Rejection Response in Canine Lung Homotransplan tation

Transcription

1 Rejection Response in Canine Lung Homotransplan tation Effects of Azathioprine and Steroids Donald R. Kahn, M.D., Murray R. Abell, M.D., Fred K. Vontz, M.D., and Herbert Sloan, M.D. A lthough host rejection of lung homografts can be delayed by inimunosuppressive drugs, permanent tolerance is rarely achieved. In the homotransplantation of kidneys in dogs and humans, some success has been achieved by administering azathioprine with steroids or other immunosuppressive drugs [3, 8, 131. In the study reported here, our purpose was to observe the serial microscopic changes in hoiiiografted canine lungs when azathioprine and steroids were used as the immunosuppressive agents, with intensive steroid therapy reserved for the rejection crises. A further objective was to determine whether reversing the rejection response, once it had appeared, would establish a type of graft tolerance. METHVLIS Homotransplantation of the left lung was performed in 10 adult mongrel dogs after removal of each animal s own lung. The donor lung was placed in the normal anatomical position by anastomosis of the left atrial cuff, the left pulmonary artery, and the left main-stein bronchus (Fig. 1); a splenectomy was also performed in the recipient animals. Azathioprine was started on the day of operation in doses of 10 mg./kg. body weight for two days, 7.5 nig./kg. for the next three days, and 5 mg./kg. thereafter. Leukocyte counts were made twice weekly. Lung biopsies were obtained every one to two weeks to detect early changes of rejection. Prednisone in doses of 200 mg. per day was started when microscopic evidence of rejection appeared and was continued until From the 1)epartments of Surgery (Section of Thoracic Surgery) and Pathology, University of Michigan hlcdical Center, Ann Arbor, Mich. This work has I)ccn supported Iiy tlic National Institutes of Health, the Michigan Heart Association, and tlic 1k:in s Fund, llicliigati Hcart Association. Presented at the First Antiiial Meeting of The Society of Thoracic Surgeons, St. Imiiis. )lo., Ian , *Kindly supplied by Burroughs Wellcome & Co., Inc. 650 THE ANNALS OF THOKACIC SlJKGERY

2 NOTE: Kejection of Lung Homografts FIG. 1. Surgical technique of homotransplantation of left lung. The donor lung is placed in the normal anatomical position by anastomosis of the left atrial ciifl, th(. left pulmonary urtery, and the left mciin-stem bronchus. iniprovernent was seen. The animals were then placed on maintenance dosages of 5 mg. of prednisone per day. When microscopic signs of rejection reappeared the 200 mg. dosage was reinstituted. All animals received 400,000 units of penicillin per day from the day of operation until their death. RESULTS CONTROL ANIMALS The sequence of microscopic findings in the homotransplanted lung in the untreated control animal had previously been worked out in this laboratory [2]. By the second day after homotransplantation of the left lung, early perivascular round-cell infiltration was seen, and by the fourth day, acute alveolar edema and more perivascular round-cell intiltration was present. Necrosis of the alveoli, with some hemorrhage, was noted by the seventh day. By the fourteenth day, necrosis was complete and histological architecture destroyed. AZATHIOPRINE AN11 STEROID THERAPY In the 10 animals treated with azathioprine and steroids, a fairly constant pattern was observed. With azathioprine alone, definite peri- VOI.. I, NO.,?, SEPT.,

3 KAHN, ABELL, VONTZ, AND SLOAN FIG. 2. Dog hro. 1. Lung biopsy 21 days after homotransplniitatioii. Definite peribronchial and perivascular mononuclear cell infiltration is present. bronchial and perivascular mononuclear cell infiltration was seen by the end of the second or third week after transplantation (Fig. 2). When 200 mg. per day of prednisone was started, the cellular infiltration disappeared, for the most part, by the fourth or fifth week (Fig. 3). Previous experience indicates that if prednisone had not been started when the rejection began, the iriicroscopic changes would increase until the lung would be totally rejected by the fourth or fifth posttransplant week. After the rejection changes regressed, prednisone was decreased to FIG. 3. Dog hro. 1. Lung biopsy 35 days nffrr homotrnnsplantation. Cellular infiltration has disappeared after 2 weeks of prednisone therapy. 652 THE ANNALS OF THORACIC SURGERY

