically produce sensitization but instead cause prolonged or complete acceptance of an allogeneic graft.38 Tolerance is a specific immunologic unresponsiveness, natural or acquired, which allows the prolonged acceptance of an allogeneic graft. 51 When considering these possible explanations for the survival of incompatible grafts one must not overlook other potentially significant modifiers of the intensity and rate of progression of rejection. These factors may include the inability of a chronically ill patient on dialysis to respond vigorously to an antigenic stimulus, the intermediate strength of the antigen in question and its susceptibility to immune suppressant drugs, the effect of cross-reactivity between certain donor and recipient antigens, and even the technical problems in specifically identifying all the histocompatibility antigens involved in the reaction. These points will be elaborated upon in the article on Histocompatibility Testing (p.1175).
IMMUNOLOGIC ASPECTS OF RENAL TRANSPLANTATION
1163
At the heart of all immunologic phenomena that develop in the course of transplanting an organ from one person to another is the confrontation between the foreign antigens residing in the graft and a recipient capable of responding to them. Focused on this prime problem of incompatibility, expressed as rejection, are a large number of preventive, diagnostic, and therapeutic techniques designed to eliminate it. However, despite the solid status of renal transplantation as one form of accepted treatment for end-stage renal disease, there remain definite inadequacies in these techniques because of incomplete information about the basic immunologic process. Consequently, for very practical reasons it seems appropriate to begin with a consideration of some fundamental questions concerning the rejection process. The major area for the prevention of rejection, histocompatibility testing, will be treated separately. The pathology of rejection and immunosuppression are discussed by other authors in this symposium.
MEDIATORS IN MODELS OF ALLOGRAFT REJECTION Cellular Response Because of modifications of the rejection process in humans brought about by genetic variability, uncertain strength of histocompatibility antigens, and differing responses to the same antigen by various hosts, a genetically defined animal model of transplantation has been chosen as the backbone for the following discussion of events in an unmodified rejection process. This model, furthermore, permits daily evaluation of renal histology in a large group of subjects-an opportunity that is impossible in humans. U sing inbred strains of rats, Guttmann, Lindquist, and their colleagues have performed over 2000 renal transplants. The rat strains were selected to provide either strong or weak genetic barriers to histocompatibility. When kidney transplants were performed across the strong barrier without the use of any immunosuppressive drugs, essentially complete rejection occurred in one week,34 though other workers have reported occasionally longer survivals. 78 The earliest changes noted were the presence of large mononuclear cells in the peritubular capillaries and the perivascular accumulation of such cells in the renal cortex. These cells, arising from the recipient, entered the graft in response to the presence of foreign antigens, and could be found, as described earlier in dogs,62 penetrating the walls of peritubular capillaries in the kidney. A thin homogeneous deposit of immunoglobulin G (IgG) and the third component of complement (beta 1 C) were found by immunofluorescence along the capillary wall, implying that an antigen-antibody reaction had taken place. 45 Thus, both cellular and humoral mechanisms, which will be discussed below, appear to have been involved. As greater numbers of cells entered the interstitium of the kidney, conspicuous perivascular infiltrates were formed. These infiltrates increased and extended diffusely into the interstitium, thus losing their vascular orientation. The origin of these cells has been debated. Their
1164
WILLIAM
E.
BRAUN
presumed derivation from the recipient was first challenged,1B and later supported. 60 Recent studies in a local graft-versus-host model have suggested that both contribute significantly to what has been called an "in vivo mixed lymphocyte culture" resembling rejection. 24 In the later stage of rejection there was glomerular hypercellularity of both endothelial and mesangial cells with occlusion of capillary lumina. Arteries showed endothelial cells which had large vesicular nuclei and swollen cytoplasm. Fibrinoid necrosis occurred later. When virtually all renal function had ceased in the allograft, the dense cellular infiltrates were accompanied by interstitial edema, tubular necrosis, and glomerular capillary occlusion with infiltration by polymorphonuclear cells. Studies of the microvasculature by latex injections showed that earlier patchy defects in cortical vasculature had progressed to a complete absence of cortical filling.2B This finding, reported nearly 20 years ago by Dempster,1B is not, however, specific for rejection. 35 Allografts in dogs and man have also been shown by xenon-133 wash-out techniques to have marked depression of outer cortical perfusion with rej ection. 65 In the interstitium was the cell most characteristic of acute rejection, a large mononuclear cell with a vesicular nucleus and pyroninophilic cytoplasm. The failure to demonstrate IgG or IgM in these cells at a time when they had abundant cytoplasmic ribosomes suggested that these changes were directed toward growth and replication rather than antibody synthesis. 45 Enzyme histochemical studies of these acutely rejected allografts were compatible with a process of progressive ischemia, indicating that the immunologic process of tissue injury worked through a common pathway usable by other modes of injury.46 Mature lymphocytes and plasma cells also composed much of these infiltrations. But only very few of these cells may have actually been specifically sensitized to the donor antigens. 49 Since the accumulation of these cell masses may obliterate tubules, one must be careful under these circumstances not to confuse the large regenerating tubular epithelial cells with the large mononuclear cells of rejection. Such cellular infiltrates, with many lymphoblastic cells ("immunoblasts") occurring both in the interstitium and vessel walls, are the primary finding in acute rejection both in animals and in man. Some of these lymphoblasts, stimulated to transformation by foreign antigen, are believed to arise from that portion of the small lymphocyte population which is thymus-dependent. 66 In contrast to the smalllymphocyte population described later (see Antibody Response, p. 1166), these lymphocytes are long-lived and constitute the majority of the circulating lymphocyte pOOP1 Both the small lymphocytes destined for cellular responses and those destined for antibody production originate from bone marrow precursors. It is uncertain whether antigens on living cells, such as transplantation antigens, can directly stimulate this thymusdependent population of lymphocytes or whether antigen-processing by a macrophage is necessary. It should be kept in mind, however, that "non-immunologic;' substances, including bacterial products, can also induce transformation in vitro. In their role as "killer" cells, this popula-
IMMUNOLOGIC ASPECTS OF RENAL TRANSPLANTATION
1165
tion of transforming lymphocytes has an arsenal of immunologic weapons to destroy tissue. These include a factor to immobilize macrophages at the site of antigen (MIF), a lymphotoxin, a chemotactic factor for monocytes,43 to mention a few, as well as lysosomes,19 a capacity to augment antibody response 13 and to mimic C ' 8 in activating earlier complement components bound to cell membranes. 58 One of these substances, macrophage inhibition factor, has provided a means of detecting allograft rej ection in animals. 26 Perhaps even closer to the problem of human transplantation was the study of rat renal allografts performed across a weaker barrier to histocompatibility. 50 In this model, again studied in precisely defined inbred strains without any therapy, there was a period of acute, spontaneously reversible rejection. Cellular infiltrates were not visible until several days after transplantation when typical large mononuclear cells accumulated in perivascular areas. These cells reached a maximum at 2 to 3 weeks and then progressively decreased. Functional improvement paralleled the diminution in cellular infiltration. About this time the character of the cells changed to include more mature lymphocytes and plasma cells and fewer large mononuclear cells. Over a similar time period in man a comparable shift in cell type has been describedY Just as in human grafts, there was a great variability in these and other changes at the time of early rejection. In some animals there was no glomerular or tubular damage, while in others there was tubular necrosis, necrosis of glomerular cells, and capillary occlusion by fibrin and platelets. As these allografts went on to long-term survival after weathering a rejection, this variability in morphology persisted. Some kidneys were completely normal by light microscopy. In others, mature lymphocytes and plasma cells could be found in perivascular locations with tubular atrophy and interstitial fibrosis. Glomerular lesions, which were composed of thickened capillary walls and mesangial enlargement, were the most significant alterations in this, model of chronic rejection. Subendothelial fibrosis developed in small arteries but not to the extent seen in humans.l1 When a donor skin graft was placed on a recipient which was sustaining at 7 months a long-surviving renal allograft, the skin graft also had a very prolonged survival time. This, along with the failure of serum from such recipients to transfer passively a protective effect for other skin grafts, suggested that the recipient had not developed enhancing antibody, but rather a state of tolerance to the donor tissue. 5o Interestingly, skin and heart allografts have short survival and renal allografts prolonged survivaP7,80 when performed on separate groups of the same inbred strains of rats without treatment. One of the most important concepts pursued in animal models 24 ,76 has been that the primary site of antigen in an allograft is not the organ parenchyma itself, but rather donor leukocytes of bone marrow origin trapped in the graft and carried over to the new host as "passenger leukocytes." The identity of these cells has been assessed further by using bone marrow chimeras (an individual of one genetic strain pos-
1166
WILLIAM
E.
BRAUN
sessing the bone marrow cells of another genetic strain). When the donor's hematopoietic system was made syngeneic with the recipient, but the kidney was not, the rejection of the kidney was impaired. 33 This indicated that when marrow-derived cells transferred within the graft were of recipient and not donor origin, a major site of foreign antigen was eliminated. Although one must be cautious in extrapolating from animal work to human beings, certain observations in the animal warrant emphasis. Even in genetically homogeneous transplant systems without any immunosuppressive drugs, whether across a strong or weak histocompatibility barrier, the rate and intensity of rejection, judged by both graft morphology and survival, may show considerable variation. This suggests that either subtle genetic differences, slight technical variables, or nonspecific inflammatory mechanisms could cause such changes. One should not be surprised, then, if similarly matched human donorrecipient pairs do not all respond identically. Spontaneous reversal of acute rejection may occur with weak histocompatibility barriers, and the allograft may survive for a prolonged time. This may be an important fact to recall when one is faced with an element of rejection in a situation dominated by a harmful factor (such as infection) which could become a fatal factor if immunosuppressive drugs were massively applied before infection was controlled. The presence of mononuclear cell infiltrates in the interstitium, in the absence of a significant component of lymphoblastic forms, does not necessarily mean acute rejection and may, as in enhanced rat renal allografts, eventually disappear without residual functional impairment. The difference between skin, heart, and kidney graft survivals indicates that the same apparent degree of incompatibility does not affect all tissues equally. Whether this is due to quantitative differences in histocompatibility antigens,3,5 tissue-specific antigens,6 or other mechanisms 80 is uncertain. Perhaps the most valuable asset of this animal model thus far has been the localization of the site of antigen and the development of therapeutic maneuvers to selectively control it.
