Transplantation of the lung

Transplantation of the lung Correlation of physiologic, immunologic, and histologic findings Nicholas A. Halasz, M.D., Antonino Catanzaro, M.D., Max J. Trummer, M.D., Gennaro M. Tisi, M.D., Sidney L. Saltzstein, M.D., Kenneth M. Moser, M.D., and Peter Hutchin, M.D., San Diego, Calif.

Compared to other organ allografts, the early appearance and severe degree of functional and histologic changes in lung allografts is striking. Blumenstock and Kahn ' described perivascular round cell infiltration seen on the second postoperative day, while Waldhausen and associates" reported a significant diminution of compliance and surfactant. On the third post-transplant day, Hutchin and Walker" found a significant anatomic shunt and decreasing ventilationperfusion ratios, and Blumenstock ' noted diffuse opacification of the chest on roentgenography. Unfortunately, the majority of reported observations were not made early enough to evaluate the full extent of these rapid changes. Many histologic studies were done at the time of autopsy, with significant resultant artifacts. Often no synchronous physiologic and histologic data were obtained, and comparisons with simultaneous auto grafts were not made. The studies to be reported were designed to fill this gapto examine the early unmodified allograft clinically, physiologically, and histologically, and to compare it with autografts. From the University of California, San Diego School of Medicine, 225 West Dickinson Street, San Diego, Calif. 92103. Supported in part by U. S. Public Health Service Grants HL-13148 and HL-14169. Received for publication June 8. 1973.

Methods Fifteen conditioned, dewormed dogs weighing 16 to 2] kilograms were used as recipients in these studies. They were anesthetized with pentobarbital and maintained with Pentothal, intubated, and ventilated with 40 per cent oxygen. Left lung autografts and allografts were performed by previously described techniques. I. " Recipients were given ].0 Om. ampicillin daily starting on the day before operation. All dogs were fully studied on the fourth postoperative day and were put to death with an overdose of pentobarbital after completion of the studies, except for 2 dogs which were put to death on Day 6. Studies were done on the fourth postoperative day using mild tranquilization with Tran-Vet. Supine anteroposterior chest roentgenograms were taken, and perfusion scanning (prone, anteroposterior) was done with Xenosol (133Xe in solution) and technetium-tagged microspheres. Scans were recorded by means of a split-crystal Anger scintillation camera. G Arterial oxygen tensions were determined on room air and after the anima] had breathed ] 00 per cent oxygen (from a valved air-tight head bag with a high flow to prevent rebreathing) for ]5 minutes. The immunodiffusion technique of Edgington' was used to quantitate the levels of

581

582

Halasz et

The Journal of

at.

Thoracic and Card iovascular Surgery

Fig. 1. Four day lung autograft showing density in mid-lung field, compatible with ateiectasis or edem a, in region underlying incision.

Fig. 2. Four day lung allograft showing typical diffuse alveolar infiltrative filling process involving entire graft.

fibrinogen and of fibrin-split products (nonclottable fibrinogen antigen). Antisera to dog fibrinogen were prepared in rabbits. Venous blood was collected in ethylenediaminetetraacetic acid. The amount of fibrinogen antigen present before and after the addition of 10 U. of thrombin was determined. The result was expressed as milligrams per cent fibrinogen antigen (mg. per cent FA). Radial immunodiffusion" was utilized to determine the amount of IgG and complement (B,C) present. Antisera to each component were prepared in rabbits and demonstrated to be specific. Each was compared to a pool of normal plasma and expressed as per cent of the pool. Postmortem examinations were performed within 1 hour of death , on completion of the physiologic studies. Adhesions at the thoracotomy site were divided and the thoracic organs removed en bloc to permit careful dissection of the suture lines. Representative tissue samples were taken from each lobe of the reimplanted/transplanted left lung and the unmanipulated right lung. These were fixed in formal in and stained with hematoxylin and eosin after imbedding and sectioning.

The histologic material was reviewed by two of the authors (S. L. S. and N. A. H.) This was done with coded slides so that their origins were unknown. Each slide was graded as to the presence and extent of necrosis, alveolar edema, alveolar exudate, perivascular infiltration, peribronchial infiltration, perivascular and bronchial edema, mucosal damage , bronchitis, and pneumonia . Results One dog that received an allograft died 1 day postoperatively from a tension pneumothorax . Another, an autograft recipient, was found to have thrombosed upper and middle pulmonary veins at autopsy. These 2 animals were eliminated from the study. Plasma fibrinogen values obtained on the fourth postoperative day were comparable in autograft and allograft recipients (mean 247 ± 187 and 289 ± 146 mg. per cent FA, respectively). Fibrin-split products were not detected in any autografted dog on postoperative Day 4 or 6. All 6 allograft recipients had detectable circulating fibrinsplit products each time assayed. On the fourth postoperative day, the mean level was

Volume 66

Transplantation of lung

Number 4

583

October, 1973

Table I. Blood gas and perfusion data obtained 4 days postoperatively Room air (Flo, 0.2) pH

Autografts

Mean :t S.D. Allografts

Mean :t S.D.

