Acute Isolated Pulmonaw Reiection Following Transplantatich of the Heart and Both Lungs: Experimental and Clinical Observations D. Novitzky, M.D., D. K. C. Cooper, Ph.D., F.R.C.S., A. G. Rose, Ch.B., M.Med.(Path), M.R.C.(Path), and B. Reichart, M.D. ABSTRACT Early observations following transplantation of the heart and both lungs have suggested that acute rejection occurs simultaneously in both organs. Endomyocardial biopsy could, therefore, be used to monitor rejection in both heart and lungs. We present here our experience with heart-lung transplantation in the baboon, and in two recently observed human patients. The evidence we provide suggests that acute rejection may occur earlier in the lungs than in the heart, and that monitoring the heart alone may prove inadequate. Of 12 baboons that survived heart and lung transplantation, 11 died from acute isolated pulmonary rejection; 10 of these 11 animals showed no features of cardiac rejection. In 2 of 6 human patients who have undergone this operation, an episode of acute pulmonary rejection is believed to have occurred in the absence of cardiac rejection. There is no easy method of confirming pulmonary rejection directly (other than open-lung biopsy, which is clearly contraindicated as a routine procedure). We suggest that more attention be directed toward developing tests that indicate acute rejection but are not organ specific, rather than relying on techniques that diagnose cardiac rejection only. Transplantation of the heart and both lungs is the logical therapy for patients with primary hypertension or Eisenmenger syndrome accompanied by end-stage rightheart failure [l-61. Early observations by Reitz and colleagues [7] suggested that the heart showed evidence of rejection whenever the lungs were undergoing acute rejection. Endomyocardial biopsy [8] could, therefore, be used to monitor rejection in both heart and lungs. At least 1 human patient, however, has been reported in whom severe pulmonary rejection was not associated with cardiac rejection [9], and a similar observation has been made in 2 experimental animals [lo]. We present here our experience with heart-lung transplantation in the baboon, and in 2 recently observed From the Departments of Cardiothoracic Surgery and Pathology, Groote Schuur Hospital and the University of Cape Town Medical School, Cape Town, South Africa. Accepted for publication Nov 19, 1985. Address reprint requests to Dr. Novitzky, Department of Cardiothoracic Surgery, University of Cape Town Medical School, Cape Town, South Africa. human patients. We also provide evidence to suggest that acute rejection occurs earlier in the lungs than in the heart, and that monitoring of the heart alone may therefore prove inadequate. Experimental Study Fourteen chacma baboons, weighing 25 to 27 kg each, underwent transplantation of the heart and both lungs from donor baboons matched for size and AB blood group. The techniques of excision of the donor and recipient organs and of implantation of the allografts were as described by Reitz and by Jamieson and colleagues [ 1, 21 and have been fully described elsewhere. In both experimental studies and clinical cases, the lungs were preserved by low-pressure (50 cm H20) infusion of Euro Collins solution with 50 ml of 50% dextrose added, and the heart was preserved by infusion of the cardioplegic solution used by Wicomb and co-workers [ll]; both organs were topically cooled with saline at 4 C. The 14 baboons received one of three immunosuppressive regimens (Table); cyclosporin A was given daily, either orally or by a single intravenous (IV) injection, or it was given twice daily by intravenous injections. Cyclosporin trough levels were measured in whole blood and were found to be below the ideal therapeutic levels in all animals (less than 100 ng/ml). No attempt was made to treat suspected acute rejection. Monitoring consisted of daily recording of electrocardiogram data, rectal temperature, arterial blood gas tension, complete blood count, and liver function test results. Chest roentgenograms were taken at intervals in selected animals. Elevation of the temperature, a fall in the arterial oxygen tension (Pa02), and patchy shadowing on chest roentgenograms were thought to indicate acute rejection of the lungs. No monitoring for rejection of the heart by endomyocardial biopsy or radionuclide scanning [12] was performed. Res u 1 ts Twelve of the 14 baboons survived between 4 and 29 days (Table). When death occurred, it resulted from acute pulmonary rejection in 11 and from bronchopneumonia in 1. In 11 animals, the lungs showed histologic evidence of severe acute rejection, including prominent perivascular and interstitial infiltrates of lymphocytes and plasma cells (Figs 1-3). Interstitial and intraalveolar edema was also noted. These changes resulted in greatly thickened 180 Ann Thorac Surg 42:180-184, Aug 1986