Antibody Response Humoral antibody is produced by plasma cells derived from bone marrow precursors whose evolution through a small lymphocyte and subsequent blast stage is independent of thyriIus control. These small lymphocytes are short-lived, represent the minority of the circulating lymphocyte pool, and are more confined to lymphoid organs. 31 In contrast to the classical cellular mechanism of rejection which had early support from the passive transfer of tumor allograft immunity with cells,52 the appreciation of humoral mechanisms, seemingly limited to leukotic grafts,30, 51 has been relatively slow to develop since Simonsen's early description of hemagglutinins in allografted dogs. 69 This may have been due to the fact that complement-fixing antibodies were not found 69 and that passive transfer of renal allograft immunity with serum proved difficult to accomplish unless cross-reacting renal tissues were reduced 56 or the serum was injected directly into the renal artery. I, 23 Direct renal
IMMUNOLOGIC ASPECTS OF RENAL TRANSPLANTATION
1167
artery injections for several days of the globulin fraction of donorspecific sensitized serum caused severe allograft damage which paralleled the serum's cytotoxic titer.23 The changes were primarily vascular wall and parenchymal necrosis, but cellular proliferation and mesangial condensation were also seen in the glomerulus. Of particular interest was the presence of perivascular mononuclear cell infiltrations believed to be so typical of purely cell-mediated injury. 16, 23 Since the kidney being injected was the donor's own kidney, the cells could not have arisen because of any allogeneic incompatibility. Because the kidney had never been transplanted to another animal and returned, as in Clark's experiment,t4 there could not have been any transfer of the previous host's leukocytes to initiate rejection. Elution of IgG and IgM from unmodified, rejected canine allografts further supported the direct participation of antibody in rejection. 42 Man himself became an early model for the most dramatic example of the effect of cytotoxic antibody. In patients with preformed antibody to their donor's cells, a rapid, irreversible form of renal failure occurred which was called the hyperacute rejection.4i Frequently, within minutes after transplantation, the allograft became cyanotic and urine production ceased. The extensive thrombotic lesions in the glomeruli and renal vessels have suggested a parallelism to the generalized Shwartzman reaction. 74 Circulating antibody has also been incriminated in less virulent forms of allograft injury. Within 1 hour of transplantation in man glomerular endothelial, epithelial, and basement membrane changes were apparene9 which were similar to lesions attributed to immunologic injury but seen 13/4 to 21f2 years after transplantation. 61 A probable explanation for such rapid damage would be the presence of preexisting antibodies directed against donor tissue. Antibody developing to donor cells after transplantation may also be harmful to the allograft. In unsensitized dogs cytotoxic antibody titers increased and allografts were rejected when azathioprine was stopped. 82 Jeannet37 tested 35 patients before and after transplantation for humoral antibody by three techniques: leukoagglutination, cytotoxicity, and mixed agglutination. Nine of 10 patients with antibody before transplantation and 12 of 16 who developed antibody against their donor after transplantation rejected their kidney or died. Good to excellent clinical courses accompanied the 5 patients who never developed antibody and 5 of the seven patients whose antibody was not directed against donor cells. A close association was noted between the presence of humoral antibodies and the development of vascular lesions in the allograft. There is also abundant immunofluorescent data in humans to document the deposition of immunoglobulins, presumably acting as antibody, in both renal lO , 11,29,47,59 and heart68 , 75, 81 allografts. The diagnostic value of circulating cytotoxic antibody has been seen primarily in cardiac allografts. 25 Cytotoxic antibodies to renal allografts belong to the IgG class of immunoglobulins but have not yet been classified as to their subclass .
1168
WILLIAM
E.
BRAUN
This information would help to assess its avidity for fixing complement and its potential to produce cellular damage by this means. An IgM anti-gamma globulin rheumatoid-like factor has also been found in renal transplant recipients, but its clinical significance is presently uncertain. 39 Perhaps the most stimulating work involving antibody has recently come from animal models in which enhancement was produced. This phenomenon was extensively explored in mouse tumor allografts by Kaliss. 38 Its potential value to other transplants was shown in the rat renal allograft model by Stuart, who obtained marked prolongation of kidney survival by pretreatment with donor antigen plus antiserum to donor cells. 78 The use of both substances complicated the interpretation of the impressive results. Ockner et al., using a single intravenous dose of 10 7 donor bone marrow cells one to two weeks before transplantation, also produced enhancement and achieved essentially indefinite survival of rat kidney allografts. 57 The rejection of a subsequent skin graft and the passive transfer of the protective effect by the recipient's serum indicated that enhancement was achieved. By ingenious manipulation of the genetic strains involved and the establishment of bone marrow chimeras, it was then possible to show that the enhancing antibody was directed specifically against bonemarrow-derived cells located in the graft, but having a genetic source different from that of the renal parenchyma. It has been shown, too, that this method of enhancement can overcome a state of pre sensitization. When Lucas produced enhancement with homologous anti-donor lymphocyte serum, there was localization of enhancing serum in the transplanted kidney and a decreased availability of antigen sites on the vascular endothelium. 48 These data suggested "coating" of crucial antigenic sites by the serum and a subsequent modulation of these antigens to permit prolonged survival in a state of "immunologic adaption." After achieving enhancement in the rat model with antigraft alloantibody, Batchelor has recently reported a provocative study using "enhancing" antibody in a human parent-child transplant. 4 The delayed function of the allograft was a disturbing feature, though the ability to withdraw later azathioprine and prednisone was encouraging.
Complement, the Polymorphonuclear Leukocyte, and Coagulation Other major mediators of tissue injury in allograft rejection include the complement system, polymorphonuclear leukocytes, and the coagulation system. Lower levels of C'2 were found in allograft rejection both in rats and man. 32 Numerous studies in animals and in man have documented tissue deposition of C'3 in the rejected kidney by immunofluorescence, implying fixation by antigen-antibody complexes. 10, 11,29,47,59 Unstable serum complement levels and increased catabolism of C'3 and C'4 have accompanied rejections of human allografts. 12 Of several substances produced in the complement cascade that might induce allograft injury, the chemotactic factors that attract polymorphonuclear leukocytes have received the most attention. The importance of the polymorphonuclear leukocyte was appreciated by its
IMMUNOLOGIC ASPECTS OF RENAL TRANSPLANTATION
1169
proposed role in producing the glomerular basement membrane damage found in experimental forms of glomerulonephritis. 20 However, electron microscopic documentation of damage by polymorphonuclear leukocytes in human glomerulonephritis has come only recently. 8 The appearance of these cells in glomeruli within 1 hour of transplantation in man is one finding different from that in animal work. 79 The severity of polymorphonuclear leukocyte infiltration has correlated with a poorer prognosis for the survival of the allograft.40 Increased numbers of polymorphonuclear leukocytes in the glomeruli were a prominent feature of the hyperacute form of allograft rejection, though the picture was dominated by the coagulation process. Emphasis on the former finding suggested its relation to the Arthus phenomenon,54 while dominance of the intravascular clotting events related it to the generalized Shwartzman reaction. 74 Unlike the generalized Shwartzman reaction clotting is usually confined to the kidney.9, 17 The role of preformed antibody in the induction of this form of rejection has been well documented (see Histocompatibility Testing, p. 000). Evidence of recurring participation of clotting in acute and chronic rejection was shown by the urinary excretion of fibrin degradation products. 7, 15 There is interweaving of the clotting system, at the level of plasmin,63 and Hageman factor 22 with the complement system. In addition there is a mutual inhibitor of C'l esterase and plasmin. 64 These relationships may help to explain the triggering of the clotting system at times when complement has been activated by antigen-antibody complexes. Despite the immediate involvement of the clotting system in the obliteration of xenografts,67 anticoagulant and defibrinating agents did not prolong survival of xenografts beyond 10 minutes. 53 ,71 The longest survivals, which still were less than 4 hours, were achieved using donor liver as an immunoabsorbant. 53 , 71 Further progress will require suppression of antibody synthesis rather than temporary lowering of circulating antibody.
RECURRENT GLOMERULONEPHRITIS This concluding section is concerned with the recurrence of glomerulonephritis in the transplanted kidney. In Glassock's study of 22 identical twin transplants, 11 of 17 whose original disease was glomerulonephritis had recurrence of a similar disease in the isograft. 29 The mesangial and focal linear immunofluorescent deposits of IgG and /31C were not typical of nephritis induced by circulating anti-glomerular basement membrane antibody. Further work with renal allografts led to the description of the first human case of transplant glomerulonephritis caused by circulating anti-glomerular basement membrane antibody.44 Several important points were evident in this case: the clinical course of the original disease was rapidly progressive (less than 1 year); the original kidneys had a linear fluorescent pattern of IgG and /31C on the glomerular basement membrane; circulating anti-glomerular basement
1170
WILLIAM
E.
