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Complete replacement of tracheal epithelia by the host promotes spontaneous acceptance of orthotopic tracheal allografts in rats
Complete Replacement of Tracheal Epithelia by the Host Promotes Spontaneous Acceptance of Orthotopic Tracheal Allografts in Rats Y. Ito, H. Suzuki, Y. Hattori, B.A.H. Muhammad, T. Takahashi, K. Suzuki, and T. Kazui ABSTRACT Background. Tracheal immunogenicity has been controversial. Although replacement of allotracheal epithelia by the host epithelia has been reported in rat orthotopic tracheal grafting, the immunological effect of epithelial replacement is still uncertain. Methods. We performed orthotopic tracheal grafting of nine cartilage rings in the following groups: 1, Lewis 3 Lewis (n ⫽ 30); 2, ACI 3 DA (n ⫽ 25); 3, Lewis 3 F344 (n ⫽ 23); 4-A, DA 3 Lewis (n ⫽ 41); 4-B, DA 3 Lewis with tacrolimus therapy (1 mg/kg/d for 10 days) starting from the day of the operation (n ⫽ 31); 4-C, retransplantation of DA allografts to secondary naive Lewis rats 10 or 15 days after primary grafting (n ⫽ 11); 4-D, DA 3 Lewis with tacrolimus therapy starting from postoperative day 10 (n ⫽ 6). Survival times and histopathology were assessed. Epithelial replacement was evaluated by immunohistochemistry. Results. All rats survived in groups 1, 2, and 3. Even in the fully histoincompatible group 4-A, survival ratio on day 120 was 15%. Epithelial replacement was in progress on day 10 in this group. However, all tacrolimus-treated rats died by day 54 and epithelial replacement did not occur on days 30 and 50 in group 4-B. In group 4-C, retransplantation after complete epithelial replacement increased the long-surviving rats. In group 4-D, all rats receiving tacrolimus therapy after complete epithelial replacement survived over 120 days. Conclusions. These results suggest that complete replacement of tracheal epithelia by the host promotes spontaneous acceptance of orthotopic tracheal allografts in rats.
T
RACHEAL ALLOGRAFT has been the subject of continued clinical interest.1– 6 Experimental murine tracheal transplantation models have been widely used for investigating the etiology of allograft stenosis in tracheal transplantation2– 6 and obliterative bronchiolitis (OB).5,7–11 However, the immunological responses after tracheal transplantation are less elucidated than those in other tissues and/or organ allografts and tracheal immunogenicity has been controversial. In 1979, Rose et al reported that tracheal allotransplants elicited only a weak graft rejection response even in the presence of a major histoincompatibility barrier because trachea might have a weak antigenicity.1 On the contrary, Beigel et al showed that systemic immunization had occurred by tracheal transplantation and the trachea was subject to the same immunological responses as other tissues.2– 4 They also demonstrated that tracheal allografts sometimes could be accepted spontaneously and that the donor tracheal epithelia of long-surviving
hosts were replaced by the host epithelia after the events in rat orthotopic transplantation models, but they did not mention morphological changes of the allografts and the process of epithelial replacement.4 Recently, Ikonen et al provided evidence that epithelial regrowth was associated with the inhibition of allograft stenosis.5 However, the process of epithelial replacement and its effect on rejection responses are still uncertain. Moreover, the effect of nonspecific immunosuppressants on epithelial replacement remains obscure. The purposes of this study were (1) to confirm the process of rejection responses and allograft From the First Department of Surgery (Y.I., T.T., K.S., T.K.), and Second Department of Anatomy, (H.S., Y.H.), Hamamatsu University School of Medicine, Hamamatsu, Japan. Address reprint requests to Dr Yasushi Ito, Department of Thoracic Surgery, Iwata City Hospital, 512-3 Ohkubo, Iwata, Shizuoka 438-8550, Japan. E-mail: [email protected]
0041-1345/04/$–see front matter doi:10.1016/j.transproceed.2004.06.028
© 2004 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
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Transplantation Proceedings, 36, 2406 –2412 (2004)
COMPLETE REPLACEMENT OF TRACHEAL EPITHELIA
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Table 1. Number of Rats That Received Orthotopic Tracheal Grafting Postoperative day of sacrifice for histopathological analysis (H&E and/or IHC) Group
For observation of survival times*
5
7
10
15
30
50
60
1 2 3 4-A 4-B 4-C 4-D
8 8 8 20 14 11 6
3 0 0 6 0 0 0
3 3 3 6 3 0 0
3 0 0 3 0 0 0
6 6 6 6 6 0 0
3 5 3 0 6 0 0
0 0 0 0 2 0 0
4 3 3 0 0 0 0
ⱖ 120 b
8 8 b 6 b 3 0 b 6 b 6 b
Total
Technical errors
30 25 23 41 31 11 6
2 3 2 4 1 1 0
Group 1, Lewis-Lewis; 2, ACI-DA; 3, Lewis-F344; 4-A, DA-Lewis without tacrolimus therapy; 4-B, DA-Lewis with tacrolimus therapy (days 0 –9); 4-C, retransplantation group; 4-D, DA-Lewis with tacrolimus therapy (days 10 –19). H&E, hematoxylin and eosin staining; IHC, immunohistochemical study. *In the rats for observation of survival times, some rats survived over 120 days were sacrificed on day 120 or over day 120 for histopathological analysis.
stenosis in orthotopic tracheal transplantation resulting from major histocompatibility complex (MHC) and/or minor histocompatibility antigen (miH) differences, and (2) to clarify the role of epithelial replacement on rejection responses and allograft stenosis.
harvested and divided into two transverse halves at the middle of the allograft, and one half was trimmed to the length of about nine cartilage rings by cutting the excessive recipient trachea. Then it was orthotopically transplanted to the secondary naive Lewis rat. The DA allograft was anastomosed to the caudal trachea of the secondary recipient.
