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Cellular characteristics of the rejection response to canine lung allotransplants
Cellular characteristics of the rejection response to canine lung allotransplants Shigeiumi Fujimura, M.D., Victor Rosen, M.D., Gerald E. Adomian, Ph.D., William W. Parmley, M.D., * Chigashi Suzuki, M.D., ** and Jack M. Matlof], M.D., Los Angeles, Calif.
DesPite continuing laboratory and clinical experience with organ transplantation, rejection remains the primary barrier to general clinical application. Studies in dogs to clarify the mechanism of allograft rejection have been limited by genetic dissimilarities between donor and recipient. operative technique, and associated toxicity of immunosuppressive therapy. Postoperative infection, a common manifestation of toxicity, often obscures the pattern of unmodified rejection. This is particularly true in pulmonary allografts. This presentation is based on past and present laboratory experience with whole lung transplants in dogs. Histologic examination of these transplants is the basis for From the Departments of Thoracic, Cardiac and Vascular Surgery, Pathology, and Cardiology, Cedars-Sinai Medical Center, Los Angeles, Calif. This work was supported by National Institutes of Health Grants HE-13297-02, HE-I0382-07, and RR-05468-09 and the Factor Foundation for Cardiovascular Surgical Research. Received for publication Aug. 23, 1972. Address for reprints: Jack M. Matlotf, M.D., Director, Department of Thoracic, Cardiac and Vascular Surgery, Cedars-Sinai Medical Center, 4833 Fountain Ave., Los Angeles, Calif. 90029. "This work was done during the tenure of an established investigator of the American Heart Association. ""From the Department of Surgery, The Research Institute for Tuberculosis, Leprosy and Cancer, Tohoku University, Send ai, Japan.
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discussion of the rejection process in lung allografts. This study will focus on the characteristics of the mononuclear cells that are observed in the transplant at the time of rejection. Materials
In 113 adult, mongrel dogs, left or right lung transplantation was performed by means of previously described techniques.v" In lung autotransplantation, the lung was excised and perfused with cold heparinized dextran solution or lactated Ringer's solution; reconstruction was accomplished by suture anastomoses of hilar elements. In allotransplantation of the lung, the donor lung was treated with 50 mg. of heparin prior to the occlusion and division of the pulmonary artery; the remaining hilar structures were divided, and the lung was excised and orthotopically transplanted to the recipient. Postoperatively, antibiotics, including penicillin, streptomycin, and tetracycline, were administered daily to all animals. The dogs were divided into three groups (Table I). Group I: Autotransplant controls. This group consisted of 8 animals that underwent right or left lung autotransplantation. All animals had serial surgical biopsies of the transplant three times postoperatively, except
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Table I
Group
No. of dogs
Time of examination (biopsy/autopsy)
Survival
I. Control autotransplants
8
4 days, 1 dog; 21 days, 7 dogs (sacrificed)
II. Control allotransplants III. Immunosuppressed allotransplants
39
4 to 20 days, 38 dogs; 100 days, 1 dog 3 to 221 days
66
Biopsy immediately and 3 to 7 days after transplantation; sacrificed at 21 days Variable biopsy and at autopsy Variable biopsy and at autopsy
Type of examination LM
I EM I FA
8
8
39
6
66
20 15
Legend: LM, Light microscopy. EM, Electron microscopy FA, Fluorescent antibody.
for 1 animal that was put to death 4 days after operation. Usually, specimens were obtained immediately after operation, 3 to 7 days postoperatively, and at sacrifice 21 days after the operation. Light and electron microscopic examinations were performed; however, immunofluorescent studies were not carried out in this group. Group II: Allotransplant controls. This group consisted of 39 animals which underwent left lung allotransplantation and received no immunosuppressive treatment. Survival time varied from 4 to 20 days in all but 1 dog. This animal survived 100 days, at which time it was put to death. Specimens of the transplanted lung were obtained by surgical biopsy in some cases and by autopsy in all cases. All specimens were examined by light microscopy. Material from twenty of the lung allotransplants was examined by the fluorescent antibody method.' Specimens removed from 6 dogs with grafted lungs were studied by electron microscopy. Group III: Allotransplant suppressed. This group consisted of 66 animals that underwent left lung allotransplantation and were treated with a variety of immunosuppressive regimens, including corticosteroids, methotrexate, azathioprine, 6-methylchlorambucil, antilymphocytic serum, antilymphocytic globulin, external irradiation, and infusion of donor blood or spleen cells in various combinations. In this group, survival varied from 3 to 221 days. Specimens of the transplanted lung obtained by surgical
biopsy or by autopsy were studied by light microscopy in all cases. Fluorescent microscopic studies were carried out in 15 animals. Graft ultrastructure was not studied in this group. Methods Light microscopic examination. The specimens obtained from the grafted lungs were fixed in 10 per cent neutral formalin and were embedded in paraffin. Sections were stained with hematoxylin and eosin. Some of these sections were studied with special stainings when indicated. Fluorescent microscopy. Fluorescent microscopic studies were obtained in 35 dogs from Groups II and III. The detailed methodology of fluorescent microscopy on pulmonary allotransplants has been reported previously.v 5 In this study, tissue blocks were fixed in 95 per cent ethanol at _70 0 C. and were embedded in paraffin. Two canine immunoglobulins were isolated by gel filtration through a column of Sephadex G-200 followed by diethylaminoethyl cellulose chromatography. One fraction showed 18.9 S (yM) and another showed 6.8 S ( yG) by analytic ultracentrifugation. Antisera against canine immunoglobulins were prepared in rabbits. Fluorescent antibody against yM was absorbed with canine yG, and that against yG was absorbed with yM before the immunofluorescence procedure. Tissue sections were stained by direct staining with absorbed fluorescent antibodies. Electron microscopy. Electron microscopic
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Fig. 1. Lung autotransplant (dog No . X·7) , 3 weeks after operation, shows a perivascular collection of mononuclear cells consisting mainly of plasma cells. (Hematoxylin and eosin; original magnification x250.)