BRAUN
membrane antibody was demonstrable after removal of the original kidneys; antibody found in the patient's circulation caused proteinuria and anti-glomerular basement membrane nephritis in monkeys. The sibling allograft very early demonstrated heavy proteinuria and linear fluorescent deposits of IgG, indicative of recurrence of the original disease. However, except for proteinuria (about 1 to 3 gm. per 24 hr.) for the first 6 months after transplantation, the kidney has had excellent function for at least 3 years. Extension of this work has shown that not only the anti-glomerular basement membrane form of nephritis has recurred in renal transplants, but also immune complex nephritis. The true incidence of such recurrent disease was difficult to ascertain since only a selected population was under study. However, of 11 allograft recipients whose original disease was anti-glomerular basement membrane nephritis (2 with Goodpasture's syndrome and 9 with subacute or chronic glomerulonephritis), 7 have had recurrent nephritis with impairment of renal function and 4 have had no apparent recurrence (2 with Goodpasture's syndrome and 2 with chronic glomerulonephritis).21 The latter 4 patients were managed with immunosuppressive drugs during a 1 to 2 month anephric state before being transplanted. Of 19 patients who originally had complex-induced nephritis (17 with chronic glomerulonephritis and 2 with systemic lupus erythematosus), 4 thus far have recurrent nephritis (3 with chronic glomerulonephritis and 1 with systemic lupus erythematosus).21 Such alternate immunologic mechanisms of allograft injury must be appreciated in order that appropriate management be afforded the patient and the cause of impaired allograft function be correctly interpreted.
REFERENCES 1. Altman, B.: Tissue transplantation: Circulating antibody in the homotransplantation of kidney and skin. Ann. Roy. ColI. Surg., 33 :79, 1963. 2. Barker, C. F., Billingham, R E., and Shaffer, C. F.: Further studies on the hamster's cheek pouch as a privileged site. Transplant. Proc., 1 :597, 1969. 3. Basch, R S., and Stetson, C. A.: Quantitative studies on histocompatibility antigens of the mouse. Transplantation, 1 :469, 1963. 4. Batchelor, J. R, Ellis, F., French, M. E., Bewick, M., Cameron, J. S., and Ogg, C. S.: Immunological enhancement of human kidney graft. Lancet, 2:1007,1970. 5. Berah, M., Hors, J., and Dausset, J.: A study of HL-A antigens in human organs. Transplantation, 9:185, 1970. 6. Boyse, E. A., Lance, E. M., Carswell, E. A., Cooper, S., and Old, L. J.: Rejection of skin allografts by radiation chimaeras: Selective action in the specification of cell surface structure. Nature, 227:901,1970. 7. Braun, W. E., and Merrill, J. P.: Urine fibrinogen fragments in human renal allografts: A possible mechanism of renal injury. New Eng. J. Med., 278:1366,1968. 8. Burkholder, P. M.: Ultrastructural demonstration of injury and perforation of glomerular basement membrane in acute proliferative glomerulonephritis. Amer. J. Path., 56:251,1969. 9. Busch, G. J., Braun, W. E., Carpenter, C. B., Corson, J. M., Galvanek, E. R, Reynolds, E. S., Merrill, J. P., and Dammin, G. J.: Intravascular coagulation (IVC) in human renal allograft rejection. Transplant. Proc., 1 :267, 1969. 10. Busch, G. J., Braun, W. E., Glassock, R J., and Dammin, G. J.: Immunofluorescent study of 46 human renal allograft biopsies. Proc. First Annual Meeting of Amer. Soc. of Neph., Los Angeles, 1967, p. 11.
IMMUNOLOGIC ASPECTS OF RENAL TRANSPLANTATION
1171
11. Busch, G. J., Reynolds, E. S., Galvanek, E. G., Braun, W. E., and Dammin, G. J.: Human renal allografts: The role of vascular inj ury in early graft failure. Medicine, 50: 29, 1971. 12. Carpenter, C. B., Ruddy, S., Shehadeh, I. H., Muller-Eberhard, H. J., Merrill, J. P., and Austen, K F.: Complement metabolism in man: hypercatabolism of the fourth (C4) and third (C3) components in patients with renal allograft rejection and hereditary angioedema (HAE). J. Clin. Invest., 48:1495, 1969. 13. Claman, H. N., and Chaperon, E. A.: Immunologic complementation between thymus and marrow cells-a model for the two-cell theory of immunocompetence. Transplant. Rev., 1 :92, 1969. 14. Clark, D. S., Foker, J. E., Good, R A., and Varco, R L.: Humoral factors in canine renal allograft rejection. Lancet, 1 :8, 1968. 15. Clarkson, A. R, Morton, J. B., and Cash, J. D.: Urinary fibrin/fibrinogen degradation products after renal homotransplantation. Lancet, 2: 1220, 1970. 16. Cochrum, K C., Davis, W. C., Kountz, S. L., and Fudenberg, H. H.: Renal autograft rejection initiated by passive transfer of immune plasma. Transplant. Proc., 1 :301, 1969. 17. Coleman, R W., Braun, W. E., Busch, G. J., Dammin, G. J., and Merrill, J. P.: Coagulation studies in the hyperacute and other forms of renal allograft rejection. New Eng. J. Med., 281 :685,1969. 18. Dempster, W. J.: Kidney homotransplantation. Brit. J. Surg., 40:447, 1953. 19. Diengdoh, J. V., and Turk, J. L.: Immunological significance of lysosomes within lymphocytes in vivo. Nature, 207:1405,1965. 20. Dixon, F. J.: Glomerulonephritis and immunopathology. Hosp. Pract., 2:35,1967. 21. Dixon, F. J., McPhaul, J. J., and Lerner, R A.: The contribution of kidney transplantation to the study of glomerulonephritis in renal transplants. Transplant. Proc., 1: 194, 1969. 22. Donaldson, V. H.: Mechanisms of activation of C'l esterase in hereditary angioneurotic edema plasma in vitro: Role of Hageman factor, clot-promoting agent. J. Exper. Med., 127:411, 1968. 23. Dubernard, J. M., Carpenter, C. B., Busch, G. J., Diethelm, A. G., and Murray, J. E.: Rejection of canine renal allografts by passive transfer of sensitized serum. Surgery, 64:752,1968. 24. Elkins, W. L., and Guttmann, R D.: Pathogenesis of a local graft versus host reaction: Immunogenicity of circulating host leukocytes. Science, 159: 1250, 1968. 25. Ellis, R J., Lillehei, C. W., Fischetti, V. A., and Zabriskie, J. B.: Heart-reactive antibody: An index of cardiac rejection in human heart transplantation. Circulation,41 and 42 (Supp!. 2): 91, 1970. 26. Ferraresi, R W., Gorhman-Yahr, M., and Raffel, S.: Studies of the macrophage-inhibition test. III. Use of the macrophage migration-inhibition technic in detection of active graft immunity. Transplantation, 10:237, 1970. 27. Freeman, J.' S., Reemtsma, K, and Steinmuller, D.: Comparative survival of transplanted heart and skin in inbred rats. Circ. Supp!. II 41 and 42 (Supp!. 11):86, 1970. 28. Gardner, L. B., Guttmann, R D., and Merrill, J. P.: Renal transplantation in the inbred rat. IV. Alterations in the microvasculature in acute unmodified rejection. Transplantation, 6:411,1968. 29. Glassock, R J., Feldman, D., Reynolds, E. S., Dammin, G. J., and Merrill, J. P.: Human renal isografts: A clinical and pathologic analysis. Medicine, 47:411,1968. 30. Gorer, P. A.: Some recent work on tumor immunity. Adv. Cancer Res., 4:149, 1956. 31. Gowans, J. L.: Immunobiology of the small lymphocyte. Hosp. Pract., 3:34,1968. 32. Guiney, E. J., Austen, K F., and Russell, P. S.: Measurement of serum complement during homograft rejection in man and rat. Proc. Exper. Bio!. Med., 115:1113,1964. 33. Guttman, R D., Lindquist, R R, and Ockner, S. A.: Renal transplantation in the inbred rat. IX. Hematopoietic origin of an immunogenic stimulus of rejection. Transplantation, 8:472,1969. 34. Guttmann, R D., Lindquist, R R, Parker, R M., Carpenter, C. B., and Merrill, J. P.: Renal transplantation in the inbred rat. I. Morphologic, immunologic and functional alterations during acute rejection. Transplantation, 5:668,1967. 35. Hollenberg, N. K, Epstein, M., Rosen, S. M., Basch, R I., Oken, D. E., and Merrill, J. P.: Acute oliguric renal failure in man: Evidence for preferential renal cortical ischemia. Medicine, 47:455,1968. 36. Hume, D. M., and Egdahl, R H.: Progressive destruction of renal homografts isolated from the regional lymphatics of the host. Surgery, 38:194, 1955. 37. Jeannet, M., Pinn, V. W., Flax, M. H., Winn, H. J., and Russell, P. S. Humoral antibodies in renal allotransplantation in man. New Eng. J. Med., 282:111,1970. 38. Kaliss, N.: Dynamics of immunologic enhancement. Transplant. Proc., 2:59, 1970. 39. Kano, K, and Milgrom, F.: Anti-gamma globulin factors in human allograft recipients. Transplantation, 6: 111, 1968. 40. Kincaid-Smith, P., Morris, P. J., Saker, B. M., Ting, A., and Marshall, V. C.: Immediate renal-graft biopsy and subsequent rejection. Lancet, 2:748,1968.
1172
WILLIAM
E.
