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Airway epithelium in obliterative airway diseaseQu, Ning

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AIRWAY EPITHELIUM IN OBLITERATIVE AIRWAY DISEASE

Ning Qu

Ning Qu AIRWAY EPITHELIUM IN OBLITERATIVE AIRWAY DISEASE

© Copyright 2005 Ning Qu, ISBN 90-36722-888

All rights are reserved. No part of this publication may be reproduced, stored in a retrieval

system, or transmitted in any form or by any means, mechanically, by photocopying,

recording, or ortherwise, without the written permission of the author.

Cover design by Ning Qu, Layout by Yijin Ren

Printed by

RIJKSUNIVERSITEIT GRONINGEN

AIRWAY EPITHELIUM IN OBLITERATIVE AIRWAY DISEASE

Proefschrift

ter verkrijging van het doctoraat in de

Medische Wetenschappen

aan de Rijksuniversiteit Groningen

op gezag van de

Rector Magnificus, dr. F. Zwarts,

in het openbaar te verdedigen op

woensdag 22 juni 2005

om 16.15 uur

door

Ning Qu

geboren op 8 januari 1968

te Liaoyang, China

Promotor: Prof. dr. L.F.M.H.de Leij

Co-promotor:

Dr. J.Prop

Beoordelingscommissie:

Prof. dr. T.Ebels

Prof.dr. F.G.M.Kroese Prof. dr. W.Timens

Supervisor: Jochum Prop, MD.PhD.

Co-supervisor:

Aalzen de Haan, PhD.

Paranimfen: Yvonne Drijver

Cheng Qian

The study presented in this thesis was financially supported by a grant from

J.K. de Cock Foundation and by an Ubbo Emmius Scholarship of the

University of Groningen to Ning Qu.

The publication of this thesis was kindly supported by Edwards Lifescience BV,

Norvatis Pharma BV, Johnson&Johnson Medical BV, ST.Jude Medical

Nederland BV, Sorin Group Nederland BV, Wyeth Pharmaceuticals BV,

Roche Nederland BV, Krijnen Medical, Astellas Pharma Prograft®, Medtronic

Nederland BV, Harlan Nederland BV and GUIDE research Institute Groningen.

Dedicated to:

My dear wife Yijin, our unborn baby My parents and my brother

Table of contents CHAPTER 1

General introduction 1

CHAPTER 2

Integrity of Airway Epithelium is Essential Against Obliterative Airway Disease in Transplanted Rat Tracheas

21

CHAPTER3

Specific immune responses against airway epithelial cells in a transgenic mouse trachea transplantation model for obliterative airway disease

43

CHAPTER 4

Obliterative airway disease development in mouse trachea transplants under pre-existing immunity

65

CHAPTER 5

Anti-CD45RB antibody monotherapy protects mouse trachea allograft epithelium

85

CHAPTER 6

General discussion 101

CHAPTER 7

Summary 117

CHAPTER 8

Samenvatting 123

CHAPTER 9

Summary in Chinese 129

Acknowledgement

135

Curriculum vitae 137

Chapter 1

General Introduction

CHAPTER 1

2

General introduction

3

1. Obliterative bronchiolitis and obliterative airway disease

1.1. Obliterative Bronchiolitis

Lung transplantation is currently the only available treatment for end-stage lung disease patients. Despite the success of improved modern lung transplantation with the introduction of new surgical techniques, improved immunosuppressive agents and innovations in managing of acute rejection and infection, the survival rate of recipients is 75% at 1 year and less than 50% at 5 years. (1) One of the most severe complications after lung transplantation is obliterative bronchiolitis (OB) which affect over 40% of the recipients within 5 years during the post-transplantation period.(1).

OB is a chronic disease that develops from months and mostly years after lung transplantation (2-4). It is characterized by progressive bronchial inflammation, epithelial injury and luminal fibrosis.(5-7). There is no treatment for human OB and insight into the understandings of its mechanism is still lacking. Presently, all clinical efforts are directed at slowing down the process. These efforts include pre-transplant treatment to the donor lung to reduce inflammatory inducing factors, employment of aggressive peri-transplant administration with antibiotics (3;8-11) and improved immunosuppressive regimens. Only re-transplantation appears to be a curable solution, but is mostly not possible due to the limited availability of donor organs. Clinical investigation of OB in humans is restricted by the limited amount of patient material available for research. This makes the development of new treatments a difficult and time consuming task (12). Thus, a simple animal model that resembles the development of OB in human is a desirable goal.