examinations were carried out in 14 animals from Groups I and II. In animals with autotransplanted lungs (Group I), the specimens for electron microscopy were cut into 1 by 2 mm. blocks and immersed overnight in a solution containing 3 per cent glutaraldehyde and 0.135M phosphate buffer. After a buffer rinse , the tissue was postfixed in a buffered 1 per cent osmium tetroxide solution for 2 h hours . The specimens were then rinsed , dehydrated in acetone, embedded in Vestopal W, and polymerized overnight at 60° C. Sections were prepared with an LKB Vetrotome III and were stained with uranyl acetate and lead citrate. Sections were examined with the JEM 7A electron microscope at an accelerating voltage of 80 Kv. A 200 flo condenser aperture and a 50 flo objective aperture were utilized. In animals with allotransplanted lungs (Group II) , specimens for electron microscopic examination were fixed in both glutaraldehyde and osmium tetroxide buffered with Millonig 's buffer . After fixation, the specimens were dehydrated in an ethanol series and embedded in epon 812. The blocks were sectioned with glass knives by means of the Porter-Blum MT-l microtome. Thin sections were examined with the
Hitachi HU-ll B electron microscope after being stained with uranyl acetate and lead citrate. Results Group I. The results of histologic examinations of lung autotransplants have been described in detail. 3 The unilaterally auto transplanted lung in the early postoperative period generally showed focal variable degrees of pulmonary congestion and edema . There was no evidence of mononuclear cell infiltration around the pulmonary vessels and bronchioli. Pulmonary histiocytic proliferation was shown in the grafted lung in 5 of 8 animals 3 weeks postoperatively. Pulmonary emboli, comprised of intravascular thrombi and leukocytic agglutinations, were observed in 3 animals. A perivascular collection of mononuclear cells was found in only 1 animal 3 weeks after operation. This lesion was associated with other histologic findings, including lung edema , pulmonary histiocytic proliferation, endothelial cell proliferation, and bronchopneumonia. Mononuclear cells consisted mostly of plasma cells and histiocytes with rare lymphocytes (Fig. 1) . No mononuclear cells classified as intermediate between lymphocytes
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Fig. 2. Lung allotransplant (dog No. 1006), 10 days after transplantation without immunosuppression. Intra- and extravascular mononuclear cells are seen, i.e., a lymphocyte (left), a plasma cell (right), and a macrophage (center) (x4,OOO).
and plasma cells were found in this autotransplanted specimen ultrastructurally. Group II. Pulmonary allografts in untreated dogs generally showed signs of acute rejection within 4 to 6 days postoperatively. The initial histologic characteristic of rejection in these grafts was infiltration of mononuclear cells around the small vessels and bronchi and into the subpleural space. Clusters of these mononuclear inflammatory cells often were noted to form a granuloma-like pattern around the small vessels; the nature of these vessels was not always clear. In the first week of rejection, the mononuclear infiltrate consisted of reticulum-like cells, macrophages, lymphocytes, and plasma cells. Electron microscopic examination during rejection in the untreated dogs showed clusters of mononuclear cells in the lung parenchyma, which included macrophages, lymphocytes, and plasma cells. Occasionally, cells intermediate between the lymphocyte and the plasma cell were observed in the
cell clusters, both inside and outside the capillaries (Fig. 2). The cytoplasm of the lymphocytes seen in the transplants (Fig. 3) had a fine granular structure, with small numbers of mitochondria and slightly dilated endoplasmic reticulum. It appeared almost translucent, with few organelles or inclusions. Fig. 4 shows the typical features of the plasma cells seen during the rejection period. The endoplasmic reticulum is dilated and seems to occupy virtually all of the cytoplasm except the paranuclear region (Fig. 5). Fig. 6 shows an intermediate cell type. The endoplasmic reticulum is not as well developed as the typical plasma cell. Pyronine-positive cells were observed in considerable numbers and yM-containing cells in fewer numbers (Fig. 7). yG-containing cells were not demonstrated in the perivascular infiltrates. Furthermore, none of these immunoglobulin-containing cells were seen within vascular lumina, despite the evidence of endothelial damage at this stage.
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Fig. 3. Ordinary lymphocyte in the perivascular space with a fine granular structure, mitochondria in a small number, and some slightly dilated endoplasmic reticulum. It appears to be translucent, as its organelles or inclusions are few (x28,OOO).
Fig. 4. Typical plasma cell in the perivascular space of a lung allotransplant, 10 days after transplantation. There is considerable development of the endoplasmic reticulum (xIS,OOO) .
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Fig. 5. Typical plasma cell with dilated endoplasmic reticulum (.<15,000).
Fig. 6. Representative cell type intermediate between the lymphocyte and the plasma cell. The endoplasmic reticulum is well developed, but not so well developed as the typical plasma cell (x24,OOO).
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Fig. 7. Lung allotransplant (dog No. 901), 5 days after transplantation, shows yM-positive cells (arrows) in the perivascular reaction (fluorescent antibody technique with anti-yM serum). (Original magnification x400.)