MATERIALS AND METHODS Animals and Experimental Groups
Graft Removal
Adult male DA (RT1avl), ACI (RT1avl), Fischer 344 (RT1lvl), and Lewis (RT1l) rats were used. Orthotopic tracheal grafting was performed on the following groups: syngeneic strain combination (group 1), Lewis 3 Lewis (n ⫽ 30); MHC-compatible but miH-incompatible strain combination (group 2), ACI 3 DA (n ⫽ 25); “weak” MHC- and miH-incompatible strain combination (group 3), Lewis 3 F344 (n ⫽ 23); and fully MHC- and miH-incompatible high-responder combination (group 4), DA 3 Lewis.12 Group 4 was subdivided as follows: DA 3 Lewis without any interventions (group 4-A, n ⫽ 41); DA 3 Lewis with tacrolimus therapy for 10 days starting from the day of the operation to postoperative day 9 (group 4-B, n ⫽ 31); retransplantation of DA allografts to secondary naive Lewis rats 10 or 15 days after primary grafting (group 4-C, n ⫽ 11); and DA 3 Lewis with tacrolimus therapy for 10 days starting from postoperative day 10 to 19 (group 4-D, n ⫽ 6). Tracheal rejection was assessed through survival times and histopathological analysis. All rats dying within the initial 5 days were excluded because of the possibility that the death might have been caused by technical errors.
Procedures of Orthotopic Tracheal Transplantation After a lethal dose of pentobarbital (120 mg/kg IP) was given to a donor rat, the whole trachea was harvested and stored in a saline solution on ice until the grafting which was performed within 2 hours. Recipients were anesthetized with both intraperitoneal pentobarbital (25 mg/kg) and additional ether inhalation. Orthotopic tracheal transplantation was performed while the rat was breathing spontaneously. Our technique was a modification of previously reported methods of Beigel et al4 and Ikonen et al.5 The tracheal segment from the 4th to 12th cartilage rings of the host was resected. The donor graft of nine cartilage rings was anastomosed end-to-end to the host trachea with six interrupted 7-0 polypropylene sutures. In group 4-C, half of the DA-allograft segment that was earlier transplanted to a primary Lewis rat for 10 (n ⫽ 5) or 15 days (n ⫽ 6) was retransplanted to a secondary naive Lewis recipient. The whole trachea including the allograft was
Rats were sacrificed and the whole trachea, including the graft, was removed at various points of time (Table 1). The graft was usually divided into two segments at the midpoint. One segment was fixed in 20% formalin solution for hematoxylin-eosin (H&E) staining. The other segment was placed in the OCT (Tissue-Tek), frozen in liquid nitrogen, and stored at ⫺80°C for cryostat sectioning.
Histopathology and Computerized Morphometry The specimens fixed in a formalin solution were embedded in paraffin and 4-m cross and/or sagittal sections were stained with H&E. Images of tracheal sections were taken with an HC-2000 high-resolution color camera mounted on a Vanox-58 microscope and analyzed with an NIH image analysis system. According to the method described by Reichenspurner et al,10 we defined the luminal obliteration ratio. Tracheal allograft injury was evaluated by this ratio and the pathological scoring criteria previously reported by Kelly et al.11
Immunohistochemistry (Alkaline Phosphatase Method) The primary monoclonal antibodies used in this study were MN4-91-6 (RT1Aa), OX18 (RT1A), OX6 (Ia), W3/25 (CD4), OX8 (CD8), OX39 (CD25), OX22 (CD45RC), OX62, and goat alkaline phosphatase (AP) conjugated anti-mouse IgG was used as the secondary antibody (Serotec, Kidlington, UK). Cryostat sections were cut in thickness of 5 m, air-dried at room temperature for 30 minutes, and fixed in an absolute acetone on ice for 10 minutes. Incubation with primary antibodies at room temperature for 30 minutes was followed by incubation with a secondary antibody (diluted 1:100 in 1% rat serum) at room temperature for 20 minutes. AP activity was revealed by incubation with NaphtolAS-MX phosphate and Fast Red TR salt (Sigma).
Statistical Analysis Survival curves were estimated by Kaplan-Meyer method. The data obtained by morphometric analysis were analyzed by calculating mean values and the standard errors of the mean. When the data
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ITO, SUZUKI, HATTORI ET AL
Fig 1.
Survival curves of the rats receiving orthotopic tracheal grafting.
were compared between two groups, an unpaired t test or the Mann-Whitney U test was used. A probability value of less than .05 was considered to be significant.
RESULTS Effect of MHC and miH Differences on Allograft Survival
We first observed the survival days in groups without any treatment to investigate the effect of histocompatibility differences on tracheal allograft survival (Fig 1a). All rats survived indefinitely in miH-incompatible combination of group 2 and “weak” MHC- and miH-incompatible combinations of group 3. It has been reported that ACI skin grafts had been always rejected rapidly on naive DA rats and naive F344 rats always rejected Lewis skin grafts rapidly,13 but in the case of orthotopic tracheal grafting, DA rats accepted ACI tracheas and F344 rats accepted Lewis tracheas spontaneously. The survival ratio even in the high-responder strain combination of group 4-A was 15% on day 120. Effect of Tacrolimus Therapy Starting From the Day of the Operation on Allograft Survival
For the DA-Lewis combination, we administered tacrolimus to investigate the effect of nonspecific immunosuppres-
sants on allograft survival. Tacrolimus at a dose of 1 mg/kg/d for 10 days is often used to induce allograft acceptance in several organ transplantations (ie, heart, spleen).13,14 So we applied this regimen in expectation that the transplants would be accepted indefinitely because of the Lewis rats described above accepted DA allografts spontaneously (15%, group 4-A). Unexpectedly, all rats treated with tacrolimus for the initial 10 days died by day 54 in group 4-B (Fig 1b).