BRAUN
41. Kissmeyer-Nielsen, F., Olsen, S., Petersen, V. P., Fjeldborg, 0.: Hyperacute rejection of kidney allografts, associated with pre-existing humoral antibodies against donor cells. Lancet,2:662,1966. 42. Kolker, P., Hampers, C. L., Hager, E. B., and Leon, P. E.: Characterization of immunologically active substances from canine renal allotransplants. Transplantation, 6:131, 1968. 43. Lawrence, H. S., and Landy, M., eds.: Mediators of Cellular Immunity. New York, Academic Press, 1969. 44. Lerner, R A., Glassock, R J., and Dixon, F. J.: The role of anti-glomerular basement membrane antibody in the pathogenesis of human glomerulonephritis. J. Exper. Med., 126:989, 1967. 45. Lindquist, R R, Guttmann, R D., and Merrill, J. P.: Renal transplantation in the inbred rat. II. An immunohistochemical study of acute allograft rejection. Amer. J. Path., 52:531,1968. 46. Lindquist, R R, Guttmann, RD., and Merrill, J. P.: Renal transplantation in the inbred rat. V. Histochemical studies of acute renal allograft rejection. Amer. J. Path., 52:1145, 1968. 47. Lindquist, R R, Guttmann, R D., Merrill, J. P., and Dammin, G. J.: Human renal allografts. Interpretation of morphologic and immunohistochemical observations. Amer. J. Path., 53:851, 1968. 48. Lucas, Z. J., Markley, J., and Travis, M.: Immunologic enhancement of renal allografts in rat. I. Dissociation of graft survival and antibody response. Fed. Proc., 29:2041,1970. 49. McCluskey, R T., Benacerraf, B., and McCluskey, J. W.: Studies on the specificity of the cellular infiltrate in delayed hypersensitivity reactions. J. Immunol., 90:466, 1963. 50. Mahabir, R N., Guttmann, R D., and Lindquist, R R: Renal transplantation in the inbred rat. X. A model of "weak histoincompatibility" by major locus matching. Transplantation, 8:369,1969. 51. Medawar, P. B.: The homograft reaction. Proc. Roy. Soc. B., 149:145,1958. 52. Mitchison, N. A.: Studies on the immunological response to foreign tumor transplants in the mouse. J. Exper. Med., 102:157,1955. 53. Moberg, A. W., Shons, A. R, Gewurz, H., Mozes, M., and Najarian, J. S.: Prolongation of renal xenografts by the simultaneous sequestration of preformed antibody and the inhibition of complement, coagulation, and antibody synthesis. Abstracts of Third Internat. Congr. Transplantation Society, The Hague, Netherlands, 1970, p. 185. 54. Myburgh, J. A., Cohen, I., Gecelter, L., Meyers, A. M., Abrahams, C., Furman, K I., Goldberg, B., and van Blerk, P. J. P.: Hyperacute rejections in human-kidney allograftsShwartzman or Arthus? New Eng. J. Med., 281 :685,1969. 55. Najarian, J. S., May, J., Cochrum, K. C., Baronberg, N., and Way, L. W.: Mechanism of antigen release from canine kidney homotransplants. Ann. N.Y. Acad. Sci., 129:76, 1966. 56. Najarian, J. S., and Perper, R. J.: Participation of humoral antibodies in allogeneic organ transplantation rejection. Surgery, 62:213,1967. 57. Ockner, S. A., Guttmann, R D., and Lindquist, R R: Renal transplantation in the inbred rat. XIV. Mechanism of the modified rejection produced by bone marrow cell pretreatment. Transplantation, 9:39, 1970. 58. Perlmann, P., Perlmann, H., Muller-Eberhard, H. J., and Manni, J. A.: Cytotoxic effects of leukocytes triggered by complement bound to target cells. Science, 163:937,1969. 59. Porter, K A., Andres, G. A., Calder, M. W., Dossetor, J. B., Hsu, K. C., Rendall, J. M., Seegal, B. C., Starzl, T. E.: Human renal transplants II. Immunofluorescent and immunoferritin studies. Lab. Invest., 18: 159, 1968. 60. Porter, K A., and Calne, R Y.: Origin of the infiltrating cells in skin and kidney homografts. Transplant. Bull., 7:458,1960. 61. Porter, K A., Dossetor, J. B., Marchioro, T. L., Peart, W. S., Rendall, J. M., Starzl, T. E., Terasaki, P. I.: Human renal transplants. I. Glomerular changes. Lab. Invest., 16: 153,1967. 62. Porter, K A., Joseph, N. H., Rendall, J. M., Stolinski, C., Hoehn, R J., and CaIne, R Y.: The role of lymphocytes in the rejection of canine renal homotransplants. Lab. Invest., 13:1080,1964. 63. Ratnoff, O. D., and Naff, G. B.: Conversion of C'1 s to C'1 esterase by plasmin and trypsin. J. Exper. Med., 125:337,1967. 64. Ratnoff, O. D., Pensky, J., Ogston, D., and Naff, G. B.: The inhibition of plasmin, plasma kallikrein, plasma permeability factor, and the C'1r SUbcomponent of complement by serum C'1 esterase inhibitor. J. Exper. Med., 129:315,1969. 65. Retik, A. B., Hollenberg, N. K, Rosen, S. M., Merrill, J. P., and Murray, J. E.: Cortical ischemia in renal allograft rejection. Surg. Gynec. Obstet., 124:989, 1967. 66. Roitt, I. M., Greaves, M. F., Torrigiani, G., Brostoff, J., and Playfair, J. H. L.: The cellular basis of immunological responses. Lancet, 1 :367, 1969.
IMMUNOLOGIC ASPECTS OF RENAL TRANSPLANTATION
1173
67. Rosenberg, J. C., Broersma, R J., Bullemer, G., Mammen, E. F., Lenaghan, R, and Rosenberg, B. F.: Relationship of platelets, blood coagulation, and fibrinolysis to hyperacute rejection of renal allografts. Transplantation, 8:152, 1969. 68. Rossen, R D., Butler, W. T., Reisberg, M. A., Mittal, K. K., Johnson, A. H., Brooks, D. K., Leachman, RD., Milans, J. D., Montgomery, J. R, Nora, T. J., and Rochelle, D. G.: Immunofluorescent and serologic studies of the humoral antibody response to human cardiac allografts. Abstracts of Third Int. Congr. Transplantation Society, The Hague, Netherlands, 1970, p. 251. 69. Simonsen, M.: Biological incompatibility in kidney transplantation in dogs. II. Serological investigations. Acta Pathol. Micro. Scand., 32:36,1953. 70. Simonsen, M.: Graft versus host reactions, their natural history and applicability as tools of research. Prog. Allergy, 6:349, 1962. 71. Slapak, M., Joison, J., Greenbaum, M., Saravis, C. A., and McDermott, W. V., Jr.: The effect of heparin, Arvin, liver perfusion and antiplatelet serum on the rejection of the pig kidney by the dog. Abstracts of Third Internat. Congr. Transplantation Society, The Hague, Netherlands, 1970, p. 189. 72. Snell, G. D.: The terminology of tissue transplantation. Transplantation, 2 :655, 1964. 73. Starzl, T. E., Boehmig, H. J., Amemiya, H., Wilson, C. B., Dixon, F. J., Giles, G. R, Simpson, K. M., and Halgrimson, C. G.: Clotting changes during rapid renal homograft rejection. New Eng. J. Med., 283:383,1970. 74. Starzl, T. E., Lerner, R A., Dixon, F. J., Groth, C. G., Brettschneider, L., and Terasaki, P. I.: Shwartzman reaction after human renal homotransplantation. New Eng. J. Med. 278:642, 1968. 75. Stastny, P.: Bound immunoglobulin and complement in heart allografts undergoing rejection. Transplantation, 10:248, 1970. 76. Steinmuller, D.: Immunization with skin isografts taken from tolerant mice. Science, 158:157,1967. 77. Strober, S., and Gowans, J. L.: The role of lymphocytes in the sensitization of rats to renal homografts. J. Exper. Med., 122:347,1965. 78. Stuart, F. P., Saitoh, T., Fitch, F. W., and Spargo, B. H.: Immunologic enhancement of renal allografts in the rat. Surgery, 64:17, 1968. 79. Weymouth, R J., Seibel, H. R, Lee, H. M., Hume, D. M., and Williams, G. M.: The glomerulus in man one hour after transplantation. Amer. J. Path., 58 :85, 1970. 80. White, F., and Hildemann, W. H.: Kidney versus skin allograft reaction in normal adult rats of inbred strains. Transplant. Proc., 1 :395, 1969. 81. Williams, G. M., DePlanque, B., Graham, W. H., and Lower, R R: Participation of antibodies in acute cardiac-allograft rejection in man. New Eng. J. Med., 281 :1145,1969. 82. Yamada, T., and Kay, J. H.: Kidney homotransplantation with special reference to cytotoxic antibody. Surgery, 63 :637, 1968.
Immunologic Aspects of Renal Transplantation
WilliamE. Braun, M.D.
TERMINOLOGY The most common type of transplant performed is that between two genetically disparate members of the same species - an allogeneic graft (also called an allograft or homograft).72 A graft performed between genetically identical individuals is an isogeneic graft (also called a syngeneic graft or an isograft). If grafting is done by transferring tissue from one area to another on the same individual, this is still called an autograft. When grafting crosses species lines (e.g., pig to dog, or chimpanzee to man), this is a xenogeneic graft (also called a xenograft or heterograft) . When unspecified, the direction of the immunologic assault is from recipient (host) toward donor (graft), giving the host-versus-graft reaction. 51 It occurs because the donor graft contains antigens which are lacking in the recipient and to which the recipient responds. On the other hand, if the antigen deficit is solely in the opposite direction - that is, if the donor lacks antigens contained in the recipient-the graft itself can successfully mount a reaction against the foreign antigens of a susceptible recipient, giving rise to a graft-versus-host (GVH) reaction. 70 When a graft is placed in a recipient who has not been previously sensitized to the graft's foreign antigens, the resulting rejection is called a first-set rejection. The duration of graft survival may vary according to a number of factors, including the type of graft,27, 80 and donorrecipient characteristics such as species, strain, and sex. 51 When the recipient has already been sensitized to the same foreign donor antigens which the graft contains, the duration of graft survival is considerably shorter and rejection is called a second-set rejectionY
From the Division of Research, The Cleveland Clinic Foundation This work is supported by Contract PH-43-68-1298, Public Health Service, United States Department of Health, Education, and Welfare.
Surgical Clinics of North America- Vol. 51, No.5, October 1971
1161
1162
WILLIAM
E.