I(mill.Po,Hg) I (mm. Pea, Hg)

Oxygen (Flo, 1.0) pH

I(mm.Po,Hg) I(mm. Pea, Hg)

Perfusion

I

Left Right (per cent) (per cent)

7.44 7.40 7.45 7.42 7.41 7.42

75 71 100 78 75 80 :t 12

36 33 34 37 36 35 :t I

7.40 7.40 7.45 7.41 7.40 7.41

474 468 462 480 467 470 ± 6

42 34 34 35 36 36 :t 3

14 14 21 22 19 18 :t 3

86 86 79 78 81

7.44 7.43 7.50 7.41 7.42 7.45 7.44

65 41 41 74 46 53 55 :t 12

29 21 25 30 26 28 26 :t 3

7.43 7.39 7.43 7.37 7.38 7.39 7.40

227 41 140 272 211 183 179 :t 73

31 29 34 27

20 8 7 13 11 9 11 ± 4

80 92 93 87 89 91

12 ± 7 mg. per cent FA (range 4 to 24). One dog tested on Day 6 had a level of 30 mg. per cent FA. IgG levels varied over a wide range in individual dogs (38 to 143 per cent of pool). Neither experimental group changed significantly from its base line over the period of observation. Likewise, complement levels (B1C) varied over a wide range in individual dogs (127 to 240 per cent of pool), and serial studies did not disclose a difference between autografted and allografted dogs. Radiographic changes. In the autotransplanted lungs a density developed in the mid-lung field within 48 to 72 hours of operation (Fig. 1). Its geometric confines appeared to coincide with the (overlying) thoracotomy incision; it involved 20 to 40 per cent of the left lung and had characteristics most consistent with atelectasis. By contrast, an alveolar filling process developed in the allograft lungs within 24 to 36 hours, which rapidly progressed to involve up to 90 per cent of the lung by the fourth postoperative day (Fig. 2). Arterial blood gases on the fourth postoperative day are given in Table I. They showed mild arterial hypoxemia in several of the autografted dogs breathing room air (pH 7.42, Po" 80 ± 12 mm. Hg, and reo, 35 ± 1 mm. Hg) and an average right-toleft shunt of 10 per cent breathing 100 per

32

33 31 :t 2

cent oxygen (pH 7.4, POz 470 ± 6 mm. Hg, and Pco, 36 ± 3 mm. Hg):" In contrast, the allograft recipients showed moderately severe arterial hypoxemia on room air (Po, 55 ± 12 mm. Hg), significantly differing from that of the autografted dogs (p < 0.05). This was observed in the face of significant (p < 0.001) hyperventilation (Pco, 26 ± 3 mm. Hg), which diminished somewhat on 100 per cent oxygen (Pco, 31 ± 2 mm. Hg). The allograft recipients also had a markedly widened alveolararterial oxygen gradient when breathing 100 per cent oxygen (Po" 179 ± 73 mm. Hg), significant at p < 0.001. Assuming a normal arteriovenous oxygen difference, this would indicate a right-to-left shunt in excess of 25 per cent. U Perfusion distribution as measured on the fourth postoperative day is also shown in Table I. In both groups, a major portion of the normal fraction (about 0.45) of cardiac output to the left lung had been shifted away. The fraction perfusing the autografted lungs was 0.18 ± 0.03, the shift therefore being about 60 per cent of the normal flow fraction. In the allografts, 0.11 ± 0.04 of the cardiac output went to the left lung, significantly less than in the autografts (p < 0.001) and representing a drop of 76 per cent from the normal left lung flow fraction.

The Journal of

5 84

Halasz et al.

Tho racic and Card iovascula r Surgery

"

. " .,J

It

. ...

,

f

r

-

'-

•.;,

..- ..-

'f

Fag. 3. Autograft lung at 4 days showing alveolar congestion and edema.

Fig. 4. Allograft lung at 4 days showing arteriole and vein. with periv ascular round cell infiltration and alveolar exuda tive process.

Pathologic findings

Gross examination revealed slightly wet left lungs with mild congestion in both allograft and homograft recipients. Patchy areas of atelectasis were present in both lungs of both groups. The lung weight ratio s were different in the two groups. In the auto-

grafted animals , the left lung weight was 0.52 ± 0.04 of total lung weight; in the allograft recipients it was 0.61 ± 0.03. This difference is statistically significant (p < 0.001 ). The allograft lungs were consistently congested , with purple-red discoloration. Their cut surfaces exuded pink fluid.