Effect of Retransplantation After Complete Epithelial Replacement on Allograft Survival
The discrepancy mentioned above led to the idea to retransplant DA allograft to the secondary Lewis recipients 10 or 15 days after primary grafting to investigate the effect of retransplantation after complete epithelial replacement on allograft survival. Epithelial replacement was expected to be in progress or finished around postoperative day 10 by the immunohistochemical study in group 4-A. Actually, the survival ratio on day 120 of the rats in group 4-C was higher (45.5%) than that of the nontreated rats in group 4-A (Fig 1c).
COMPLETE REPLACEMENT OF TRACHEAL EPITHELIA
Fig 2.
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Mean luminal obliteration ratios of the tracheal allografts.
Effect of Tacrolimus Therapy After Complete Epithelial Replacement on Allograft Survival
Moreover, it occurred to us that tacrolimus therapy from postoperative days 10 to 19 after complete epithelial replacement should be more effective. All rats treated with tacrolimus from postoperative days 10 to 19 survived over 120 days in group 4-D (Fig 1d). This was in sharp contrast to the survival outcome of the rats in group 4-B. Histopathology and Computerized Morphometry
Mean luminal obliteration ratios are shown in Fig 2. On day 15, according to the extent of histocompatibility antigen differences, this ratio became gradually higher. On day 120, this ratio in group 4-A was significantly higher than those in the other combinations. In group 4-B, mean luminal obliteration ratio on day 15 was almost equivalent (22.3%) to that in group 1 (19.8%). But this ratio on day 30 was 32.2% and tended to higher than that on day 15. In group 4-D, mean luminal obliteration ratio on day 120 was almost equivalent (22.8%) to that in group 1 (19.9%). The average pathological scores tended to be higher according to the extent of histocompatibility antigen differences. Epithelia of allografts in all allogeneic combinations without tacrolimus therapy changed after day 15. In group 2, single-layer
ciliated cuboidal epithelia were mainly observed after day 15. In group 3, squamous metaplasia was found on day 15, but an intact epithelium was occasionally found on day 120 (Fig 3a). In group 4-A, single-layer cuboidal ciliated epithelia were found in all rats surviving until day 120 (Fig 3b). In this group, squamous metaplasia was found in the rats that died during the survival period. Persson reported that a flat, undifferentiated epithelium covered the basement membrane after epithelial denudation in vivo shedding-restitution model using guinea-pig trachea.15 Epithelial changes in our study suggested that denudation of epithelia and sequential restitution had occurred around day 15 in histoincompatible combinations without tacrolimus therapy. In group 4-B, columnar ciliated epithelia were maintained until day 30, but infiltration of mononuclear cells was observed on day 30. Severe epithelial changes were found on day 50 (Fig 3c). In group 4-C, single-layer cuboidal or columnar ciliated epithelia were found on day 120. In group 4-D, cuboidal or columnar-ciliated epithelia were found on day 120 (Fig 3d). Immunohistochemistry
Origin of graft epithelium. In group 4-A, the immunohistochemical studies revealed that graft epithelia derived
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ITO, SUZUKI, HATTORI ET AL
Fig 3.
Morphological changes of tracheal allograft.
from donor rats on day 7. But interestingly, epithelial replacement seemed to be in progress on day 10 (Fig 4a, b, c), was completed on day 15, and graft epithelia derived from recipient rats on day 120. In group 4-B, epithelia of allografts derived from donor rats even on days 30 and 50 (Fig 4d). In group 4-C, the epithelia of the DA allografts were of recipient origin on the day of the retransplantation, and the epithelia of the retransplanted grafts were also of recipient origin 120 days after the retransplantation (Fig 4e). In group 4-D, epithelia of allografts derived from recipient rats on day 120. Distribution of MHC antigens and graft infiltrating cells. In group 4-A, MHC antigens were more strongly expressed in the epithelia of allografts in comparison with those in group 1. DA-specific RT1Aa antigens were expressed in the endothelia, epithelia, and cartilage until day 7, but after day 10, they were expressed only in cartilage. The numbers of the T-cell surface marker-positive cells (CD4, CD8, CD45RC, CD25) were at their maximum on day 5. But OX62⫹ cells were absent in the grafts on day 5, and a few OX62⫹ cells existed in the grafts on other days. In group 4-B, CD25⫹ T cells were almost totally absent but other surface marker-positive cells were observed in the grafts on day 30.