BRAUN
MECHANISMS The rejection of a foreign graft may be separated conceptually into two major areas: sensitization of the host and destruction of the donor graft. Sensitization, or the afferent limb of the immune response, begins with the recognition and processing of foreign antigens by the host. Donor antigens may leave the graft in renal venous blood,55 to produce sensitization in the lymph nodes, or host cells may be sensitized to donor antigens as they pass through the graft.77 Of fundamental importance is precisely where in the graft the antigen resides. The two prime sites are vascular endothelium and passenger leukocytes. Recent studies on this point will be summarized later. It has been shown that exclusion of lymphatic drainage from the kidney does not prevent the development of rejection, indicating that the vascular link is a sufficient route of sensitization for that organ. 36 For skin rejection to occur, however, the lymphatics must be reestablished. 2 Sensitization proceeds with the enlistment of regional lymphoid centers in a coordinated immunologic response of some magnitude following the entrapment of circulating antigen or the arrival of sensitized lymphocytes. Mitchison's demonstration that the passive transfer of regional lymph node cells from sensitized animals could confer an active immunity ("adoptive immunity") on immunologically naive animals 52 has clearly implicated lymph nodes in allograft rejection. Donor graft destruction (efferent or effector limb of the immune response) may be mediated by a cellular (see p. 1163) or antibody response (p. 1166), or both. Other mediators of tissue injury such as complement, polymorphonuclear leukocytes, the coagulation system, and certain vasoactive substances, are closely associated with these primary mechanisms (p. 1168). When a graft survives despite histoincompatibility one must consider that other mechanisms - such as enhancement or tolerance - may be responsible for sustaining it. Enhancement is an antibody-dependent phenomenon which occurs under circumstances that might typically produce sensitization but instead cause prolonged or complete acceptance of an allogeneic graft.38 Tolerance is a specific immunologic unresponsiveness, natural or acquired, which allows the prolonged acceptance of an allogeneic graft. 51 When considering these possible explanations for the survival of incompatible grafts one must not overlook other potentially significant modifiers of the intensity and rate of progression of rejection. These factors may include the inability of a chronically ill patient on dialysis to respond vigorously to an antigenic stimulus, the intermediate strength of the antigen in question and its susceptibility to immune suppressant drugs, the effect of cross-reactivity between certain donor and recipient antigens, and even the technical problems in specifically identifying all the histocompatibility antigens involved in the reaction. These points will be elaborated upon in the article on Histocompatibility Testing (p.1175).
IMMUNOLOGIC ASPECTS OF RENAL TRANSPLANTATION
1163
At the heart of all immunologic phenomena that develop in the course of transplanting an organ from one person to another is the confrontation between the foreign antigens residing in the graft and a recipient capable of responding to them. Focused on this prime problem of incompatibility, expressed as rejection, are a large number of preventive, diagnostic, and therapeutic techniques designed to eliminate it. However, despite the solid status of renal transplantation as one form of accepted treatment for end-stage renal disease, there remain definite inadequacies in these techniques because of incomplete information about the basic immunologic process. Consequently, for very practical reasons it seems appropriate to begin with a consideration of some fundamental questions concerning the rejection process. The major area for the prevention of rejection, histocompatibility testing, will be treated separately. The pathology of rejection and immunosuppression are discussed by other authors in this symposium.
MEDIATORS IN MODELS OF ALLOGRAFT REJECTION Cellular Response Because of modifications of the rejection process in humans brought about by genetic variability, uncertain strength of histocompatibility antigens, and differing responses to the same antigen by various hosts, a genetically defined animal model of transplantation has been chosen as the backbone for the following discussion of events in an unmodified rejection process. This model, furthermore, permits daily evaluation of renal histology in a large group of subjects-an opportunity that is impossible in humans. U sing inbred strains of rats, Guttmann, Lindquist, and their colleagues have performed over 2000 renal transplants. The rat strains were selected to provide either strong or weak genetic barriers to histocompatibility. When kidney transplants were performed across the strong barrier without the use of any immunosuppressive drugs, essentially complete rejection occurred in one week,34 though other workers have reported occasionally longer survivals. 78 The earliest changes noted were the presence of large mononuclear cells in the peritubular capillaries and the perivascular accumulation of such cells in the renal cortex. These cells, arising from the recipient, entered the graft in response to the presence of foreign antigens, and could be found, as described earlier in dogs,62 penetrating the walls of peritubular capillaries in the kidney. A thin homogeneous deposit of immunoglobulin G (IgG) and the third component of complement (beta 1 C) were found by immunofluorescence along the capillary wall, implying that an antigen-antibody reaction had taken place. 45 Thus, both cellular and humoral mechanisms, which will be discussed below, appear to have been involved. As greater numbers of cells entered the interstitium of the kidney, conspicuous perivascular infiltrates were formed. These infiltrates increased and extended diffusely into the interstitium, thus losing their vascular orientation. The origin of these cells has been debated. Their
1164
WILLIAM
E.
BRAUN
presumed derivation from the recipient was first challenged,1B and later supported. 60 Recent studies in a local graft-versus-host model have suggested that both contribute significantly to what has been called an "in vivo mixed lymphocyte culture" resembling rejection. 24 In the later stage of rejection there was glomerular hypercellularity of both endothelial and mesangial cells with occlusion of capillary lumina. Arteries showed endothelial cells which had large vesicular nuclei and swollen cytoplasm. Fibrinoid necrosis occurred later. When virtually all renal function had ceased in the allograft, the dense cellular infiltrates were accompanied by interstitial edema, tubular necrosis, and glomerular capillary occlusion with infiltration by polymorphonuclear cells. Studies of the microvasculature by latex injections showed that earlier patchy defects in cortical vasculature had progressed to a complete absence of cortical filling.2B This finding, reported nearly 20 years ago by Dempster,1B is not, however, specific for rejection. 35 Allografts in dogs and man have also been shown by xenon-133 wash-out techniques to have marked depression of outer cortical perfusion with rej ection. 65 In the interstitium was the cell most characteristic of acute rejection, a large mononuclear cell with a vesicular nucleus and pyroninophilic cytoplasm. The failure to demonstrate IgG or IgM in these cells at a time when they had abundant cytoplasmic ribosomes suggested that these changes were directed toward growth and replication rather than antibody synthesis. 45 Enzyme histochemical studies of these acutely rejected allografts were compatible with a process of progressive ischemia, indicating that the immunologic process of tissue injury worked through a common pathway usable by other modes of injury.46 Mature lymphocytes and plasma cells also composed much of these infiltrations. But only very few of these cells may have actually been specifically sensitized to the donor antigens. 49 Since the accumulation of these cell masses may obliterate tubules, one must be careful under these circumstances not to confuse the large regenerating tubular epithelial cells with the large mononuclear cells of rejection. Such cellular infiltrates, with many lymphoblastic cells ("immunoblasts") occurring both in the interstitium and vessel walls, are the primary finding in acute rejection both in animals and in man. Some of these lymphoblasts, stimulated to transformation by foreign antigen, are believed to arise from that portion of the small lymphocyte population which is thymus-dependent. 66 In contrast to the smalllymphocyte population described later (see Antibody Response, p. 1166), these lymphocytes are long-lived and constitute the majority of the circulating lymphocyte pOOP1 Both the small lymphocytes destined for cellular responses and those destined for antibody production originate from bone marrow precursors. It is uncertain whether antigens on living cells, such as transplantation antigens, can directly stimulate this thymusdependent population of lymphocytes or whether antigen-processing by a macrophage is necessary. It should be kept in mind, however, that "non-immunologic;' substances, including bacterial products, can also induce transformation in vitro. In their role as "killer" cells, this popula-
IMMUNOLOGIC ASPECTS OF RENAL TRANSPLANTATION
1165
tion of transforming lymphocytes has an arsenal of immunologic weapons to destroy tissue. These include a factor to immobilize macrophages at the site of antigen (MIF), a lymphotoxin, a chemotactic factor for monocytes,43 to mention a few, as well as lysosomes,19 a capacity to augment antibody response 13 and to mimic C ' 8 in activating earlier complement components bound to cell membranes. 58 One of these substances, macrophage inhibition factor, has provided a means of detecting allograft rej ection in animals. 26 Perhaps even closer to the problem of human transplantation was the study of rat renal allografts performed across a weaker barrier to histocompatibility. 50 In this model, again studied in precisely defined inbred strains without any therapy, there was a period of acute, spontaneously reversible rejection. Cellular infiltrates were not visible until several days after transplantation when typical large mononuclear cells accumulated in perivascular areas. These cells reached a maximum at 2 to 3 weeks and then progressively decreased. Functional improvement paralleled the diminution in cellular infiltration. About this time the character of the cells changed to include more mature lymphocytes and plasma cells and fewer large mononuclear cells. Over a similar time period in man a comparable shift in cell type has been describedY Just as in human grafts, there was a great variability in these and other changes at the time of early rejection. In some animals there was no glomerular or tubular damage, while in others there was tubular necrosis, necrosis of glomerular cells, and capillary occlusion by fibrin and platelets. As these allografts went on to long-term survival after weathering a rejection, this variability in morphology persisted. Some kidneys were completely normal by light microscopy. In others, mature lymphocytes and plasma cells could be found in perivascular locations with tubular atrophy and interstitial fibrosis. Glomerular lesions, which were composed of thickened capillary walls and mesangial enlargement, were the most significant alterations in this, model of chronic rejection. Subendothelial fibrosis developed in small arteries but not to the extent seen in humans.l1 When a donor skin graft was placed on a recipient which was sustaining at 7 months a long-surviving renal allograft, the skin graft also had a very prolonged survival time. This, along with the failure of serum from such recipients to transfer passively a protective effect for other skin grafts, suggested that the recipient had not developed enhancing antibody, but rather a state of tolerance to the donor tissue. 5o Interestingly, skin and heart allografts have short survival and renal allografts prolonged survivaP7,80 when performed on separate groups of the same inbred strains of rats without treatment. One of the most important concepts pursued in animal models 24 ,76 has been that the primary site of antigen in an allograft is not the organ parenchyma itself, but rather donor leukocytes of bone marrow origin trapped in the graft and carried over to the new host as "passenger leukocytes." The identity of these cells has been assessed further by using bone marrow chimeras (an individual of one genetic strain pos-
1166
WILLIAM
E.