Volume 66

Transplantation of lung

Numbe r 4

585

October, 1973

Fig, 5. Allograft lung at 4 days showing bronchitis, mucosal destruction, and inflammatory exudate.

The bronchi contained bloody mucus and the bronchial mucosa distal to the anastomosis was congested and edematous. Histologic examination. Dogs sacrificed at 4 days. Autografted lungs showed mild-to-moderate perivascular and peribronchial edema and moderate peripheral (alveolar) congestion (Fig. 3). Both these changes were most evident in the region underlying the thoracotomy incision. Contralateral lungs in the autograft recipients were unremarkable except for the presence of patchy zones of mild alveolar congestion. Allografted lungs showed the changes described above, although often masked by other, more striking alterations. Every lung showed moderate-to-marked diffuse and generalized perivascular infiltration by mononuclear cells (Fig. 4). This was maximal around vessels 500jl or smaller in diameter and involved pulmonary veins and arteries alike. In two of the lungs, the infiltrate was also present in the alveoli, within a matrix of basophilic material. Peribronchial mononuclear infiltration was present in one lung peripherally. In contrast, more centrally, the larger bronchi (greater than

2 or 3 mm.) of all allograft lungs showed inflammatory changes, with polymorphonuclear leukocytes predominating, as well as mucosal damage and focal necrosis (Fig. 5) . Dogs sacrificed at 6 days . The two allografted lungs showed the changes observed at 4 days , but in more advanced stages. Perivascular infiltration was severe, diffuse , and extended centripetally, involving larger vessels as well at this time. Alveolar exudate was present in both animals. A new finding, scattered focal necrosis of alveolar walls, was evident and coalescent in a few regions. Peribronchial infiltration was marked, with moderate focal necrotic changes again being noted.

Discussion In their early studies, Barnes and colleagues' ? described perivascular infiltration, alveolar edema, and bronchiolitis in 4 to 5 day allografts. Strieder and associates" found a 60 per cent diminution in the total lung capacity of the allograft lung with a ventilation-perfusion (V / 0 ) relationship of 0.76 at 5 days. Hutchin and Walker" found a 50 per cent shunt in allograft lungs at 7 or

The Journal of

586

Halasz et al.

Thoracic and Cardiovascular Surgery

8 days. Becker and co-workers'> described an alveolar, fibrinous exudative reaction in 2 to 3 day dog allografts, a finding confirmed by Kondo's group> between 4 and 9 days. Joseph and Morton> made similar observations in the baboon autograft, but they believed that the exudate was secondary to ischemia. Blumenstock and Kahn' and Yeh and others" emphasized the early appearance of radiographic opacification in allograft lungs. In our studies, functional, radiographic, and anatomic features of the early autograft and allograft were examined simultaneously and compared. In both the autografted and allografted dogs there was a marked shift of perfusion away from the transplanted lung. Although ventilation scans were not performed, the absence of significant pathologic changes in the contralateral lungs, combined with the chest radiographs and the arterial oxygen tensions on 100 per cent oxygen, allow us to infer the V/0 relationships in both auto- and allografted lungs. The chest radiographs revealed a more diffuse alveolar filling process in the allografted lung, and the arterial oxygen tensions on 100 per cent oxygen ventilation revealed a larger right-to-Ieft shunt in these dogs. These two findings would suggest a better match of V/0 relationships in the autografted lungs and the presence of more lung zones with V /0 ratios of zero in allografted lungs. Based on these observations, identification and quantification of right-toleft shunting (V /0 = 0) in allografts may provide a simple, noninvasive method of defining the adequacy of immunosuppressive therapy. Complement, immunoglobulin, and fibrinogen studies were not revealing. However, the consistent presence of fibrin-split products in unmodified allograft recipients is of note, indicating the presence of vascular damage and an on-going thrombotic process at the early time of these observations. De novo sensitization beginning at the time of lung transplantation is unlikely to explain these findings adequately on immunologic

grounds-it is quite likely that a state of presensitization or heightened responsiveness is present in the allograft lung in comparison to other organ transplants. The histologic changes observed are striking in the earliness of their onset and diffuseness of distribution. At 4 days, when other organ allografts are unchanged microscopically or are just beginning to show signs of an immune response, a florid allograft rejection is taking place in the lung, consistent and compatible with the radiographic and functional changes observed. The extensiveness of the histologic changes is no less surprising. One of the hallmarks of the rejection reaction in dog kidneys, liver, and heart is its focal histologic distribution and asynchrony. Essentially normal zones alternate with regions of severe damage and all stages in between. This is not observed in the lung. Alterations are homogeneous and consistent throughout the transplanted organ, synchronous and parallel in development. It is perhaps for this reason that functional deterioration is as early and severe as it is. There is no reserve of undamaged tissue to maintain some function even while part of the organ is undergoing rejection and destruction; instead, it is all attacked simultaneously. This fact, combined with the remarkably early appearance of "rejection," raises the question of why and even how these changes can be mediated. For full-blown cellular immune changes to be evident at 4 days, there must be some sort of presensitization (perhaps to cross-reacting antigens) to account for the accelerated process or some mechanism not involved in the rejection of other allografts. The lung, with its large reservoir of macrophages and lymphoid tissue, certainly represents a different graft qualitatively from other parenchymatous organs. It also represents an interface between the body and the antigenically alien outside world and, as such, has local immune mechanisms not present in other organs thus far studied. Finally, it is not a sterile structure and is therefore susceptible to early bacterial dam-

Volume 66 Number 4

Transplantation of lung

587

October, 1973

age, particularly when under immunologic attack or damaged by ischemia.