DISCUSSION
Although allograft stenosis is the major obstacle in tracheal transplantation, it is uncertain to what extent histocompatibility antigen differences influence this stenosis. In our study, tracheal allografts in not only miH-incompatible but “weak” MHC- and miH-incompatible strain combination were spontaneously accepted indefinitely. Furthermore, 15% of spontaneous tracheal allograft acceptance in “strong” MHC- and miH-incompatible strain combination is associated with epithelial replacement derived from recipients. Ikonen et al postulated that the putative time of complete epithelial replacement was between 10 and 20 days after transplantation.5 Our results show that epithelial replacement actually occurred and it had just been completed around day 10 in group 4-A. The degree of histopathological injury became higher according to the extent of histocompatibility antigen differences despite the fact that all rats were dealt with in the same way. These outcomes suggest that the main cause of tracheal allograft stenosis is a result of rejection responses and that other possible causes (eg, failure of revascularization, infection) may play a moderate role. If tracheal rejection responses were essentially weak, it would be easier to induce indefinite allograft survival under
COMPLETE REPLACEMENT OF TRACHEAL EPITHELIA
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Fig 4. Findings concerning epithelial replacement. (a– c) Group 4-A, DA-Lewis without any intervention. (d) Group 4-B, DA-Lewis ⫹ tacrolimus. (e) Group 4-C, retransplantation of DA trachea to secondary Lewis 10 days after primary grafting.
limited nonspecific immunosupressants. Tacrolimus has been used successfully in various organ transplantations, and it reportedly has an advantage over cyclosporine in reducing the risk of OB.16 However, all rats treated with tacrolimus for the initial 10 days died because of severe allograft stenosis. Allograft epithelia originated from donor rats even on day 50 in this group; epithelial replacement did not occur. According to previous data at our institution, DA heart grafts (n ⫽ 4) were accepted over 100 days but DA skin grafts (n ⫽ 6) were rejected within 60 days in Lewis recipients treated with tacrolimus for the initial 10 days. In comparison with these results, tracheal epithelia seem to have as strong immunogenicity as skin. As a result, it was suggested that tacrolimus therapy for the initial 10 days seemed to inhibit epithelial replacement and spontaneous acceptance of tracheal allografts. Thus, we believe that tracheal immunogenicity was not as weak as previously descrived.1 We hypothesized that epithelial replacement was one active immunological defensive, or tolerogenic mechanism of the host that enabled allograft acceptance. To investigate whether epithelial replacement is really requisite for spontaneous tracheal allo-
graft acceptance or not, we added another experiment. Retransplantation of allografts after complete epithelial replacement increased the number of long-surviving rats. This fact indicates the importance of epithelial replacement for spontaneous acceptance of allograft. In comparison with mean luminal obliteration ratio of retransplanted graft on day 10 and day 15, allograft stenosis seemed to still progress from days 10 to 15. We considered that complete epithelial replacement and prevention of rejection responses after epithelial replacement were necessary for indefinite allograft acceptance. In group 4-D, tacrolimus was administered from postoperative day 10 to 19 to keep continuing epithelial replacement and to inhibit further stenosis. The result was just as we had expected. All rats survived indefinitely without severe stenosis. This fact strongly supports that complete epithelial replacement by the host promotes acceptance of orthotopic tracheal allografts in rats. Some investigators reported the successful effects of nonspecific immunosuppressants that maintained epithelia and prevented allograft stenosis using the heterotopic tracheal transplantation models, but they did not refer to the
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origin of graft epithelia.6 –10 Our results showed that, at the early stage, tacrolimus therapy for the initial 10 days could maintain the epithelia. However, severe rejection responses occurred later and tracheal epithelia could not be replaced by the host until at least on day 50. It is important to take it into consideration that nonspecific immunosuppressants, such as tacrolimus, influence the regulation of epithelial replacement, resulting in a potential risk factor for allograft survival due to maintenance of donor epithelia with a strong immunogenicity. We suspected that there was some kind of immmunological player promoting epithelial replacement and/or spontaneous acceptance of allografts. So we investigated whether there was any special player regulating the immunological responses in the graft infiltrating cells or not. In group 4-A, the maximum numbers of T cells were found on day 5 and CD4⫹ T cells were recognized to be dominant. CD25⫹ T cells were also found. In group 4-B, mononuclear cell infiltration appeared on day 30, and the decrease in only CD25⫹ T cells was different from the results in group 4-A. Recently, immunoregulatory T cells have attracted attention as the key players in the toleranogenic pathway.17 So we separated the graft infiltrating cells using a modification of the method of Sertl et al18 and investigated them by flowcytometry (data are not shown). In group 4-A, coexpression of CD4/CD8 and CD4/CD25 molecules were observed on graft infiltrating lymphocytes on day 5. Fujihashi et al19 showed that CD4⫹/CD8⫹ T cells existed and exhibited cytokine synthesis with helper function in the epithelium of the gastrointestinal tract. On the other hand, a potent CD4⫹ immunoregulatory T-cell population can be defined by expression of the IL-2 receptor ␣-chain (CD25).17 Hence, it is supposed that regulatory CD4⫹ T cells play some role in the spontaneous acceptance of tracheal allografts. Further investigation will be necessary to clarify the role of dendritic cells and T-cell populations or various cytokines to understand the immunological mechanisms involved in tracheal transplantation. CONCLUSIONS
Tracheal allografts can be accepted across the histocompatibility differences. However, tracheal epithelia seem to have a strong immunogenicity because MHC antigens were strongly expressed on the epithelia, and the tacrolimus therapy for the initial 10 days could not induce acceptance
ITO, SUZUKI, HATTORI ET AL
of tracheal allografts. Epithelial replacement occurs around 10 days after grafting in the fully histoincompatible combination. The timing for starting nonspecific immunosuppressants without interfering with epithelial replacement is pivotal to induce indefinite acceptance of tracheal allografts. Complete epithelial replacement by the host promotes spontaneous acceptance of orthotopic tracheal allografts in rats. ACKNOWLEDGMENTS The authors gratefully acknowledge Dr T. Hoshino, Dr K. Ohashi, Dr A. Yamashita, and Dr T. Uezato for their critical comments and useful suggestions.