After the first postoperative week, there were increasing numbers of mononuclear cells around bronchioli and the pulmonary vasculature, with narrowing of the vascular lumina. In addition, there was an infiltration or emigration of inflammatory cells into the intra-alveolar spaces, along with exudation of red cells and proliferation and swelling of the endothelial cells (Fig. 8). In some cases, there were manifestations of panvasculitis, intravascular thrombus formation, and infarction necrosis. During this second week, yM-containing cells were often detected around the pulmonary vasculature (Fig. 9). Occasionally, yM-positive cells were seen in the intravascular space.' At times, cells that infiltrated the lung parenchyma also contained cytoplasmic yM. Additionally, there were cell remnants with positive I'M in the lung parenchyma.' In this period, yG was found to be localized especially in the perivascular spaces. Group III. In the immunosuppressed dogs, pulmonary infection was the most common feature to appear in the first 2 weeks after transplantation. In animals that survived their infection, the onset of the rejection reaction, as defined for the Group II animals,
was generally delayed until 3 to 5 weeks after operation. This was consistent with the observation that immunoglobulin-containing cells were rarely seen in the grafts before 3 weeks. After 3 weeks, features of rejection were disclosed by hematoxylin and eosin staining, and "1M-positive cells were often observed in the transplants (Fig. 10). Even with maximum immunosuppressive therapy, treated dogs with transplanted lungs survived for only 5 weeks. In the 18 that survived beyond 5 weeks, the subsequent course was relatively stable; the animals survived to 32 weeks, at which time they were put to death. In 5 dogs that survived 25 to 32 weeks, the transplant showed marked granulomatous formation, which consisted of lymphocytes and macrophages around vessels and bronchioli. Occasionally, the walls of the arterioles were markedly thickened and were stenotic or obstructed. In addition, there was conspicuous migration of histiocytes and epithelial cells into the alveoli (Fig. 11). In more advanced states of rejection, the walls of the small vessels were destroyed, and the alveolar structure lost its normal appearance. There were extensive signs of infarction necrosis and
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Fig. 8. Lung allotransplant (dog No. 840), 9 days after grafting, shows a small pulmonary vessel with endothelial destruction and perivascular mononuclear cell infiltration. (Hematoxylin and eosin; original magnification x400.)
Fig. 9. Lung allotransplant (dog No. 840), 9 days after grafting, shows yM-containing cells (fluorescent antibody technique with anti-yM serum). (Original magnification x400.)
(arrows) in the perivascular space
fibrosis in these lungs. In this chronic period, immunofluorescence studies showed no specific patterns in the lung transplant. Discussion
Perivascular infiltration of mononuclear cells has been accepted generally as one of
the dominant histologic features of the rejection process in allotransplanted organs. This change has been described as an integral aspect of early rejection of the kidney," heart; and liver." In the heart and liver, chronic rejection has been manifested by narrowing of the coronary and hepatic
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Fig. 10. Lung allotransplant (dog No . 926), 40 days after grafting. This dog was treated with antil ymphocyte globulin and azathioprine. yM-positive cells are seen in the per ivascular space as well as in the pulmonary vasculature (fluorescent antibody technique with anti-yM serum ). (Original magnification x400.)
Fig. 11. Lung allotransplant (dog No. 601), 32 weeks after grafting with immunosuppression. There is perivascular fibrosis with the infiltration of small mononuclear cells and a thickened vascular wall . (Hematoxylin and eosin ; original magnification x lOO.)
arteries due to internal proliferation and/or thrombus formation. In regard to pulmonary allografts, a similar histologic picture of early rejection has been verified in studies by Flax and Barnes" and in the present study. In un-
treated pulmonary allografts, the earliest evidence for rejection is the appearance of mononuclear cells around small vessels and bronchioli and also in the subpleural space within 4 to 6 days. Flax and Barnes identified these small vessels as veins. After the
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first postoperative week, increasing numbers of mononuclear cells, which compromise the vascular lumina, are noted around the pulmonary vessels and bronchioli. During this time, these mononuclear cells are often observed remote from the perivascular spaces, in the lung parenchyma. In treated animals, the early stages after transplantation are distinguished by evidence of infection. In the animals that survive, the onset of rejection is retarded until 3 to 6 weeks after the operation, at which time changes similar to those seen during earlier, acute rejection of untreated allografts predominate. In long-term, modified grafts, subendothelial inflammation, hyperplasia, and fibrosis are present. 9 In view of the findings of perivascular mononuclear cell collections in pulmonary autotransplants 3 weeks after surgery, it could be suggested that this perivascular mononuclear cell infiltration is a nonspecific histologic pattern in lung transplants. However, the fact remains that there are striking differences in the composition of cell populations between autotransplants and allotransplants. In allografts, the mononuclear, perivascular collections consist mainly of lymphocytes and plasma cells, whereas lymphocytes rarely appear in the autografts. These findings might suggest that in pulmonary allotransplants the majority of the cells are of recipient origin whereas in autotransplants they are of graft origin. Therefore, from these results and those described for other organs, it would appear that early rejection is generally effected by mononuclear cells, whether the event of rejection is modified by altered histocompatibility between donor and recipient. A number of investigations'? have acknowledged the role of antibody-mediated immunity in allograft rejection. Antibodies have been detected in experimental and clinical transplants. To further define the characteristics of the mononuclear cells in allografts undergoing rejection, fluorescent antibody and electron microscopic techniques have been used to study these cells. Porter" demonstrated that 15 per cent of