BRAUN
sessing the bone marrow cells of another genetic strain). When the donor's hematopoietic system was made syngeneic with the recipient, but the kidney was not, the rejection of the kidney was impaired. 33 This indicated that when marrow-derived cells transferred within the graft were of recipient and not donor origin, a major site of foreign antigen was eliminated. Although one must be cautious in extrapolating from animal work to human beings, certain observations in the animal warrant emphasis. Even in genetically homogeneous transplant systems without any immunosuppressive drugs, whether across a strong or weak histocompatibility barrier, the rate and intensity of rejection, judged by both graft morphology and survival, may show considerable variation. This suggests that either subtle genetic differences, slight technical variables, or nonspecific inflammatory mechanisms could cause such changes. One should not be surprised, then, if similarly matched human donorrecipient pairs do not all respond identically. Spontaneous reversal of acute rejection may occur with weak histocompatibility barriers, and the allograft may survive for a prolonged time. This may be an important fact to recall when one is faced with an element of rejection in a situation dominated by a harmful factor (such as infection) which could become a fatal factor if immunosuppressive drugs were massively applied before infection was controlled. The presence of mononuclear cell infiltrates in the interstitium, in the absence of a significant component of lymphoblastic forms, does not necessarily mean acute rejection and may, as in enhanced rat renal allografts, eventually disappear without residual functional impairment. The difference between skin, heart, and kidney graft survivals indicates that the same apparent degree of incompatibility does not affect all tissues equally. Whether this is due to quantitative differences in histocompatibility antigens,3,5 tissue-specific antigens,6 or other mechanisms 80 is uncertain. Perhaps the most valuable asset of this animal model thus far has been the localization of the site of antigen and the development of therapeutic maneuvers to selectively control it.
Antibody Response Humoral antibody is produced by plasma cells derived from bone marrow precursors whose evolution through a small lymphocyte and subsequent blast stage is independent of thyriIus control. These small lymphocytes are short-lived, represent the minority of the circulating lymphocyte pool, and are more confined to lymphoid organs. 31 In contrast to the classical cellular mechanism of rejection which had early support from the passive transfer of tumor allograft immunity with cells,52 the appreciation of humoral mechanisms, seemingly limited to leukotic grafts,30, 51 has been relatively slow to develop since Simonsen's early description of hemagglutinins in allografted dogs. 69 This may have been due to the fact that complement-fixing antibodies were not found 69 and that passive transfer of renal allograft immunity with serum proved difficult to accomplish unless cross-reacting renal tissues were reduced 56 or the serum was injected directly into the renal artery. I, 23 Direct renal
IMMUNOLOGIC ASPECTS OF RENAL TRANSPLANTATION
1167
artery injections for several days of the globulin fraction of donorspecific sensitized serum caused severe allograft damage which paralleled the serum's cytotoxic titer.23 The changes were primarily vascular wall and parenchymal necrosis, but cellular proliferation and mesangial condensation were also seen in the glomerulus. Of particular interest was the presence of perivascular mononuclear cell infiltrations believed to be so typical of purely cell-mediated injury. 16, 23 Since the kidney being injected was the donor's own kidney, the cells could not have arisen because of any allogeneic incompatibility. Because the kidney had never been transplanted to another animal and returned, as in Clark's experiment,t4 there could not have been any transfer of the previous host's leukocytes to initiate rejection. Elution of IgG and IgM from unmodified, rejected canine allografts further supported the direct participation of antibody in rejection. 42 Man himself became an early model for the most dramatic example of the effect of cytotoxic antibody. In patients with preformed antibody to their donor's cells, a rapid, irreversible form of renal failure occurred which was called the hyperacute rejection.4i Frequently, within minutes after transplantation, the allograft became cyanotic and urine production ceased. The extensive thrombotic lesions in the glomeruli and renal vessels have suggested a parallelism to the generalized Shwartzman reaction. 74 Circulating antibody has also been incriminated in less virulent forms of allograft injury. Within 1 hour of transplantation in man glomerular endothelial, epithelial, and basement membrane changes were apparene9 which were similar to lesions attributed to immunologic injury but seen 13/4 to 21f2 years after transplantation. 61 A probable explanation for such rapid damage would be the presence of preexisting antibodies directed against donor tissue. Antibody developing to donor cells after transplantation may also be harmful to the allograft. In unsensitized dogs cytotoxic antibody titers increased and allografts were rejected when azathioprine was stopped. 82 Jeannet37 tested 35 patients before and after transplantation for humoral antibody by three techniques: leukoagglutination, cytotoxicity, and mixed agglutination. Nine of 10 patients with antibody before transplantation and 12 of 16 who developed antibody against their donor after transplantation rejected their kidney or died. Good to excellent clinical courses accompanied the 5 patients who never developed antibody and 5 of the seven patients whose antibody was not directed against donor cells. A close association was noted between the presence of humoral antibodies and the development of vascular lesions in the allograft. There is also abundant immunofluorescent data in humans to document the deposition of immunoglobulins, presumably acting as antibody, in both renal lO , 11,29,47,59 and heart68 , 75, 81 allografts. The diagnostic value of circulating cytotoxic antibody has been seen primarily in cardiac allografts. 25 Cytotoxic antibodies to renal allografts belong to the IgG class of immunoglobulins but have not yet been classified as to their subclass .
1168
WILLIAM
E.
BRAUN
This information would help to assess its avidity for fixing complement and its potential to produce cellular damage by this means. An IgM anti-gamma globulin rheumatoid-like factor has also been found in renal transplant recipients, but its clinical significance is presently uncertain. 39 Perhaps the most stimulating work involving antibody has recently come from animal models in which enhancement was produced. This phenomenon was extensively explored in mouse tumor allografts by Kaliss. 38 Its potential value to other transplants was shown in the rat renal allograft model by Stuart, who obtained marked prolongation of kidney survival by pretreatment with donor antigen plus antiserum to donor cells. 78 The use of both substances complicated the interpretation of the impressive results. Ockner et al., using a single intravenous dose of 10 7 donor bone marrow cells one to two weeks before transplantation, also produced enhancement and achieved essentially indefinite survival of rat kidney allografts. 57 The rejection of a subsequent skin graft and the passive transfer of the protective effect by the recipient's serum indicated that enhancement was achieved. By ingenious manipulation of the genetic strains involved and the establishment of bone marrow chimeras, it was then possible to show that the enhancing antibody was directed specifically against bonemarrow-derived cells located in the graft, but having a genetic source different from that of the renal parenchyma. It has been shown, too, that this method of enhancement can overcome a state of pre sensitization. When Lucas produced enhancement with homologous anti-donor lymphocyte serum, there was localization of enhancing serum in the transplanted kidney and a decreased availability of antigen sites on the vascular endothelium. 48 These data suggested "coating" of crucial antigenic sites by the serum and a subsequent modulation of these antigens to permit prolonged survival in a state of "immunologic adaption." After achieving enhancement in the rat model with antigraft alloantibody, Batchelor has recently reported a provocative study using "enhancing" antibody in a human parent-child transplant. 4 The delayed function of the allograft was a disturbing feature, though the ability to withdraw later azathioprine and prednisone was encouraging.
Complement, the Polymorphonuclear Leukocyte, and Coagulation Other major mediators of tissue injury in allograft rejection include the complement system, polymorphonuclear leukocytes, and the coagulation system. Lower levels of C'2 were found in allograft rejection both in rats and man. 32 Numerous studies in animals and in man have documented tissue deposition of C'3 in the rejected kidney by immunofluorescence, implying fixation by antigen-antibody complexes. 10, 11,29,47,59 Unstable serum complement levels and increased catabolism of C'3 and C'4 have accompanied rejections of human allografts. 12 Of several substances produced in the complement cascade that might induce allograft injury, the chemotactic factors that attract polymorphonuclear leukocytes have received the most attention. The importance of the polymorphonuclear leukocyte was appreciated by its
IMMUNOLOGIC ASPECTS OF RENAL TRANSPLANTATION
1169
proposed role in producing the glomerular basement membrane damage found in experimental forms of glomerulonephritis. 20 However, electron microscopic documentation of damage by polymorphonuclear leukocytes in human glomerulonephritis has come only recently. 8 The appearance of these cells in glomeruli within 1 hour of transplantation in man is one finding different from that in animal work. 79 The severity of polymorphonuclear leukocyte infiltration has correlated with a poorer prognosis for the survival of the allograft.40 Increased numbers of polymorphonuclear leukocytes in the glomeruli were a prominent feature of the hyperacute form of allograft rejection, though the picture was dominated by the coagulation process. Emphasis on the former finding suggested its relation to the Arthus phenomenon,54 while dominance of the intravascular clotting events related it to the generalized Shwartzman reaction. 74 Unlike the generalized Shwartzman reaction clotting is usually confined to the kidney.9, 17 The role of preformed antibody in the induction of this form of rejection has been well documented (see Histocompatibility Testing, p. 000). Evidence of recurring participation of clotting in acute and chronic rejection was shown by the urinary excretion of fibrin degradation products. 7, 15 There is interweaving of the clotting system, at the level of plasmin,63 and Hageman factor 22 with the complement system. In addition there is a mutual inhibitor of C'l esterase and plasmin. 64 These relationships may help to explain the triggering of the clotting system at times when complement has been activated by antigen-antibody complexes. Despite the immediate involvement of the clotting system in the obliteration of xenografts,67 anticoagulant and defibrinating agents did not prolong survival of xenografts beyond 10 minutes. 53 ,71 The longest survivals, which still were less than 4 hours, were achieved using donor liver as an immunoabsorbant. 53 , 71 Further progress will require suppression of antibody synthesis rather than temporary lowering of circulating antibody.