181 Novitzky, Cooper, Rose, et al: Pulmonary Rejection after Heart-Lung Transplant lmmunosuppressive Regimens and Survival Times in 12 Chacma Baboons Undergoing Allotransplantation of the Heart and Both Lungs Immunosuppressive Therapy (mg/kg) Allograft Survival No. of Baboons Cyclosporin A Azathioprine Methylprednisolone (days) 4a 4 4 Wday, orally 18/day, by single IV injection 2-4iday orally for first 2 weeks 2iday from beginning of week 3 18, by twice daily IV injection "Two further baboons died as a result of technical problems. IV = intravenous. 6, 6, 19, 29 (mean, 15) 7, 12, 19, 20 (mean 15) 4, 5, 20, 28 (mean, 14) Fig I. Acute rejection of donor lung is manifested by a perivascular cellular infiltrate composed of lymphocytes and plasma cells that are spreading out into the adjacent alveolar septa. (H6E; x 250). Fig 3. Higher-power magnification of the lymphoid infiltrate in an alveolar septum of the rejection lung shown in Figure 2. (HBE; X 600). Clinical Experience The early clinical course of 2 of our 6 human patients (4 in Cape Town and 2 in Munich) who underwent transplantation of the heart and both lungs is described briefly as follows. Fig 2. Photomicrograph showing acute pulmonary rejection. Severe widening of the alveolar septa by the cellular infiltrate has led to reduction in size of the air spaces. (H&E; x 250). alveolar septa and, consequently, reduced alveolar aircarrying capacity (Fig 2). Only 1 of these animals showed any features of acute cardiac rejection; this baboon's heart had sparse focal infiltrates of pyroninophilic lymphocytes in the edematous interstitium, indicating mild rejection. The remaining 10 animals showed no features of cardiac rejection. Patient 1 This patient was a 27-year-old woman with underlying primary pulmonary hypertension who underwent transplantation in June, 1985. Initial immunosuppression consisted of oral cyclosporin A to maintain trough levels at approximately 400 ng/ml and oral azathioprine at 1.5 to 2.0 mg/kg daily. Ten days after transplantation a pyrexia of 38 C developed, associated with a reticular pattern visible in the lung fields on chest roentgenograms (Fig 4). Cytological monitoring, using a method developed in Munich [13], showed the presence of activated lymphoblasts, which suggests acute rejection. One day later she became moderately dyspneic, although her blood gas tension remained normal. Klebsiella spp. were cultured from her sputum, and she was given a course of tobramycin and subsequently amikacin. Within 4 days her sputum became sterile, but her symptoms of pyrexia, dyspnea, and cough persisted, and the
182 The Annals of Thoracic Surgery Vol 42 No 2 August 1986 A B Fig 4. Posteroanterior chest roentgenogram of patient I: (A) 27 days after transplantation of the heart and both lungs and (B) 4 days after ant ireject ion therapy. shadowing on the chest roentgenogram became more marked. On day 17 posttransplant her respiratory rate increased and Pa02 fell to 9 kpa (67.5 mm Hg) from a previous level of 10 to 12 kpa when she was breathing room air. She required oxygen therapy by mask. Radionuclide scanning (121 and endomyocardial biopsy (Fig 5) showed no features of myocardial rejection. Monoclonal antibodies used on frozen sections of the biopsy to investigate lymphocyte subpopulations revealed only a few T8 cytotoxic/suppressor lymphocytes, no inducer1 Fig 5. Histological section of endomyocardial biopsy done on patient I on day 17 after transplantation of the heart and both lungs, showing no signs of cardiac rejection. helper T cells, and no B cells, macrophages, or neutrophils. Intensive investigations for a source of bacterial, fungal, or viral infection proved negative. The patient's clinical condition was deteriorating. This fact, together with the findings on the chest roentgenogram and the presence of activated lymphocytes on cytological monitoring [ 131, suggested the possibility of acute rejection of the lungs, despite the absence of any features of rejection of the heart. She was therefore given a course of IV methylprednisolone (MP), 1 gm daily for 4 days, beginning on day 17. Within 6 hours of the first 1-gm infusion, she showed symptomatic improvement, becoming less dyspneic; her Pa02 rose to 12 kpa. Within 24 hours she was afebrile. On day 18, administration of oral MP was begun (0.3 mg/kg daily). Within 5 days the lung fields as