the infiltrating mononuclear cells in treated canine hepatic allografts contain yG in their cytoplasma as compared with 5 per cent of those in untreated grafts, thereby suggesting the presence of greater numbers of plasma cells in the infiltrate. Stastny!' also described large, mononuclear, yG-containing cells that infiltrated the myocardium of both late canine and human heart transplants. In the present study, some mononuclear cells containing yM in their cytoplasm, both in the early unmodified and late treated allografts, were seen. yG-containing cells were much less common. Occasionally, these cells were seen in intravascular collections, suggesting a recipient origin. Despite double staining for fluorescent antibodies and with hematoxylin and eosin, morphologic identification of these cells was not successfully carried out in this study; however, some of these cells were considered to be Russell body plasma cells. In a recent study of renal allotransplant histology,t- local liberation of antibodies by cellular disruption of plasma cells was suggested as one aspect of the rejection process. In the present study, electron microscopy in unmodified lung allografts demonstrated macrophages, lymphocytes, plasma cells, and cells intermediate between lymphocytes and plasma cells. The latter three cell types contained the fine structure of antibodyproducing cells." These intermediate cells have been termed graft rejection cells.v Although there has been no evidence to support transformation of lymphocytes into plasma cells in pulmonary allografts, the concept now arises that some of the mononuclear cells, including plasma cells and socalled graft rejection cells, are of recipient origin and may produce antibodies which lead to disruption of the graft. The degree of antibody specificity by these cells is speculative at best. Therefore, it would seem that, as rejection proceeds and enters the chronic phase, antibody exposure is continuous and the described reaction extends from its perivascular location to the vessel wall, associated with internal proliferation, vascular fibrosis, infarction necrosis, and extensive
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pulmonary parenchymal fibrosis. The cellular characteristics of the mononuclear cells in relation to the changes of chronic rejection remain to be defined. Summary Histologic changes occurring in lung autotransplants and allotransplants have been characterized by means of light, fluorescent antibody, and electron microscopy. Vascular lesions due to mononuclear cell infiltrates initially appear around small vessels and bronchioli. These mononuclear cells include macrophages, lymphocytes, and plasma cells. yM-containing cells have been identified in lung transplants by fluorescent microscopy. Findings of electron microscopy are discussed in relation to those by fluorescent antibody microscopy in unmodified lung allografts. Ultrastructurally, macrophages, lymphocytes, plasma cells, and cells intermediate between lymphocyte and plasma cell, i.e., graft rejection cells, were demonstrated among infiltrates of the mononuclear cells. The latter three types of cells contained the ultrastructural features of antibody-producing cells. These antibody-producing cells in the lung allograft that is undergoing rejection are considered to be among the mediators of the rejection phenomenon. REFERENCES Suzuki, C., Nakada, T., Furusawa, T., Watanabe, A., and Hirose, M.: Die Experimentelle Lungenreimplantation, Z. Tuberk. 123: 195, 1965. 2 Suzuki, C, Nakada, T., Watanabe, A., Fujimura, S., and Kawakami, T.: Experimental Studies of Canine Lung Allotransplantation: The Effect of Cytotoxic Agents and Donor Spleen Cells for Immunosuppressive Treatment, 1. THORAC. CARDIOVASC. SURG. 55: 200, 1968.
3 Fujimura, S., Rosen, V., Adomian, G. E., Parmley, W. W., Norman, 1. R., and Matloff, J. M.: Early Morphological Observations in Canine Pulmonary Autotransplants in Dogs: The Similarity to Shock Lung. Submitted for publication. 4 Fujimura, S., Yoneti, M., Kagami, Y., Kawakami, M., Chiba, S., and Okaniwa, G.: Immunohistological Studies on Canine Lung Allotransplantation. I. Preparation of Anti-canine Immunoglobulins (y M and yG), Ishoku 3: 414, 1969 (in Japanese). 5 Fujimura, S., Nakada, T., Kawakami, M., Sukeno, T., Yoneti, M., Okaniwa, G., Kagami, W., and Suzuki, C: Detection of Immunoglobulins (yM and yG) by Fluorescent Antibody Method in Canine Lung Allotransplantation, Tohoku J. Exp. Med. 101: 183, 1970. 6 Porter, K. A., Caine, R. Y., and Zukoski, C. F.: Vascular and Other Changes in 200 Canine Renal Homotransplants Treated With Immunosuppressive Drugs, Lab. Invest. 13: 809, 1964. 7 Lower, R. R., Kosek, J. C, Kemp, V. R., Graham, W. H., Sewell, D. M., and Lim, F.: Rejection of the Cardiac Transplant, Am. J. Cardio!. 24: 492, 1969. 8 Porter, K. A.: Pathology of Liver Transplantation, Transplant. Rev. 2: 129, 1969. 9 Flax, M. H., and Barnes, B. A.: The Role of Vascular Injury in Pulmonary Allograft Rejection, Transplantation 4: 66, 1966. 10 Najarian, 1. A., and Foker, 1. E.: Mechanisms of Kidney Allograft Rejection, Transplant. Proc. 1: 184, 1969. II Stastny, P.: Bound Immunoglobulin and Complement in Heart Allografts Undergoing Rejection, Transplantation 10: 248, 1970. 12 Mitchell, R. E.: The Evolution of Plasma Cells in Canine Renal Allografts, Bib!. Haemato!. 29: 667, 1968. l3 Harris, T. N., Hummeler, K., and Harris, S.: Electron Microscopic Observations on Antibody-Producing Cells of Lymph Node, Lymph and Blood, ill Yoffey, J. M., editor: The Lymphocyte in Immunology and Haemopoiesis, London, 1966, Edward Arnold (Publishers), Ltd. 14 Wiener, J., Spiro, D., and Russell, P. S.: An Electron Microscopic Study of the Homograft Reaction, Am. J. Patho!' 44: 319, 1964.