REFERENCES 1. Rose KG, Sesterhenn K, Wustrow F: Lancet 1:433, 1979 2. Beigel A, Mu ¨ller-Ruchholtz W: Arch Otorhinolaryngol 240: 185, 1984 3. Beigel A, Mu ¨ller-Ruchholtz W: Arch Otorhinolaryngol 240: 217, 1984 4. Beigel A, Steffens-Knutzen R, Mu ¨ller B, et al: Arch Otorhinolaryngol 241:1, 1984 5. Ikonen TS, Brazelton TR, Berry GJ, et al: Transplantation 70:857, 2000 6. Hashimoto M, Nakanishi R, Muranaka H, et al: J Cardiovasc Surg (Torino) 41:487, 2000 7. Hertz MI, Jessurun J, King MB, et al: Am J Pathol 142:1945, 1993 8. Adams BF, Berry GJ, Huang X, et al: Transplantation 69:2260, 2000 9. Morikawa M, Brazelton TR, Berry GJ, et al: Transplantation 71:1616, 2001 10. Reichenspurner H, Soni V, Nitscheke M, et al: Transplantation 64:373, 1997 11. Kelly KE, Hertz MJ, Mueller DL: Transplantation 66:764, 1998 12. Gill TJ 3rd, Kunz HW, Misra DN, et al: Transplantation 43:773, 1987 13. Suzuki H, Li XH, Miyamoto M, et al: Transplantation 64:650, 1997 14. Ochiai T, Nakajima K, Nagata M, et al: Transplantation 44:734, 1987 15. Persson CGA, Bando K, Hardesty RL, et al: Am J Respir Crit Care Med 153:S9, 1996 16. Griffith BP, Bando K, Hardesty RL, et al: Transplantation 57:848, 1994 17. Sakaguchi S: Cell 101:455, 2000 18. Sertl K, Takemura T, Tschachler E, et al: J Exp Med 163:436, 1986 19. Fujihashi K, Yamamoto M, McGhee JR, et al: Int Immunol 5:1473, 1993
T
RACHEAL ALLOGRAFT has been the subject of continued clinical interest.1– 6 Experimental murine tracheal transplantation models have been widely used for investigating the etiology of allograft stenosis in tracheal transplantation2– 6 and obliterative bronchiolitis (OB).5,7–11 However, the immunological responses after tracheal transplantation are less elucidated than those in other tissues and/or organ allografts and tracheal immunogenicity has been controversial. In 1979, Rose et al reported that tracheal allotransplants elicited only a weak graft rejection response even in the presence of a major histoincompatibility barrier because trachea might have a weak antigenicity.1 On the contrary, Beigel et al showed that systemic immunization had occurred by tracheal transplantation and the trachea was subject to the same immunological responses as other tissues.2– 4 They also demonstrated that tracheal allografts sometimes could be accepted spontaneously and that the donor tracheal epithelia of long-surviving
hosts were replaced by the host epithelia after the events in rat orthotopic transplantation models, but they did not mention morphological changes of the allografts and the process of epithelial replacement.4 Recently, Ikonen et al provided evidence that epithelial regrowth was associated with the inhibition of allograft stenosis.5 However, the process of epithelial replacement and its effect on rejection responses are still uncertain. Moreover, the effect of nonspecific immunosuppressants on epithelial replacement remains obscure. The purposes of this study were (1) to confirm the process of rejection responses and allograft From the First Department of Surgery (Y.I., T.T., K.S., T.K.), and Second Department of Anatomy, (H.S., Y.H.), Hamamatsu University School of Medicine, Hamamatsu, Japan. Address reprint requests to Dr Yasushi Ito, Department of Thoracic Surgery, Iwata City Hospital, 512-3 Ohkubo, Iwata, Shizuoka 438-8550, Japan. E-mail: [email protected]
0041-1345/04/$–see front matter doi:10.1016/j.transproceed.2004.06.028
© 2004 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
2406
Transplantation Proceedings, 36, 2406 –2412 (2004)
COMPLETE REPLACEMENT OF TRACHEAL EPITHELIA
2407
Table 1. Number of Rats That Received Orthotopic Tracheal Grafting Postoperative day of sacrifice for histopathological analysis (H&E and/or IHC) Group
For observation of survival times*
5
7
10
15
30
50
60
1 2 3 4-A 4-B 4-C 4-D
8 8 8 20 14 11 6
3 0 0 6 0 0 0
3 3 3 6 3 0 0
3 0 0 3 0 0 0
6 6 6 6 6 0 0
3 5 3 0 6 0 0
0 0 0 0 2 0 0
4 3 3 0 0 0 0
ⱖ 120 b
8 8 b 6 b 3 0 b 6 b 6 b
Total
Technical errors
30 25 23 41 31 11 6
2 3 2 4 1 1 0
Group 1, Lewis-Lewis; 2, ACI-DA; 3, Lewis-F344; 4-A, DA-Lewis without tacrolimus therapy; 4-B, DA-Lewis with tacrolimus therapy (days 0 –9); 4-C, retransplantation group; 4-D, DA-Lewis with tacrolimus therapy (days 10 –19). H&E, hematoxylin and eosin staining; IHC, immunohistochemical study. *In the rats for observation of survival times, some rats survived over 120 days were sacrificed on day 120 or over day 120 for histopathological analysis.
stenosis in orthotopic tracheal transplantation resulting from major histocompatibility complex (MHC) and/or minor histocompatibility antigen (miH) differences, and (2) to clarify the role of epithelial replacement on rejection responses and allograft stenosis.
harvested and divided into two transverse halves at the middle of the allograft, and one half was trimmed to the length of about nine cartilage rings by cutting the excessive recipient trachea. Then it was orthotopically transplanted to the secondary naive Lewis rat. The DA allograft was anastomosed to the caudal trachea of the secondary recipient.