Summary Two groups of dogs with unmodified lung autografts and allografts were studied physiologically, immunologically, and histologically 4 and 6 days postoperatively. Consistent severe hypoxemia and wide alveolar-arterial gradients were observed in the allograft group, with only minor changes in the autografts, even though perfusion was diminished on the operated side in both groups. All allograft recipients had significant levels of circulating fibrin-split products, indicating an active thrombotic process at 4 days. Radiograms and histologic findings correlated well with the physiologic studies, showing diffuse opacification along with florid, advanced changes of homograft rejection, in contrast to mild changes in the autografts. It is concluded that the degree of rightto-left shunting (V /0 ratio of zero) is greatly increased in the rejecting allograft and that its determination may provide a simple, noninvasive method for defining the adequacy of immunosuppressant therapy. Second, the allograft response in the lung is unique, both in earliness of onset and severity of degree. Presensitization or altered responsiveness due to the inclusion of large components of lymphoid tissue and macrophages in the allograft lung may account for this finding. REFERENCES Blumenstock, D. A., and Kahn, D. R.: Replantation and Transplantation of the Canine Lung, J. Surg. Res. 1: 40, 1961. 2 Waldhausen, J. A., Giammona, S. T., Kilman, J. W., and Daly, W. J.: Effect of Transplantation of Canine Lung on Pulmonary Compliance and Surfactant, J. A. M. A. 191: 1002, 1965.

3 Hutchin, P., and Walker, E. L.: Ventilatory and Circulatory Adjustments After Transplantation of the Lung, Ann. Surg. 175: 349, 1972. 4 Trummer, M. J., and Berg, P.: Lung Transplantation, Springfield, Ill., 1969, Charles C Thomas, Publisher. 5 Hutchin, P.: Pulmonary Transplantation, ill Peters, R. M., editor: The Mechanical Basis of Respiration, Boston, 1969, Little, Brown & Company, p. 305. 6 Harbert, J. C., Ashburn, W. L., and Davidson, J. D.: An Improved Method of Renography Using the Split Crystal Scintillation Camera, J. Urol. 99: 681, 1968. 7 Edgington, T. S.: Radical Diffusion Method for Fibrinogen and Split Products, ill Williams, W. J., Beutler, E., Erslev, A. J., and Rundles, R. W., editors: Hematology, New York, 1972, McGraw-Hili Book Company, p. 1410. 8 Mancini, G., Carbonara, A. 0., and Heremans, J. F.: Immunochemical Quantitation of Antigens by Single Radial Immunodiffusion, Immunochemistry 2: 235, 1965. 9 Chiang, S. T.: A Nomogram for Venous Shunt QjQ, Calculation, Thorax 23: 563, 1968. 10 Barnes, B. A., Flax, M. H., Burke, J. F., and Barr, G.: Experimental Pulmonary Homografts in the Dog. I. Morphological Studies, Transplantation 1: 351,1963. II Strieder, D. J., Barnes, B. A., Aronow, S., Russell, P. S., and Kazemi, H.: Xenon 133 Study of Ventilation and Perfusion in Normal and Transplanted Dog Lungs, J. Appl. Physiol. 22: 359, 1967. 12 Becker, N. H., Sinha, S. B. P., Hagstrom, 1. W. c., Blumcke, S., and Veith, F. 1.: Fine Structure Alterations in Canine Lung Transplants, J. THORAC. CARDIOVASC. SURG. 63: 81, 1972. 13 Kondo, Y., Isin, E., Cockrell, J. V., and Hardy, J. D.: One-Stage Bilateral Allotransplantation of Canine Lungs: Further Studies, J. THoRAc. CARDIOVASC. SURG. 64: 897, 1972. 14 Joseph, W. L., and Morton, D. L.: Morphologic Alterations in the Transplanted Primate Lung, Surg. Gynecol, Obstet. 133: 821, 1971. 15 Yeh, T. J., Toyohara, H., Ellison, L. T., Parker, J. L., and Ellison, R. G.: Pulmonary Function in Dogs After Lung Homotransplantation, Ann. Thorac. Surg. 2: 195, 1966.