DesPite continuing laboratory and clinical experience with organ transplantation, rejection remains the primary barrier to general clinical application. Studies in dogs to clarify the mechanism of allograft rejection have been limited by genetic dissimilarities between donor and recipient. operative technique, and associated toxicity of immunosuppressive therapy. Postoperative infection, a common manifestation of toxicity, often obscures the pattern of unmodified rejection. This is particularly true in pulmonary allografts. This presentation is based on past and present laboratory experience with whole lung transplants in dogs. Histologic examination of these transplants is the basis for From the Departments of Thoracic, Cardiac and Vascular Surgery, Pathology, and Cardiology, Cedars-Sinai Medical Center, Los Angeles, Calif. This work was supported by National Institutes of Health Grants HE-13297-02, HE-I0382-07, and RR-05468-09 and the Factor Foundation for Cardiovascular Surgical Research. Received for publication Aug. 23, 1972. Address for reprints: Jack M. Matlotf, M.D., Director, Department of Thoracic, Cardiac and Vascular Surgery, Cedars-Sinai Medical Center, 4833 Fountain Ave., Los Angeles, Calif. 90029. "This work was done during the tenure of an established investigator of the American Heart Association. ""From the Department of Surgery, The Research Institute for Tuberculosis, Leprosy and Cancer, Tohoku University, Send ai, Japan.
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discussion of the rejection process in lung allografts. This study will focus on the characteristics of the mononuclear cells that are observed in the transplant at the time of rejection. Materials
In 113 adult, mongrel dogs, left or right lung transplantation was performed by means of previously described techniques.v" In lung autotransplantation, the lung was excised and perfused with cold heparinized dextran solution or lactated Ringer's solution; reconstruction was accomplished by suture anastomoses of hilar elements. In allotransplantation of the lung, the donor lung was treated with 50 mg. of heparin prior to the occlusion and division of the pulmonary artery; the remaining hilar structures were divided, and the lung was excised and orthotopically transplanted to the recipient. Postoperatively, antibiotics, including penicillin, streptomycin, and tetracycline, were administered daily to all animals. The dogs were divided into three groups (Table I). Group I: Autotransplant controls. This group consisted of 8 animals that underwent right or left lung autotransplantation. All animals had serial surgical biopsies of the transplant three times postoperatively, except
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Table I
Group
No. of dogs
Time of examination (biopsy/autopsy)
Survival
I. Control autotransplants
8
4 days, 1 dog; 21 days, 7 dogs (sacrificed)
II. Control allotransplants III. Immunosuppressed allotransplants
39
4 to 20 days, 38 dogs; 100 days, 1 dog 3 to 221 days
66
Biopsy immediately and 3 to 7 days after transplantation; sacrificed at 21 days Variable biopsy and at autopsy Variable biopsy and at autopsy
Type of examination LM
I EM I FA
8
8
39
6
66
20 15
Legend: LM, Light microscopy. EM, Electron microscopy FA, Fluorescent antibody.
for 1 animal that was put to death 4 days after operation. Usually, specimens were obtained immediately after operation, 3 to 7 days postoperatively, and at sacrifice 21 days after the operation. Light and electron microscopic examinations were performed; however, immunofluorescent studies were not carried out in this group. Group II: Allotransplant controls. This group consisted of 39 animals which underwent left lung allotransplantation and received no immunosuppressive treatment. Survival time varied from 4 to 20 days in all but 1 dog. This animal survived 100 days, at which time it was put to death. Specimens of the transplanted lung were obtained by surgical biopsy in some cases and by autopsy in all cases. All specimens were examined by light microscopy. Material from twenty of the lung allotransplants was examined by the fluorescent antibody method.' Specimens removed from 6 dogs with grafted lungs were studied by electron microscopy. Group III: Allotransplant suppressed. This group consisted of 66 animals that underwent left lung allotransplantation and were treated with a variety of immunosuppressive regimens, including corticosteroids, methotrexate, azathioprine, 6-methylchlorambucil, antilymphocytic serum, antilymphocytic globulin, external irradiation, and infusion of donor blood or spleen cells in various combinations. In this group, survival varied from 3 to 221 days. Specimens of the transplanted lung obtained by surgical
biopsy or by autopsy were studied by light microscopy in all cases. Fluorescent microscopic studies were carried out in 15 animals. Graft ultrastructure was not studied in this group. Methods Light microscopic examination. The specimens obtained from the grafted lungs were fixed in 10 per cent neutral formalin and were embedded in paraffin. Sections were stained with hematoxylin and eosin. Some of these sections were studied with special stainings when indicated. Fluorescent microscopy. Fluorescent microscopic studies were obtained in 35 dogs from Groups II and III. The detailed methodology of fluorescent microscopy on pulmonary allotransplants has been reported previously.v 5 In this study, tissue blocks were fixed in 95 per cent ethanol at _70 0 C. and were embedded in paraffin. Two canine immunoglobulins were isolated by gel filtration through a column of Sephadex G-200 followed by diethylaminoethyl cellulose chromatography. One fraction showed 18.9 S (yM) and another showed 6.8 S ( yG) by analytic ultracentrifugation. Antisera against canine immunoglobulins were prepared in rabbits. Fluorescent antibody against yM was absorbed with canine yG, and that against yG was absorbed with yM before the immunofluorescence procedure. Tissue sections were stained by direct staining with absorbed fluorescent antibodies. Electron microscopy. Electron microscopic
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Fig. 1. Lung autotransplant (dog No . X·7) , 3 weeks after operation, shows a perivascular collection of mononuclear cells consisting mainly of plasma cells. (Hematoxylin and eosin; original magnification x250.)