MATERIALS AND METHODS Animals and Experimental Groups
Graft Removal
Adult male DA (RT1avl), ACI (RT1avl), Fischer 344 (RT1lvl), and Lewis (RT1l) rats were used. Orthotopic tracheal grafting was performed on the following groups: syngeneic strain combination (group 1), Lewis 3 Lewis (n ⫽ 30); MHC-compatible but miH-incompatible strain combination (group 2), ACI 3 DA (n ⫽ 25); “weak” MHC- and miH-incompatible strain combination (group 3), Lewis 3 F344 (n ⫽ 23); and fully MHC- and miH-incompatible high-responder combination (group 4), DA 3 Lewis.12 Group 4 was subdivided as follows: DA 3 Lewis without any interventions (group 4-A, n ⫽ 41); DA 3 Lewis with tacrolimus therapy for 10 days starting from the day of the operation to postoperative day 9 (group 4-B, n ⫽ 31); retransplantation of DA allografts to secondary naive Lewis rats 10 or 15 days after primary grafting (group 4-C, n ⫽ 11); and DA 3 Lewis with tacrolimus therapy for 10 days starting from postoperative day 10 to 19 (group 4-D, n ⫽ 6). Tracheal rejection was assessed through survival times and histopathological analysis. All rats dying within the initial 5 days were excluded because of the possibility that the death might have been caused by technical errors.
Procedures of Orthotopic Tracheal Transplantation After a lethal dose of pentobarbital (120 mg/kg IP) was given to a donor rat, the whole trachea was harvested and stored in a saline solution on ice until the grafting which was performed within 2 hours. Recipients were anesthetized with both intraperitoneal pentobarbital (25 mg/kg) and additional ether inhalation. Orthotopic tracheal transplantation was performed while the rat was breathing spontaneously. Our technique was a modification of previously reported methods of Beigel et al4 and Ikonen et al.5 The tracheal segment from the 4th to 12th cartilage rings of the host was resected. The donor graft of nine cartilage rings was anastomosed end-to-end to the host trachea with six interrupted 7-0 polypropylene sutures. In group 4-C, half of the DA-allograft segment that was earlier transplanted to a primary Lewis rat for 10 (n ⫽ 5) or 15 days (n ⫽ 6) was retransplanted to a secondary naive Lewis recipient. The whole trachea including the allograft was
Rats were sacrificed and the whole trachea, including the graft, was removed at various points of time (Table 1). The graft was usually divided into two segments at the midpoint. One segment was fixed in 20% formalin solution for hematoxylin-eosin (H&E) staining. The other segment was placed in the OCT (Tissue-Tek), frozen in liquid nitrogen, and stored at ⫺80°C for cryostat sectioning.
Histopathology and Computerized Morphometry The specimens fixed in a formalin solution were embedded in paraffin and 4-m cross and/or sagittal sections were stained with H&E. Images of tracheal sections were taken with an HC-2000 high-resolution color camera mounted on a Vanox-58 microscope and analyzed with an NIH image analysis system. According to the method described by Reichenspurner et al,10 we defined the luminal obliteration ratio. Tracheal allograft injury was evaluated by this ratio and the pathological scoring criteria previously reported by Kelly et al.11
Immunohistochemistry (Alkaline Phosphatase Method) The primary monoclonal antibodies used in this study were MN4-91-6 (RT1Aa), OX18 (RT1A), OX6 (Ia), W3/25 (CD4), OX8 (CD8), OX39 (CD25), OX22 (CD45RC), OX62, and goat alkaline phosphatase (AP) conjugated anti-mouse IgG was used as the secondary antibody (Serotec, Kidlington, UK). Cryostat sections were cut in thickness of 5 m, air-dried at room temperature for 30 minutes, and fixed in an absolute acetone on ice for 10 minutes. Incubation with primary antibodies at room temperature for 30 minutes was followed by incubation with a secondary antibody (diluted 1:100 in 1% rat serum) at room temperature for 20 minutes. AP activity was revealed by incubation with NaphtolAS-MX phosphate and Fast Red TR salt (Sigma).
Statistical Analysis Survival curves were estimated by Kaplan-Meyer method. The data obtained by morphometric analysis were analyzed by calculating mean values and the standard errors of the mean. When the data
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ITO, SUZUKI, HATTORI ET AL
Fig 1.
Survival curves of the rats receiving orthotopic tracheal grafting.
were compared between two groups, an unpaired t test or the Mann-Whitney U test was used. A probability value of less than .05 was considered to be significant.