4 NOTE: Rejection of Lung Homografts FIG. 4. Dog No. 10. Lung biopsy 64 days after homotransplantation. The peribronchial and periuascrilar mononuclear cell inliltration has reappeared. maintenance levels of 5 mg. per day. Even though the azathioprine therapy was continued with the maintenance prednisone, the cellular infiltration reappeared by the ninth to tenth week (Fig. 4), and progressed to focal areas of necrosis and patchy interstitial and alveolar edema by the eleventh or twelfth week (Fig. 5). With resumption of 200 mg. per day of prednisone, even though the rejection response had progressed this far, the cellular infiltration and edema regressed and the focal areas of necrosis calcified by the fifteenth to sixteenth posttransplant week (Fig. 6). The regimen of 200 nig. per day of prednisone and FIG. 5. Dog No. 8. Lung biopsy 80 days after homotransplantation. There are focal arens of necrosis and patchy interstitial and alveolar edema present. VOL. I, NO. 5, SEPT.,

5 KAHN, ABELL, VONTZ, AND SLOAN FIG. 6. Dog No. 8. Lung biopsy I08 days after homotransplantation. The cellular in filtration and edema haue rc,grmed and the focal rtrcws of necrosis hone calcified. 5 mg./kg. of azathioprine was continued. The animals died by the seventeenth to twentieth week from nonspecific systemic infections, without further microscopic changes in the transplanted lung. Leukocyte counts never dropped below 5,000. DISCUSSION Only a few years ago a homograft rejection was thought to be an all-or-none phenomenon that must inevitably proceed to complete tissue necrosis once the process was started. It was discovered, however, that the process could be reversed, as shown by the retransplantation of a rejected kidney back to its original host [l], and also by drug therapy alone [lo]. Now it appears that lung homograft rejection can also be reversed, even if far advanced. Azathioprine alone delayed the onset and slowed the rate of rejection; with prednisone added in massive doses, rejection changes stopped. When prednisone was reduced to maintenance levels rejection began again. Hardy and his associates found that azathioprine alone increased lung homograft survival in dogs from 7.4 days to 30.4 days [6], confirming our impression that azathioprine will not control the rejection response by itself. The possibility that tolerance to the homograft may result from allowing rejection to begin and then reversing it was not borne out. When drug dosage was decreased the rejection changes recurred. However, some drug-treated dogs with kidney homografts [l l, 121 and lung homografts [9] have been eventually withdrawn from drug therapy with continued survival of the transplant. 654 THE ANNALS OF THOKACIC SURGERY

6 NOTE: Rejection of Lung Hornografts The exact niechanisrn of action of immunosuppressive drugs is not known. The drug s effect in preventing rejection is exerted on the host without any change in the antigenicity of the graft-a kidney transplant [ll] or skin homograft [4] survives when transplanted back to its original host. In addition, stopping the drugs usually reinitiates the rejection response. The corticosteroids probably act on the host at a later point in the immune reaction, preventing the antigen-antibody union and destroying lymphocytes [71. Because the rejection response with organ transplants is easier to control than with skin transplants, the possibility that antigenic overloading produces an immune paralysis has been considered. This appears to be disproved, however, by the observation that a second kidney transplant from the same donor, constituting a double dose of antigen, may be rejected while the first continues to survive [ 111. After extensive work with kidney transplants, Murray and associates feel that the tolerated kidney most likely is protected in its new environment by antibody coating which nullifies the efferent action of the irninunological reflex arc [l 11. Even though the homotransplanted lung looked relatively normal microscopically when the animals died after 16 to 20 weeks, we cannot be certain that more subtle changes were not present that interfered with function. Function studies are presently being performed. Splenectomy was performed simultaneously on the recipient animals to decrease the immunological response to antigenic stimuli [5]. SUMMARY Hornotransplantation of the left lung was performed in 10 mongrel dogs after removal of each animal s own lung. Azathioprine was started on the day of operation. Periodic lung biopsies showed definite peribronchial and perivascular mononuclear cell infiltration after two or three weeks. Yrednisone, 200 nig. per day, was started, and the cellular infiltration disappeared by the fourth to fifth posttransplant week. When the prednisone dosage was decreased to niaintenance levels the cellular intiltration reappeared and again was reversed by 200 Ing. dosages. The high dosage level of prednisone was continued, along with a maintenance dosage of azathioprine, until the animals died, by the seventeenth to twentieth week, froni nonspecific systemic infections. No further microscopic changes in the transplanted lungs were noted. The results indicate that azathioprine alone delays but does not prevent the rejection process occurring in homotransplantation. The addition of large doses of prednisone not only prevents cellular changes but also reverses them once they have occurred. Reversal of an established rejection process, however, does not produce tolerance of the VOL.. 1, NO. fj, SEPT.,