seen on the chest roentgenogram were almost clear. The activated lymphocytes disappeared from her blood. She then remained afebrile and asymptomatic for 2 days, after which she again had a pyrexia of 39 C and had a pleuritic rub. Radionuclide scanning and endomyocardial biopsy were again normal, and on this occasion both the chest roentgenogram and cytological monitoring showed no abnormalities. Because acute pulmonary rejection was thought to be continuing, she was given a course of IV rabbit antithymocyte globulin (RATG; 2 mg/kg daily for 3 days) and MP (1 gm on the first day and 500 mg daily for 2 further days). Throughout this period cyclosporin A levels remained in the low therapeutic range. Patient 2 This patient, a 20-year-old man, underwent transplantation in August, 1985, for underlying idiopathic pulmonary hypertension and right ventricular failure.the basic immunosuppressive regimen was the same as that for patient 1. His initial progress was uneventful, although
183 Novitzky, Cooper, Rose, et al: Pulmonary Rejection after Heart-Lung Transplant he required vigorous physiotherapy and repeated therapy with continuous positive airway pressure to expand both lung bases. On day 6 postoperation, a pyrexia of 39"C, sweating, and a cough developed that continued intermittently for the next 24 hours, during which all investigations for a source of infection proved negative. He became dyspneic, and his PaOz fell from 12 to 9 kpa during the course of 24 hours. A chest roentgenogram showed patchy shadowing throughout both lungs. In the absence of any confirmation of infection, he was treated with MP (1 gm IV) and with k4tg (2.5 mglkg IV), and then with further MP (1 gm) and RATG (1 to 2 mg/kg) therapy on days 7 and 8. Within hours of the initial antirejection therapy, his symptoms had markedly improved. By day 8 his PaOz had risen to 11 kpa, and his chest roentgenogram had virtually cleared. It was not until day 9 that an endomyocardial biopsy was performed. This showed no substantial features of acute rejection whatsoever. (It was our opinion that if cardiac rejection had been taking place during the previous 3 days, some histopathological features would still have been present on day 9. In particular we would expect the presence of residual mononuclear cells.) On day 10 he once again became tachypneic and febrile (38.5"C). On this occasion, however, his chest roentgenogram showed no deterioration, and he became cold peripherally, which suggests a low cardiac output. He was given a further 3-day course of MP (1 gm daily IV), and on day 11 he also received RATG (2 mg/kg IV). Once again his symptoms subsided. Radionuclide scanning of the heart, performed during this suspected episode of cardiac rejection, showed a reduction in stroke volume, which would strongly support this diagnosis. Subsequent scanning on day 17 showed recovery of left ventricular volumes and function. He then remained well until day 27, when again a pyrexia developed and showed a generalized reticular pattern in both lung fields on a chest roentgenogram. Radionuclide scanning at this stage again showed reduced stroke volume, and it was thought that on this occasion the patient showed features of both pulmonary and cardiac acute rejection. He received a further course of MP to which he responded well. His subsequent course has been uneventful. Comment The difficulty of maintaining adequate blood levels of cyclosporin A in the baboon has hindered our transplantation research program, as exemplified by the high incidence of acute rejection in the baboons in the present study. Even the twice-daily administration of intravenous cyclosporin in relatively high doses was insufficient to maintain whole-blood trough levels in the desired therapeutic range (200 to 400 ng/ml). The low blood level achieved by the oral administration of this drug is probably species related, because good blood levels are achieved in most other animal species, including humans. In the baboon it seems likely that gastrointestinal tract absorption is impaired, or there is rapid excretion of the drug by the liver, or both. Recent work in our laboratory has shown, however, that adequate trough levels can be attained by daily intramuscular injection of cyclosporin A, administered in a mixture of alcohol and intralipid substances [14]; this development will greatly facilitate transplantation studies in the baboon. The experimental observations presented here clearly indicate that, in the inadequately immunosuppressed baboon, acute rejection of the lung occurs earlier than rejection of the heart. Pulmonary rejection in the absence of cardiac