RESULTS Effect of MHC and miH Differences on Allograft Survival
We first observed the survival days in groups without any treatment to investigate the effect of histocompatibility differences on tracheal allograft survival (Fig 1a). All rats survived indefinitely in miH-incompatible combination of group 2 and “weak” MHC- and miH-incompatible combinations of group 3. It has been reported that ACI skin grafts had been always rejected rapidly on naive DA rats and naive F344 rats always rejected Lewis skin grafts rapidly,13 but in the case of orthotopic tracheal grafting, DA rats accepted ACI tracheas and F344 rats accepted Lewis tracheas spontaneously. The survival ratio even in the high-responder strain combination of group 4-A was 15% on day 120. Effect of Tacrolimus Therapy Starting From the Day of the Operation on Allograft Survival
For the DA-Lewis combination, we administered tacrolimus to investigate the effect of nonspecific immunosuppres-
sants on allograft survival. Tacrolimus at a dose of 1 mg/kg/d for 10 days is often used to induce allograft acceptance in several organ transplantations (ie, heart, spleen).13,14 So we applied this regimen in expectation that the transplants would be accepted indefinitely because of the Lewis rats described above accepted DA allografts spontaneously (15%, group 4-A). Unexpectedly, all rats treated with tacrolimus for the initial 10 days died by day 54 in group 4-B (Fig 1b).
Effect of Retransplantation After Complete Epithelial Replacement on Allograft Survival
The discrepancy mentioned above led to the idea to retransplant DA allograft to the secondary Lewis recipients 10 or 15 days after primary grafting to investigate the effect of retransplantation after complete epithelial replacement on allograft survival. Epithelial replacement was expected to be in progress or finished around postoperative day 10 by the immunohistochemical study in group 4-A. Actually, the survival ratio on day 120 of the rats in group 4-C was higher (45.5%) than that of the nontreated rats in group 4-A (Fig 1c).
COMPLETE REPLACEMENT OF TRACHEAL EPITHELIA
Fig 2.
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Mean luminal obliteration ratios of the tracheal allografts.
Effect of Tacrolimus Therapy After Complete Epithelial Replacement on Allograft Survival
Moreover, it occurred to us that tacrolimus therapy from postoperative days 10 to 19 after complete epithelial replacement should be more effective. All rats treated with tacrolimus from postoperative days 10 to 19 survived over 120 days in group 4-D (Fig 1d). This was in sharp contrast to the survival outcome of the rats in group 4-B. Histopathology and Computerized Morphometry
Mean luminal obliteration ratios are shown in Fig 2. On day 15, according to the extent of histocompatibility antigen differences, this ratio became gradually higher. On day 120, this ratio in group 4-A was significantly higher than those in the other combinations. In group 4-B, mean luminal obliteration ratio on day 15 was almost equivalent (22.3%) to that in group 1 (19.8%). But this ratio on day 30 was 32.2% and tended to higher than that on day 15. In group 4-D, mean luminal obliteration ratio on day 120 was almost equivalent (22.8%) to that in group 1 (19.9%). The average pathological scores tended to be higher according to the extent of histocompatibility antigen differences. Epithelia of allografts in all allogeneic combinations without tacrolimus therapy changed after day 15. In group 2, single-layer
ciliated cuboidal epithelia were mainly observed after day 15. In group 3, squamous metaplasia was found on day 15, but an intact epithelium was occasionally found on day 120 (Fig 3a). In group 4-A, single-layer cuboidal ciliated epithelia were found in all rats surviving until day 120 (Fig 3b). In this group, squamous metaplasia was found in the rats that died during the survival period. Persson reported that a flat, undifferentiated epithelium covered the basement membrane after epithelial denudation in vivo shedding-restitution model using guinea-pig trachea.15 Epithelial changes in our study suggested that denudation of epithelia and sequential restitution had occurred around day 15 in histoincompatible combinations without tacrolimus therapy. In group 4-B, columnar ciliated epithelia were maintained until day 30, but infiltration of mononuclear cells was observed on day 30. Severe epithelial changes were found on day 50 (Fig 3c). In group 4-C, single-layer cuboidal or columnar ciliated epithelia were found on day 120. In group 4-D, cuboidal or columnar-ciliated epithelia were found on day 120 (Fig 3d). Immunohistochemistry
Origin of graft epithelium. In group 4-A, the immunohistochemical studies revealed that graft epithelia derived
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Fig 3.
Morphological changes of tracheal allograft.
from donor rats on day 7. But interestingly, epithelial replacement seemed to be in progress on day 10 (Fig 4a, b, c), was completed on day 15, and graft epithelia derived from recipient rats on day 120. In group 4-B, epithelia of allografts derived from donor rats even on days 30 and 50 (Fig 4d). In group 4-C, the epithelia of the DA allografts were of recipient origin on the day of the retransplantation, and the epithelia of the retransplanted grafts were also of recipient origin 120 days after the retransplantation (Fig 4e). In group 4-D, epithelia of allografts derived from recipient rats on day 120. Distribution of MHC antigens and graft infiltrating cells. In group 4-A, MHC antigens were more strongly expressed in the epithelia of allografts in comparison with those in group 1. DA-specific RT1Aa antigens were expressed in the endothelia, epithelia, and cartilage until day 7, but after day 10, they were expressed only in cartilage. The numbers of the T-cell surface marker-positive cells (CD4, CD8, CD45RC, CD25) were at their maximum on day 5. But OX62⫹ cells were absent in the grafts on day 5, and a few OX62⫹ cells existed in the grafts on other days. In group 4-B, CD25⫹ T cells were almost totally absent but other surface marker-positive cells were observed in the grafts on day 30.