RECURRENT GLOMERULONEPHRITIS This concluding section is concerned with the recurrence of glomerulonephritis in the transplanted kidney. In Glassock's study of 22 identical twin transplants, 11 of 17 whose original disease was glomerulonephritis had recurrence of a similar disease in the isograft. 29 The mesangial and focal linear immunofluorescent deposits of IgG and /31C were not typical of nephritis induced by circulating anti-glomerular basement membrane antibody. Further work with renal allografts led to the description of the first human case of transplant glomerulonephritis caused by circulating anti-glomerular basement membrane antibody.44 Several important points were evident in this case: the clinical course of the original disease was rapidly progressive (less than 1 year); the original kidneys had a linear fluorescent pattern of IgG and /31C on the glomerular basement membrane; circulating anti-glomerular basement
1170
WILLIAM
E.
BRAUN
membrane antibody was demonstrable after removal of the original kidneys; antibody found in the patient's circulation caused proteinuria and anti-glomerular basement membrane nephritis in monkeys. The sibling allograft very early demonstrated heavy proteinuria and linear fluorescent deposits of IgG, indicative of recurrence of the original disease. However, except for proteinuria (about 1 to 3 gm. per 24 hr.) for the first 6 months after transplantation, the kidney has had excellent function for at least 3 years. Extension of this work has shown that not only the anti-glomerular basement membrane form of nephritis has recurred in renal transplants, but also immune complex nephritis. The true incidence of such recurrent disease was difficult to ascertain since only a selected population was under study. However, of 11 allograft recipients whose original disease was anti-glomerular basement membrane nephritis (2 with Goodpasture's syndrome and 9 with subacute or chronic glomerulonephritis), 7 have had recurrent nephritis with impairment of renal function and 4 have had no apparent recurrence (2 with Goodpasture's syndrome and 2 with chronic glomerulonephritis).21 The latter 4 patients were managed with immunosuppressive drugs during a 1 to 2 month anephric state before being transplanted. Of 19 patients who originally had complex-induced nephritis (17 with chronic glomerulonephritis and 2 with systemic lupus erythematosus), 4 thus far have recurrent nephritis (3 with chronic glomerulonephritis and 1 with systemic lupus erythematosus).21 Such alternate immunologic mechanisms of allograft injury must be appreciated in order that appropriate management be afforded the patient and the cause of impaired allograft function be correctly interpreted.
REFERENCES 1. Altman, B.: Tissue transplantation: Circulating antibody in the homotransplantation of kidney and skin. Ann. Roy. ColI. Surg., 33 :79, 1963. 2. Barker, C. F., Billingham, R E., and Shaffer, C. F.: Further studies on the hamster's cheek pouch as a privileged site. Transplant. Proc., 1 :597, 1969. 3. Basch, R S., and Stetson, C. A.: Quantitative studies on histocompatibility antigens of the mouse. Transplantation, 1 :469, 1963. 4. Batchelor, J. R, Ellis, F., French, M. E., Bewick, M., Cameron, J. S., and Ogg, C. S.: Immunological enhancement of human kidney graft. Lancet, 2:1007,1970. 5. Berah, M., Hors, J., and Dausset, J.: A study of HL-A antigens in human organs. Transplantation, 9:185, 1970. 6. Boyse, E. A., Lance, E. M., Carswell, E. A., Cooper, S., and Old, L. J.: Rejection of skin allografts by radiation chimaeras: Selective action in the specification of cell surface structure. Nature, 227:901,1970. 7. Braun, W. E., and Merrill, J. P.: Urine fibrinogen fragments in human renal allografts: A possible mechanism of renal injury. New Eng. J. Med., 278:1366,1968. 8. Burkholder, P. M.: Ultrastructural demonstration of injury and perforation of glomerular basement membrane in acute proliferative glomerulonephritis. Amer. J. Path., 56:251,1969. 9. Busch, G. J., Braun, W. E., Carpenter, C. B., Corson, J. M., Galvanek, E. R, Reynolds, E. S., Merrill, J. P., and Dammin, G. J.: Intravascular coagulation (IVC) in human renal allograft rejection. Transplant. Proc., 1 :267, 1969. 10. Busch, G. J., Braun, W. E., Glassock, R J., and Dammin, G. J.: Immunofluorescent study of 46 human renal allograft biopsies. Proc. First Annual Meeting of Amer. Soc. of Neph., Los Angeles, 1967, p. 11.
IMMUNOLOGIC ASPECTS OF RENAL TRANSPLANTATION
1171
11. Busch, G. J., Reynolds, E. S., Galvanek, E. G., Braun, W. E., and Dammin, G. J.: Human renal allografts: The role of vascular inj ury in early graft failure. Medicine, 50: 29, 1971. 12. Carpenter, C. B., Ruddy, S., Shehadeh, I. H., Muller-Eberhard, H. J., Merrill, J. P., and Austen, K F.: Complement metabolism in man: hypercatabolism of the fourth (C4) and third (C3) components in patients with renal allograft rejection and hereditary angioedema (HAE). J. Clin. Invest., 48:1495, 1969. 13. Claman, H. N., and Chaperon, E. A.: Immunologic complementation between thymus and marrow cells-a model for the two-cell theory of immunocompetence. Transplant. Rev., 1 :92, 1969. 14. Clark, D. S., Foker, J. E., Good, R A., and Varco, R L.: Humoral factors in canine renal allograft rejection. Lancet, 1 :8, 1968. 15. Clarkson, A. R, Morton, J. B., and Cash, J. D.: Urinary fibrin/fibrinogen degradation products after renal homotransplantation. Lancet, 2: 1220, 1970. 16. Cochrum, K C., Davis, W. C., Kountz, S. L., and Fudenberg, H. H.: Renal autograft rejection initiated by passive transfer of immune plasma. Transplant. Proc., 1 :301, 1969. 17. Coleman, R W., Braun, W. E., Busch, G. J., Dammin, G. J., and Merrill, J. P.: Coagulation studies in the hyperacute and other forms of renal allograft rejection. New Eng. J. Med., 281 :685,1969. 18. Dempster, W. J.: Kidney homotransplantation. Brit. J. Surg., 40:447, 1953. 19. Diengdoh, J. V., and Turk, J. L.: Immunological significance of lysosomes within lymphocytes in vivo. Nature, 207:1405,1965. 20. Dixon, F. J.: Glomerulonephritis and immunopathology. Hosp. Pract., 2:35,1967. 21. Dixon, F. J., McPhaul, J. J., and Lerner, R A.: The contribution of kidney transplantation to the study of glomerulonephritis in renal transplants. Transplant. Proc., 1: 194, 1969. 22. Donaldson, V. H.: Mechanisms of activation of C'l esterase in hereditary angioneurotic edema plasma in vitro: Role of Hageman factor, clot-promoting agent. J. Exper. Med., 127:411, 1968. 23. Dubernard, J. M., Carpenter, C. B., Busch, G. J., Diethelm, A. G., and Murray, J. E.: Rejection of canine renal allografts by passive transfer of sensitized serum. Surgery, 64:752,1968. 24. Elkins, W. L., and Guttmann, R D.: Pathogenesis of a local graft versus host reaction: Immunogenicity of circulating host leukocytes. Science, 159: 1250, 1968. 25. Ellis, R J., Lillehei, C. W., Fischetti, V. A., and Zabriskie, J. B.: Heart-reactive antibody: An index of cardiac rejection in human heart transplantation. Circulation,41 and 42 (Supp!. 2): 91, 1970. 26. Ferraresi, R W., Gorhman-Yahr, M., and Raffel, S.: Studies of the macrophage-inhibition test. III. Use of the macrophage migration-inhibition technic in detection of active graft immunity. Transplantation, 10:237, 1970. 27. Freeman, J.' S., Reemtsma, K, and Steinmuller, D.: Comparative survival of transplanted heart and skin in inbred rats. Circ. Supp!. II 41 and 42 (Supp!. 11):86, 1970. 28. Gardner, L. B., Guttmann, R D., and Merrill, J. P.: Renal transplantation in the inbred rat. IV. Alterations in the microvasculature in acute unmodified rejection. Transplantation, 6:411,1968. 29. Glassock, R J., Feldman, D., Reynolds, E. S., Dammin, G. J., and Merrill, J. P.: Human renal isografts: A clinical and pathologic analysis. Medicine, 47:411,1968. 30. Gorer, P. A.: Some recent work on tumor immunity. Adv. Cancer Res., 4:149, 1956. 31. Gowans, J. L.: Immunobiology of the small lymphocyte. Hosp. Pract., 3:34,1968. 32. Guiney, E. J., Austen, K F., and Russell, P. S.: Measurement of serum complement during homograft rejection in man and rat. Proc. Exper. Bio!. Med., 115:1113,1964. 33. Guttman, R D., Lindquist, R R, and Ockner, S. A.: Renal transplantation in the inbred rat. IX. Hematopoietic origin of an immunogenic stimulus of rejection. Transplantation, 8:472,1969. 34. Guttmann, R D., Lindquist, R R, Parker, R M., Carpenter, C. B., and Merrill, J. P.: Renal transplantation in the inbred rat. I. Morphologic, immunologic and functional alterations during acute rejection. Transplantation, 5:668,1967. 35. Hollenberg, N. K, Epstein, M., Rosen, S. M., Basch, R I., Oken, D. E., and Merrill, J. P.: Acute oliguric renal failure in man: Evidence for preferential renal cortical ischemia. Medicine, 47:455,1968. 36. Hume, D. M., and Egdahl, R H.: Progressive destruction of renal homografts isolated from the regional lymphatics of the host. Surgery, 38:194, 1955. 37. Jeannet, M., Pinn, V. W., Flax, M. H., Winn, H. J., and Russell, P. S. Humoral antibodies in renal allotransplantation in man. New Eng. J. Med., 282:111,1970. 38. Kaliss, N.: Dynamics of immunologic enhancement. Transplant. Proc., 2:59, 1970. 39. Kano, K, and Milgrom, F.: Anti-gamma globulin factors in human allograft recipients. Transplantation, 6: 111, 1968. 40. Kincaid-Smith, P., Morris, P. J., Saker, B. M., Ting, A., and Marshall, V. C.: Immediate renal-graft biopsy and subsequent rejection. Lancet, 2:748,1968.