examinations were carried out in 14 animals from Groups I and II. In animals with autotransplanted lungs (Group I), the specimens for electron microscopy were cut into 1 by 2 mm. blocks and immersed overnight in a solution containing 3 per cent glutaraldehyde and 0.135M phosphate buffer. After a buffer rinse , the tissue was postfixed in a buffered 1 per cent osmium tetroxide solution for 2 h hours . The specimens were then rinsed , dehydrated in acetone, embedded in Vestopal W, and polymerized overnight at 60° C. Sections were prepared with an LKB Vetrotome III and were stained with uranyl acetate and lead citrate. Sections were examined with the JEM 7A electron microscope at an accelerating voltage of 80 Kv. A 200 flo condenser aperture and a 50 flo objective aperture were utilized. In animals with allotransplanted lungs (Group II) , specimens for electron microscopic examination were fixed in both glutaraldehyde and osmium tetroxide buffered with Millonig 's buffer . After fixation, the specimens were dehydrated in an ethanol series and embedded in epon 812. The blocks were sectioned with glass knives by means of the Porter-Blum MT-l microtome. Thin sections were examined with the
Hitachi HU-ll B electron microscope after being stained with uranyl acetate and lead citrate. Results Group I. The results of histologic examinations of lung autotransplants have been described in detail. 3 The unilaterally auto transplanted lung in the early postoperative period generally showed focal variable degrees of pulmonary congestion and edema . There was no evidence of mononuclear cell infiltration around the pulmonary vessels and bronchioli. Pulmonary histiocytic proliferation was shown in the grafted lung in 5 of 8 animals 3 weeks postoperatively. Pulmonary emboli, comprised of intravascular thrombi and leukocytic agglutinations, were observed in 3 animals. A perivascular collection of mononuclear cells was found in only 1 animal 3 weeks after operation. This lesion was associated with other histologic findings, including lung edema , pulmonary histiocytic proliferation, endothelial cell proliferation, and bronchopneumonia. Mononuclear cells consisted mostly of plasma cells and histiocytes with rare lymphocytes (Fig. 1) . No mononuclear cells classified as intermediate between lymphocytes
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Fig. 2. Lung allotransplant (dog No. 1006), 10 days after transplantation without immunosuppression. Intra- and extravascular mononuclear cells are seen, i.e., a lymphocyte (left), a plasma cell (right), and a macrophage (center) (x4,OOO).
and plasma cells were found in this autotransplanted specimen ultrastructurally. Group II. Pulmonary allografts in untreated dogs generally showed signs of acute rejection within 4 to 6 days postoperatively. The initial histologic characteristic of rejection in these grafts was infiltration of mononuclear cells around the small vessels and bronchi and into the subpleural space. Clusters of these mononuclear inflammatory cells often were noted to form a granuloma-like pattern around the small vessels; the nature of these vessels was not always clear. In the first week of rejection, the mononuclear infiltrate consisted of reticulum-like cells, macrophages, lymphocytes, and plasma cells. Electron microscopic examination during rejection in the untreated dogs showed clusters of mononuclear cells in the lung parenchyma, which included macrophages, lymphocytes, and plasma cells. Occasionally, cells intermediate between the lymphocyte and the plasma cell were observed in the
cell clusters, both inside and outside the capillaries (Fig. 2). The cytoplasm of the lymphocytes seen in the transplants (Fig. 3) had a fine granular structure, with small numbers of mitochondria and slightly dilated endoplasmic reticulum. It appeared almost translucent, with few organelles or inclusions. Fig. 4 shows the typical features of the plasma cells seen during the rejection period. The endoplasmic reticulum is dilated and seems to occupy virtually all of the cytoplasm except the paranuclear region (Fig. 5). Fig. 6 shows an intermediate cell type. The endoplasmic reticulum is not as well developed as the typical plasma cell. Pyronine-positive cells were observed in considerable numbers and yM-containing cells in fewer numbers (Fig. 7). yG-containing cells were not demonstrated in the perivascular infiltrates. Furthermore, none of these immunoglobulin-containing cells were seen within vascular lumina, despite the evidence of endothelial damage at this stage.
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Fig. 3. Ordinary lymphocyte in the perivascular space with a fine granular structure, mitochondria in a small number, and some slightly dilated endoplasmic reticulum. It appears to be translucent, as its organelles or inclusions are few (x28,OOO).
Fig. 4. Typical plasma cell in the perivascular space of a lung allotransplant, 10 days after transplantation. There is considerable development of the endoplasmic reticulum (xIS,OOO) .
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Fig. 5. Typical plasma cell with dilated endoplasmic reticulum (.<15,000).
Fig. 6. Representative cell type intermediate between the lymphocyte and the plasma cell. The endoplasmic reticulum is well developed, but not so well developed as the typical plasma cell (x24,OOO).
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Fig. 7. Lung allotransplant (dog No. 901), 5 days after transplantation, shows yM-positive cells (arrows) in the perivascular reaction (fluorescent antibody technique with anti-yM serum). (Original magnification x400.)