rejection, and vice versa, has also been documented in a small number of cynomolgus monkeys [lo]. Lung rejection has been shown by Prop and colleagues [15] to occur at an earlier stage than heart rejection in rats receiving no immunosuppression; their study suggests that lymphoid tissue transplanted within the lungs is an important stimulus that makes the unmodified rejection of lungs more vigorous than that of hearts. The two case histories described here also indicate that, on occasion, pulmonary rejection may be more vigorous than cardiac rejection. A similar observation has been made by others in 1 patient [9]. From these experimental and clinical observations we conclude that, where pneumonia has been excluded (as far as possible) by investigation, acute rejection of the lungs must always be strongly suspected if there are any suggestive features. One such feature of particular importance is pulmonary shadowing seen on chest roentgenograms despite normal results of endomyocardial biopsy (indicating no acute rejection of the heart). It seems possible that episodes of pulmonary shadowing on chest roentgenograms, previously termed a "reimplantation response" because of the absence of any evidence of heart rejection in the results of endomyocardial biopsy, may in reality be episodes of reversible acute rejection of the lungs, particularly if they occur after the first postoperative week. Possibly a greater degree of suspicion with regard to the development of acute pulmonary rejection is necessary than was previously thought. (These observations agree with those made at Stanford Medical Center, Stanford, CA; [S. Jamieson, personal communication].) In the absence of any easy method of confirming pulmonary rejection directly (other than open-lung biopsy, which is clearly contraindicated as a routine procedure), more attention should be directed toward developing tests that indicate acute rejection but are not organ specific. Such tests include cytological immunological monitoring [13] or the neopterin test [16], as opposed to techniques that diagnose cardiac rejection only. References 1. Reitz BA: Heart-lung transplantation: a review. Heart Transplant 1:291, 1982 2. Jamieson SW, Baldwin JC, Reitz BA, et al: Combined heart and lung transplantation. Lancet 1:1130, 1983 3. Novitzky D, Cooper DKC, Wicomb WN, et al: Transplanta-
1% The Annals of Thoracic Surgery Vol 42 No 2 August 1986 tion of the heart and both lungs: experimental and clinical experience and review of the literature. S Afr Med J 67:575, 1985 4. Reichart B, Reble B, Kemkes BM, et al: The first two heartlung transplants in Germany. MMW 126:1355, 1984 5. McGregor CGA, Jamieson SW: Clinical heart and lung transplantation. Br Med J 290:1682, 1985 6. Novitzky D, Cooper DKC: Transplantation of the heart and both lungs. In Cooper DKC, Lanza RP (eds): Heart Transplantation. Lancaster, England, MTP Press, 1984, pp 321-340 7. Reitz BA, Gaudiani VA, Hunt SA, et al: Diagnosis and treatment of allograft rejection in heart-lung transplant recipients. J Thorac Cardiovasc Surg 85:354, 1983 8. Caves PK, Stinson EB, Graham AF, et al: Percutaneous transvenous endomyocardial biopsy. JAMA 225:288, 1973 9. McGregor CGA, Baldwin JC, Jamieson SW, et al: Isolated pulmonary rejection after combined heart-lung transplantation. J Thorac Cardiovasc Surg (in press) 10. Scott WC, Haverich A, Billingham ME, et al: Lethal rejection of the lung without significant cardiac rejection in primate heart-lung allotransplants. Heart Transplant 4:33, 1984 11. Wicomb WN, Cooper DKC, Novitzky D, Barnard CN: Cardiac transplantation following storage of the donor heart by a portable hypothermic perfusion system. Ann Thorac Surg 37:243, 1984 12. Novitzky D, Boniaszczuk J, Cooper DKC, et al: Prediction of acute cardiac rejection using radionuclide techniques. S Afr Med J 65:5, 1984 13. Hammer C, Reichenspurner H, Ertel W, et al: Cytological and immunologic monitoring of cyclosporine-treated human heart recipients. Heart Transplant 3:228, 1984 14. Novitzky D, Cooper DKC, Wicomb WN: A successful method of administering cyclosporin A to the chacma baboon. S Afr Med J 66:737, 1985 15. Prop J, Kuijpers K, Nieuwenhuis P, Wildevuur CRH: Why are lung grafts rejected more vigorously than heart grafts? Paper presented at the 5th Annual Scientific Session of the International Society for Heart Transplantation, March, 1985. Heart Transplant 4:143, 1985 (Abstract) 16. Margreiter R, Fuchs D, Husen A, et al: Neopterin as a new biochemical marker for diagnosis of allograft rejection: experience based upon evaluation of 100 consecutive cases. Transplantation 36:650, 1983