1172
WILLIAM
E.
BRAUN
41. Kissmeyer-Nielsen, F., Olsen, S., Petersen, V. P., Fjeldborg, 0.: Hyperacute rejection of kidney allografts, associated with pre-existing humoral antibodies against donor cells. Lancet,2:662,1966. 42. Kolker, P., Hampers, C. L., Hager, E. B., and Leon, P. E.: Characterization of immunologically active substances from canine renal allotransplants. Transplantation, 6:131, 1968. 43. Lawrence, H. S., and Landy, M., eds.: Mediators of Cellular Immunity. New York, Academic Press, 1969. 44. Lerner, R A., Glassock, R J., and Dixon, F. J.: The role of anti-glomerular basement membrane antibody in the pathogenesis of human glomerulonephritis. J. Exper. Med., 126:989, 1967. 45. Lindquist, R R, Guttmann, R D., and Merrill, J. P.: Renal transplantation in the inbred rat. II. An immunohistochemical study of acute allograft rejection. Amer. J. Path., 52:531,1968. 46. Lindquist, R R, Guttmann, RD., and Merrill, J. P.: Renal transplantation in the inbred rat. V. Histochemical studies of acute renal allograft rejection. Amer. J. Path., 52:1145, 1968. 47. Lindquist, R R, Guttmann, R D., Merrill, J. P., and Dammin, G. J.: Human renal allografts. Interpretation of morphologic and immunohistochemical observations. Amer. J. Path., 53:851, 1968. 48. Lucas, Z. J., Markley, J., and Travis, M.: Immunologic enhancement of renal allografts in rat. I. Dissociation of graft survival and antibody response. Fed. Proc., 29:2041,1970. 49. McCluskey, R T., Benacerraf, B., and McCluskey, J. W.: Studies on the specificity of the cellular infiltrate in delayed hypersensitivity reactions. J. Immunol., 90:466, 1963. 50. Mahabir, R N., Guttmann, R D., and Lindquist, R R: Renal transplantation in the inbred rat. X. A model of "weak histoincompatibility" by major locus matching. Transplantation, 8:369,1969. 51. Medawar, P. B.: The homograft reaction. Proc. Roy. Soc. B., 149:145,1958. 52. Mitchison, N. A.: Studies on the immunological response to foreign tumor transplants in the mouse. J. Exper. Med., 102:157,1955. 53. Moberg, A. W., Shons, A. R, Gewurz, H., Mozes, M., and Najarian, J. S.: Prolongation of renal xenografts by the simultaneous sequestration of preformed antibody and the inhibition of complement, coagulation, and antibody synthesis. Abstracts of Third Internat. Congr. Transplantation Society, The Hague, Netherlands, 1970, p. 185. 54. Myburgh, J. A., Cohen, I., Gecelter, L., Meyers, A. M., Abrahams, C., Furman, K I., Goldberg, B., and van Blerk, P. J. P.: Hyperacute rejections in human-kidney allograftsShwartzman or Arthus? New Eng. J. Med., 281 :685,1969. 55. Najarian, J. S., May, J., Cochrum, K. C., Baronberg, N., and Way, L. W.: Mechanism of antigen release from canine kidney homotransplants. Ann. N.Y. Acad. Sci., 129:76, 1966. 56. Najarian, J. S., and Perper, R. J.: Participation of humoral antibodies in allogeneic organ transplantation rejection. Surgery, 62:213,1967. 57. Ockner, S. A., Guttmann, R D., and Lindquist, R R: Renal transplantation in the inbred rat. XIV. Mechanism of the modified rejection produced by bone marrow cell pretreatment. Transplantation, 9:39, 1970. 58. Perlmann, P., Perlmann, H., Muller-Eberhard, H. J., and Manni, J. A.: Cytotoxic effects of leukocytes triggered by complement bound to target cells. Science, 163:937,1969. 59. Porter, K A., Andres, G. A., Calder, M. W., Dossetor, J. B., Hsu, K. C., Rendall, J. M., Seegal, B. C., Starzl, T. E.: Human renal transplants II. Immunofluorescent and immunoferritin studies. Lab. Invest., 18: 159, 1968. 60. Porter, K A., and Calne, R Y.: Origin of the infiltrating cells in skin and kidney homografts. Transplant. Bull., 7:458,1960. 61. Porter, K A., Dossetor, J. B., Marchioro, T. L., Peart, W. S., Rendall, J. M., Starzl, T. E., Terasaki, P. I.: Human renal transplants. I. Glomerular changes. Lab. Invest., 16: 153,1967. 62. Porter, K A., Joseph, N. H., Rendall, J. M., Stolinski, C., Hoehn, R J., and CaIne, R Y.: The role of lymphocytes in the rejection of canine renal homotransplants. Lab. Invest., 13:1080,1964. 63. Ratnoff, O. D., and Naff, G. B.: Conversion of C'1 s to C'1 esterase by plasmin and trypsin. J. Exper. Med., 125:337,1967. 64. Ratnoff, O. D., Pensky, J., Ogston, D., and Naff, G. B.: The inhibition of plasmin, plasma kallikrein, plasma permeability factor, and the C'1r SUbcomponent of complement by serum C'1 esterase inhibitor. J. Exper. Med., 129:315,1969. 65. Retik, A. B., Hollenberg, N. K, Rosen, S. M., Merrill, J. P., and Murray, J. E.: Cortical ischemia in renal allograft rejection. Surg. Gynec. Obstet., 124:989, 1967. 66. Roitt, I. M., Greaves, M. F., Torrigiani, G., Brostoff, J., and Playfair, J. H. L.: The cellular basis of immunological responses. Lancet, 1 :367, 1969.
IMMUNOLOGIC ASPECTS OF RENAL TRANSPLANTATION
1173
67. Rosenberg, J. C., Broersma, R J., Bullemer, G., Mammen, E. F., Lenaghan, R, and Rosenberg, B. F.: Relationship of platelets, blood coagulation, and fibrinolysis to hyperacute rejection of renal allografts. Transplantation, 8:152, 1969. 68. Rossen, R D., Butler, W. T., Reisberg, M. A., Mittal, K. K., Johnson, A. H., Brooks, D. K., Leachman, RD., Milans, J. D., Montgomery, J. R, Nora, T. J., and Rochelle, D. G.: Immunofluorescent and serologic studies of the humoral antibody response to human cardiac allografts. Abstracts of Third Int. Congr. Transplantation Society, The Hague, Netherlands, 1970, p. 251. 69. Simonsen, M.: Biological incompatibility in kidney transplantation in dogs. II. Serological investigations. Acta Pathol. Micro. Scand., 32:36,1953. 70. Simonsen, M.: Graft versus host reactions, their natural history and applicability as tools of research. Prog. Allergy, 6:349, 1962. 71. Slapak, M., Joison, J., Greenbaum, M., Saravis, C. A., and McDermott, W. V., Jr.: The effect of heparin, Arvin, liver perfusion and antiplatelet serum on the rejection of the pig kidney by the dog. Abstracts of Third Internat. Congr. Transplantation Society, The Hague, Netherlands, 1970, p. 189. 72. Snell, G. D.: The terminology of tissue transplantation. Transplantation, 2 :655, 1964. 73. Starzl, T. E., Boehmig, H. J., Amemiya, H., Wilson, C. B., Dixon, F. J., Giles, G. R, Simpson, K. M., and Halgrimson, C. G.: Clotting changes during rapid renal homograft rejection. New Eng. J. Med., 283:383,1970. 74. Starzl, T. E., Lerner, R A., Dixon, F. J., Groth, C. G., Brettschneider, L., and Terasaki, P. I.: Shwartzman reaction after human renal homotransplantation. New Eng. J. Med. 278:642, 1968. 75. Stastny, P.: Bound immunoglobulin and complement in heart allografts undergoing rejection. Transplantation, 10:248, 1970. 76. Steinmuller, D.: Immunization with skin isografts taken from tolerant mice. Science, 158:157,1967. 77. Strober, S., and Gowans, J. L.: The role of lymphocytes in the sensitization of rats to renal homografts. J. Exper. Med., 122:347,1965. 78. Stuart, F. P., Saitoh, T., Fitch, F. W., and Spargo, B. H.: Immunologic enhancement of renal allografts in the rat. Surgery, 64:17, 1968. 79. Weymouth, R J., Seibel, H. R, Lee, H. M., Hume, D. M., and Williams, G. M.: The glomerulus in man one hour after transplantation. Amer. J. Path., 58 :85, 1970. 80. White, F., and Hildemann, W. H.: Kidney versus skin allograft reaction in normal adult rats of inbred strains. Transplant. Proc., 1 :395, 1969. 81. Williams, G. M., DePlanque, B., Graham, W. H., and Lower, R R: Participation of antibodies in acute cardiac-allograft rejection in man. New Eng. J. Med., 281 :1145,1969. 82. Yamada, T., and Kay, J. H.: Kidney homotransplantation with special reference to cytotoxic antibody. Surgery, 63 :637, 1968.