DISCUSSION
Although allograft stenosis is the major obstacle in tracheal transplantation, it is uncertain to what extent histocompatibility antigen differences influence this stenosis. In our study, tracheal allografts in not only miH-incompatible but “weak” MHC- and miH-incompatible strain combination were spontaneously accepted indefinitely. Furthermore, 15% of spontaneous tracheal allograft acceptance in “strong” MHC- and miH-incompatible strain combination is associated with epithelial replacement derived from recipients. Ikonen et al postulated that the putative time of complete epithelial replacement was between 10 and 20 days after transplantation.5 Our results show that epithelial replacement actually occurred and it had just been completed around day 10 in group 4-A. The degree of histopathological injury became higher according to the extent of histocompatibility antigen differences despite the fact that all rats were dealt with in the same way. These outcomes suggest that the main cause of tracheal allograft stenosis is a result of rejection responses and that other possible causes (eg, failure of revascularization, infection) may play a moderate role. If tracheal rejection responses were essentially weak, it would be easier to induce indefinite allograft survival under
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Fig 4. Findings concerning epithelial replacement. (a– c) Group 4-A, DA-Lewis without any intervention. (d) Group 4-B, DA-Lewis ⫹ tacrolimus. (e) Group 4-C, retransplantation of DA trachea to secondary Lewis 10 days after primary grafting.
limited nonspecific immunosupressants. Tacrolimus has been used successfully in various organ transplantations, and it reportedly has an advantage over cyclosporine in reducing the risk of OB.16 However, all rats treated with tacrolimus for the initial 10 days died because of severe allograft stenosis. Allograft epithelia originated from donor rats even on day 50 in this group; epithelial replacement did not occur. According to previous data at our institution, DA heart grafts (n ⫽ 4) were accepted over 100 days but DA skin grafts (n ⫽ 6) were rejected within 60 days in Lewis recipients treated with tacrolimus for the initial 10 days. In comparison with these results, tracheal epithelia seem to have as strong immunogenicity as skin. As a result, it was suggested that tacrolimus therapy for the initial 10 days seemed to inhibit epithelial replacement and spontaneous acceptance of tracheal allografts. Thus, we believe that tracheal immunogenicity was not as weak as previously descrived.1 We hypothesized that epithelial replacement was one active immunological defensive, or tolerogenic mechanism of the host that enabled allograft acceptance. To investigate whether epithelial replacement is really requisite for spontaneous tracheal allo-
graft acceptance or not, we added another experiment. Retransplantation of allografts after complete epithelial replacement increased the number of long-surviving rats. This fact indicates the importance of epithelial replacement for spontaneous acceptance of allograft. In comparison with mean luminal obliteration ratio of retransplanted graft on day 10 and day 15, allograft stenosis seemed to still progress from days 10 to 15. We considered that complete epithelial replacement and prevention of rejection responses after epithelial replacement were necessary for indefinite allograft acceptance. In group 4-D, tacrolimus was administered from postoperative day 10 to 19 to keep continuing epithelial replacement and to inhibit further stenosis. The result was just as we had expected. All rats survived indefinitely without severe stenosis. This fact strongly supports that complete epithelial replacement by the host promotes acceptance of orthotopic tracheal allografts in rats. Some investigators reported the successful effects of nonspecific immunosuppressants that maintained epithelia and prevented allograft stenosis using the heterotopic tracheal transplantation models, but they did not refer to the
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origin of graft epithelia.6 –10 Our results showed that, at the early stage, tacrolimus therapy for the initial 10 days could maintain the epithelia. However, severe rejection responses occurred later and tracheal epithelia could not be replaced by the host until at least on day 50. It is important to take it into consideration that nonspecific immunosuppressants, such as tacrolimus, influence the regulation of epithelial replacement, resulting in a potential risk factor for allograft survival due to maintenance of donor epithelia with a strong immunogenicity. We suspected that there was some kind of immmunological player promoting epithelial replacement and/or spontaneous acceptance of allografts. So we investigated whether there was any special player regulating the immunological responses in the graft infiltrating cells or not. In group 4-A, the maximum numbers of T cells were found on day 5 and CD4⫹ T cells were recognized to be dominant. CD25⫹ T cells were also found. In group 4-B, mononuclear cell infiltration appeared on day 30, and the decrease in only CD25⫹ T cells was different from the results in group 4-A. Recently, immunoregulatory T cells have attracted attention as the key players in the toleranogenic pathway.17 So we separated the graft infiltrating cells using a modification of the method of Sertl et al18 and investigated them by flowcytometry (data are not shown). In group 4-A, coexpression of CD4/CD8 and CD4/CD25 molecules were observed on graft infiltrating lymphocytes on day 5. Fujihashi et al19 showed that CD4⫹/CD8⫹ T cells existed and exhibited cytokine synthesis with helper function in the epithelium of the gastrointestinal tract. On the other hand, a potent CD4⫹ immunoregulatory T-cell population can be defined by expression of the IL-2 receptor ␣-chain (CD25).17 Hence, it is supposed that regulatory CD4⫹ T cells play some role in the spontaneous acceptance of tracheal allografts. Further investigation will be necessary to clarify the role of dendritic cells and T-cell populations or various cytokines to understand the immunological mechanisms involved in tracheal transplantation. CONCLUSIONS
Tracheal allografts can be accepted across the histocompatibility differences. However, tracheal epithelia seem to have a strong immunogenicity because MHC antigens were strongly expressed on the epithelia, and the tacrolimus therapy for the initial 10 days could not induce acceptance
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of tracheal allografts. Epithelial replacement occurs around 10 days after grafting in the fully histoincompatible combination. The timing for starting nonspecific immunosuppressants without interfering with epithelial replacement is pivotal to induce indefinite acceptance of tracheal allografts. Complete epithelial replacement by the host promotes spontaneous acceptance of orthotopic tracheal allografts in rats. ACKNOWLEDGMENTS The authors gratefully acknowledge Dr T. Hoshino, Dr K. Ohashi, Dr A. Yamashita, and Dr T. Uezato for their critical comments and useful suggestions.
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