7 KAHN, ABELL, VONTZ, AND SLOAN transplant. Moreover, the mortality rate for canine subjects is extremely high when these immunosuppressive drugs are given in sufficient amounts to prevent most of the rejection changes. REFERENCES Balankura, O., Goodwin, W. E., Murray, J. E., and Dammin, G. J. Study of the homograft reaction by retransplantation of the canine kidney. Surg. Forum 1124, Blumenstock, D. A., and Kahn, D. R. Replantation of the canine lung. J. Surg. Res. 1:40, Calne, R. Y., Alexandre, G. P., and Murray, J. E. A study of the effects of drugs in prolonging survival of homologous renal transplants in dogs. Ann. ht.y. Arad. Sci. 99:743, Fisher, B., and Schewe, E. Further observations on skin homografts in pyridoxine deficient animals. Ann. Surg. 155:457, Guntlerson, C. M., Juras, D., La Via, M. F., and Wissler, R. W. Tissue and cellular changes associated with antibody formation in the rat spleen. J.A.M.A. 180:1038, Hardy, J. D., Eraslun, S., Dalton, M. L., Abian, F., and Turner, M. D. Reimplantation and homotransplantion of the lung. Ann. Surg. 157:707, Hitchings, G., and Elion, G. Chemical suppression of the immune response. Pharmacol. Rev. 15:365, Hume, D. M., Magee, J. H., Kauffman, H. M., Kittenburg, M. S., and Pront, G. R. Renal homotransplantation in man in modified recipients. Ann. Surg. 158:608, Kahn, D. R. Discussion autotransplantation and homotransplantation of the lung: Further studies. J. Thorar. Cardiov. Surg. 46:625, Murray, J. E., Balankura, O., Greenberg, J. B., and Dammin, G. J. Reversibility of the kidney homograft reaction by retransplantation and drug therapy. Ann. N.Y. Acad. Sci. 99:768, Murray, J. E., Sheil, A. G. R., Moseley, R., Knight, P., McGavic, J. D., and Dammin, G. J. Analysis of mechanism of immunosuppressive drugs in renal homotransplantation. Ann. Surg. 160:449, Pierce, J. C., and Varco, R. L. Effects of long term 6-mercapto-purine treatment upon kidney homotransplants in dogs. Surgery 54: 124, Starzl, T. E., Marchioro, T. L., and Waddell, W. R. The reversal of rejection in human renal homografts with subsequent development of homograft tolerance. Surg. Gynec. Obslet. 117:385, DISCUSSION# Dn. THOMAS J. YEH (Augusta, Ga.): I wish to compliment the two previous essayists on their excellent presentations and results. There are a few comments I would like to make on the choice of immunosuppressants. The best results in lung homotransplantation, I believe, were reported by Blumenstock. Some of his dogs survived for two years or longer. He used Methotrexate in combination with irradiation. His results have been difficult to duplicate. Recently we have done comparative studies on efficacy of Methotrexate and Imuran. Like Hardy s group, we found that Imuran is somewhat better than Methotrexate. The average survival duration of dogs with lung homograft in the Methotrexate group was This and thc preceding paper, Suppression of Kejcction Crises in the Cardiac Homograft, by Richard B. Lowcr et nl., p. 645, were tliscusscd together. 656 THE ANNALS OF IHOKACIC SURGERY

8 NOTE: Rejection of Lung Homografts seven weeks, and in the Imuran group it was nine weeks. Some of our Imurantreated dogs are still living at nearly two years. Survival, however, cannot be equated with function of the grafted lung. Most of the homografts had lost function by the end of three weeks, and only in occasional dogs could function be demonstrated by bronchospirometry at the end of three months. We have found that x-ray appearance of the lung is a very good indication of the function capacity. I would like to ask Dr. Kahn whether he had studied the function of the grafts, and also if he thinks the x-ray changes might be useful in detecting early rejection. DR. SHUMWAY: I just want to say that we do not think clinical application of either of these transplantation processes (heart or lung) is yet in order. Considerably more work needs to be done, and I think some of these therapeutic efforts have been ill-advised, to say the least. DR. KAHN: Dr. Samson, I want first to reiterate Dr. Shumway s belief that this technique is not yet ready for clinical trial when one considers the massive amounts of drugs necessary to stop the rejection response and the high mortality rate associated with them. Doctor Yeh questioned the value of chest x-rays in detecting the rejection phenomena. Chest x-rays were obtained on a number of these dogs. It is true that one can detect the rejection phenomenon by chest x-rays after rejection has progressed quite a bit. However, the rejection response must be much more advanced in order to detect it roentgenographically than it is in the method we are employing. VOL. I, NO. 5, SEPT.,