After the first postoperative week, there were increasing numbers of mononuclear cells around bronchioli and the pulmonary vasculature, with narrowing of the vascular lumina. In addition, there was an infiltration or emigration of inflammatory cells into the intra-alveolar spaces, along with exudation of red cells and proliferation and swelling of the endothelial cells (Fig. 8). In some cases, there were manifestations of panvasculitis, intravascular thrombus formation, and infarction necrosis. During this second week, yM-containing cells were often detected around the pulmonary vasculature (Fig. 9). Occasionally, yM-positive cells were seen in the intravascular space.' At times, cells that infiltrated the lung parenchyma also contained cytoplasmic yM. Additionally, there were cell remnants with positive I'M in the lung parenchyma.' In this period, yG was found to be localized especially in the perivascular spaces. Group III. In the immunosuppressed dogs, pulmonary infection was the most common feature to appear in the first 2 weeks after transplantation. In animals that survived their infection, the onset of the rejection reaction, as defined for the Group II animals,
was generally delayed until 3 to 5 weeks after operation. This was consistent with the observation that immunoglobulin-containing cells were rarely seen in the grafts before 3 weeks. After 3 weeks, features of rejection were disclosed by hematoxylin and eosin staining, and "1M-positive cells were often observed in the transplants (Fig. 10). Even with maximum immunosuppressive therapy, treated dogs with transplanted lungs survived for only 5 weeks. In the 18 that survived beyond 5 weeks, the subsequent course was relatively stable; the animals survived to 32 weeks, at which time they were put to death. In 5 dogs that survived 25 to 32 weeks, the transplant showed marked granulomatous formation, which consisted of lymphocytes and macrophages around vessels and bronchioli. Occasionally, the walls of the arterioles were markedly thickened and were stenotic or obstructed. In addition, there was conspicuous migration of histiocytes and epithelial cells into the alveoli (Fig. 11). In more advanced states of rejection, the walls of the small vessels were destroyed, and the alveolar structure lost its normal appearance. There were extensive signs of infarction necrosis and
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Fig. 8. Lung allotransplant (dog No. 840), 9 days after grafting, shows a small pulmonary vessel with endothelial destruction and perivascular mononuclear cell infiltration. (Hematoxylin and eosin; original magnification x400.)
Fig. 9. Lung allotransplant (dog No. 840), 9 days after grafting, shows yM-containing cells (fluorescent antibody technique with anti-yM serum). (Original magnification x400.)
(arrows) in the perivascular space
fibrosis in these lungs. In this chronic period, immunofluorescence studies showed no specific patterns in the lung transplant. Discussion
Perivascular infiltration of mononuclear cells has been accepted generally as one of
the dominant histologic features of the rejection process in allotransplanted organs. This change has been described as an integral aspect of early rejection of the kidney," heart; and liver." In the heart and liver, chronic rejection has been manifested by narrowing of the coronary and hepatic
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Fig. 10. Lung allotransplant (dog No . 926), 40 days after grafting. This dog was treated with antil ymphocyte globulin and azathioprine. yM-positive cells are seen in the per ivascular space as well as in the pulmonary vasculature (fluorescent antibody technique with anti-yM serum ). (Original magnification x400.)
Fig. 11. Lung allotransplant (dog No. 601), 32 weeks after grafting with immunosuppression. There is perivascular fibrosis with the infiltration of small mononuclear cells and a thickened vascular wall . (Hematoxylin and eosin ; original magnification x lOO.)
arteries due to internal proliferation and/or thrombus formation. In regard to pulmonary allografts, a similar histologic picture of early rejection has been verified in studies by Flax and Barnes" and in the present study. In un-
treated pulmonary allografts, the earliest evidence for rejection is the appearance of mononuclear cells around small vessels and bronchioli and also in the subpleural space within 4 to 6 days. Flax and Barnes identified these small vessels as veins. After the
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first postoperative week, increasing numbers of mononuclear cells, which compromise the vascular lumina, are noted around the pulmonary vessels and bronchioli. During this time, these mononuclear cells are often observed remote from the perivascular spaces, in the lung parenchyma. In treated animals, the early stages after transplantation are distinguished by evidence of infection. In the animals that survive, the onset of rejection is retarded until 3 to 6 weeks after the operation, at which time changes similar to those seen during earlier, acute rejection of untreated allografts predominate. In long-term, modified grafts, subendothelial inflammation, hyperplasia, and fibrosis are present. 9 In view of the findings of perivascular mononuclear cell collections in pulmonary autotransplants 3 weeks after surgery, it could be suggested that this perivascular mononuclear cell infiltration is a nonspecific histologic pattern in lung transplants. However, the fact remains that there are striking differences in the composition of cell populations between autotransplants and allotransplants. In allografts, the mononuclear, perivascular collections consist mainly of lymphocytes and plasma cells, whereas lymphocytes rarely appear in the autografts. These findings might suggest that in pulmonary allotransplants the majority of the cells are of recipient origin whereas in autotransplants they are of graft origin. Therefore, from these results and those described for other organs, it would appear that early rejection is generally effected by mononuclear cells, whether the event of rejection is modified by altered histocompatibility between donor and recipient. A number of investigations'? have acknowledged the role of antibody-mediated immunity in allograft rejection. Antibodies have been detected in experimental and clinical transplants. To further define the characteristics of the mononuclear cells in allografts undergoing rejection, fluorescent antibody and electron microscopic techniques have been used to study these cells. Porter" demonstrated that 15 per cent of
the infiltrating mononuclear cells in treated canine hepatic allografts contain yG in their cytoplasma as compared with 5 per cent of those in untreated grafts, thereby suggesting the presence of greater numbers of plasma cells in the infiltrate. Stastny!' also described large, mononuclear, yG-containing cells that infiltrated the myocardium of both late canine and human heart transplants. In the present study, some mononuclear cells containing yM in their cytoplasm, both in the early unmodified and late treated allografts, were seen. yG-containing cells were much less common. Occasionally, these cells were seen in intravascular collections, suggesting a recipient origin. Despite double staining for fluorescent antibodies and with hematoxylin and eosin, morphologic identification of these cells was not successfully carried out in this study; however, some of these cells were considered to be Russell body plasma cells. In a recent study of renal allotransplant histology,t- local liberation of antibodies by cellular disruption of plasma cells was suggested as one aspect of the rejection process. In the present study, electron microscopy in unmodified lung allografts demonstrated macrophages, lymphocytes, plasma cells, and cells intermediate between lymphocytes and plasma cells. The latter three cell types contained the fine structure of antibodyproducing cells." These intermediate cells have been termed graft rejection cells.v Although there has been no evidence to support transformation of lymphocytes into plasma cells in pulmonary allografts, the concept now arises that some of the mononuclear cells, including plasma cells and socalled graft rejection cells, are of recipient origin and may produce antibodies which lead to disruption of the graft. The degree of antibody specificity by these cells is speculative at best. Therefore, it would seem that, as rejection proceeds and enters the chronic phase, antibody exposure is continuous and the described reaction extends from its perivascular location to the vessel wall, associated with internal proliferation, vascular fibrosis, infarction necrosis, and extensive
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pulmonary parenchymal fibrosis. The cellular characteristics of the mononuclear cells in relation to the changes of chronic rejection remain to be defined. Summary Histologic changes occurring in lung autotransplants and allotransplants have been characterized by means of light, fluorescent antibody, and electron microscopy. Vascular lesions due to mononuclear cell infiltrates initially appear around small vessels and bronchioli. These mononuclear cells include macrophages, lymphocytes, and plasma cells. yM-containing cells have been identified in lung transplants by fluorescent microscopy. Findings of electron microscopy are discussed in relation to those by fluorescent antibody microscopy in unmodified lung allografts. Ultrastructurally, macrophages, lymphocytes, plasma cells, and cells intermediate between lymphocyte and plasma cell, i.e., graft rejection cells, were demonstrated among infiltrates of the mononuclear cells. The latter three types of cells contained the ultrastructural features of antibody-producing cells. These antibody-producing cells in the lung allograft that is undergoing rejection are considered to be among the mediators of the rejection phenomenon. REFERENCES Suzuki, C., Nakada, T., Furusawa, T., Watanabe, A., and Hirose, M.: Die Experimentelle Lungenreimplantation, Z. Tuberk. 123: 195, 1965. 2 Suzuki, C, Nakada, T., Watanabe, A., Fujimura, S., and Kawakami, T.: Experimental Studies of Canine Lung Allotransplantation: The Effect of Cytotoxic Agents and Donor Spleen Cells for Immunosuppressive Treatment, 1. THORAC. CARDIOVASC. SURG. 55: 200, 1968.
3 Fujimura, S., Rosen, V., Adomian, G. E., Parmley, W. W., Norman, 1. R., and Matloff, J. M.: Early Morphological Observations in Canine Pulmonary Autotransplants in Dogs: The Similarity to Shock Lung. Submitted for publication. 4 Fujimura, S., Yoneti, M., Kagami, Y., Kawakami, M., Chiba, S., and Okaniwa, G.: Immunohistological Studies on Canine Lung Allotransplantation. I. Preparation of Anti-canine Immunoglobulins (y M and yG), Ishoku 3: 414, 1969 (in Japanese). 5 Fujimura, S., Nakada, T., Kawakami, M., Sukeno, T., Yoneti, M., Okaniwa, G., Kagami, W., and Suzuki, C: Detection of Immunoglobulins (yM and yG) by Fluorescent Antibody Method in Canine Lung Allotransplantation, Tohoku J. Exp. Med. 101: 183, 1970. 6 Porter, K. A., Caine, R. Y., and Zukoski, C. F.: Vascular and Other Changes in 200 Canine Renal Homotransplants Treated With Immunosuppressive Drugs, Lab. Invest. 13: 809, 1964. 7 Lower, R. R., Kosek, J. C, Kemp, V. R., Graham, W. H., Sewell, D. M., and Lim, F.: Rejection of the Cardiac Transplant, Am. J. Cardio!. 24: 492, 1969. 8 Porter, K. A.: Pathology of Liver Transplantation, Transplant. Rev. 2: 129, 1969. 9 Flax, M. H., and Barnes, B. A.: The Role of Vascular Injury in Pulmonary Allograft Rejection, Transplantation 4: 66, 1966. 10 Najarian, 1. A., and Foker, 1. E.: Mechanisms of Kidney Allograft Rejection, Transplant. Proc. 1: 184, 1969. II Stastny, P.: Bound Immunoglobulin and Complement in Heart Allografts Undergoing Rejection, Transplantation 10: 248, 1970. 12 Mitchell, R. E.: The Evolution of Plasma Cells in Canine Renal Allografts, Bib!. Haemato!. 29: 667, 1968. l3 Harris, T. N., Hummeler, K., and Harris, S.: Electron Microscopic Observations on Antibody-Producing Cells of Lymph Node, Lymph and Blood, ill Yoffey, J. M., editor: The Lymphocyte in Immunology and Haemopoiesis, London, 1966, Edward Arnold (Publishers), Ltd. 14 Wiener, J., Spiro, D., and Russell, P. S.: An Electron Microscopic Study of the Homograft Reaction, Am. J. Patho!' 44: 319, 1964.