1.2. Animal transplant models of obliterative airway disease (OAD) for human OB As pointed out above an animal model is needed for studying the pathophysiology and possible treatment of OB. A rat lung transplantation model was developed in Groningen and described by Jochum Prop and colleagues in 1984 (13). Using this technique they

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were the first to show that OB was secondary to acute pulmonary rejection (14). Also, OB-like airway damage was seen during chronic rejection and viral infections in this rat lung transplantation model. (15) Hertz and his colleagues published a much simpler model in 1993, where they transplanted murine heterotopic tracheas subcutaneously (16), and found that allogeneic tracheas were rejected with massive cellular infiltration and epithelium loss as opposed to isografts which did not show these changes. This led by day 21 to obliteration of the transplanted trachea lumen by the fibroproliferation. This pathological process is called the obliterative airway disease (OAD) and is supposed to reflect the changes seen in OB. Large animal OAD models have also been studied in the past decades. Porcine, canine or even primate tracheas (17) and lungs (18-21) were transplanted and shown to mimic the human OB, since similar histological findings such as graft infiltration, epithelium loss and fibrotic luminal occlusion were observed. Although large animals may be close to humans in size and body structure, the limitations of these large animal models are also obvious: the lack of inbred strains, the cost of purchasing large animals and the peri-transplantation management. The biggest obstacle, however, is the unstable results (22). In summary, OAD in small animal models share histopathological similarities to human OB and is presently the most useful tool for human OB study.

1.3. The histopathology and immunology of OB/OAD

Clinical OB is a chronic inflammatory disease of the airways, involving the bronchial epithelium and leading to the gradual obliteration of small and large airways by inflammatory infiltrates, proliferating fibroblasts, mature collagen and extracellular matrix (3). Although the mechanisms underlying the development of OB are not clear yet, the syndrome may be divided into two distinct phases; an acute alloimmune phase with lymphocytic infiltration of the bronchiolar structures followed by a chronic fibroproliferative phase leading to partial or total occlusion of the airway lumen (3;4;10). These pathogenic phases appear to mimic a tissue 'injury-repair' type of pattern in which episodes of potentially reversible acute rejection (injury) lead to the irreversible chronic state of rejection (insufficient repair leading to scar tissue formation).


5

Transplant injury is caused by the repeated immune-mediated damage inflicted to the airways through allorecognition during acute rejection involving activation of inflammatory mediators (23). Both the humoral and cellular arm of the immune system may be involved. Donor-HLA-specific alloantibodies can be present in the recipient serum as evidence of a humoral response (24;25). More importantly, allo-specific T cells and a plethora of other immune cells observed in the donor lung indicating a cellular response (26-29). These immune processes may lead to the injury of airways. If the injury is not properly balanced by repair processes, excessive migration and proliferation of pulmonary mesenchymal cells, smooth muscle cells and fibroblasts may occur. These processes are driven, at least in part, by the growth factors platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF). PDGF and bFGF have been shown to be upregulated in bronchoalveolar lavage fluid from OB patients in the clinic (30). Fibroproliferation indicates a repair phase of the injured grafts, though it may eventually lead to an overgrowth of fibrotic tissue.

In animal OAD models, a number of pathophysiology features are observed that mimic human OB. A growing body of evidence suggests that development of OAD after trachea or lung transplantation is caused by the allogeneic immune response against cell surface antigens of the allospecific major histocompatablility complex (MHC) antigens, expressed on parenchymal cells of the allograft (24;29;31;32). In different animal OAD models, T lymphocytes, macrophages and granulocytes were found in the allografts preceding epithelium loss and complete luminal obliteration at day 21-28 were observed (CD4+, CD8+ T cells) (5;33-35). Several studies in recent years have indicated that indirect allorecognition of donor MHC-derived peptides by CD4 T cells is one of the most important factors leading to the development of chronic allograft rejection. (36-39) As a result of alloantigen recognition by CD4 (T-helper) cells, which recognize class II MHC antigens expressed by the graft's cells, an alloimmune response is started. Cytotoxic T cells (CTLs), mainly CD8+ cells which recognize class I MHC antigens, may directly kill target cells (40). But CD4+ cells may be more harmful by recruiting also the cells belonging to the innate immune system.

The airway epithelium has been mentioned several times already in the pathogenesis of OB/OAD. We think that it may play a

CHAPTER 1

6

role both in the induction of the injury and in the repair process. In the following part we will focus on this aspect.

2. Airway epithelium in transplantation

2.1. Airway epithelium structure and function

ANATOMY Mammalian airways are lined by a pseudostratified layer of epithelial cells. These include the highly differentiated ciliated cells that cover the airway luminal surface and the less differentiated basal cells bearing a high capacity for proliferation and regeneration (41;42). Other types of cells in the epithelium layer are present in lower number. Some of these are only present at scattered locations, such as goblet cells and Clara cells. These epithelial cells are involved in a number of critical functions related to normal homeostasis.

FUNCTION Classically, epithelium was considered to be a passive barrier between the external environment and the inner tissues of the lung. However, it is now clear that the epithelium plays a pivotal role in controlling many airway functions. Its function as a barrier is essential through tight junctions (zonula occludens) located between the apices of adjacent cells, which restrict paracellular diffusion of electrolytes and other molecules. Desmosomes, intermediate and gap junctions are also involved in maintaining the structural integrity of the epithelium (43-45). The epithelium has also a secretory function and can produce a diverse array of lipid mediators, growth factors, and bronchoconstricting peptides as well as chemokines and cytokines (45). In addition, the epithelium is a major source of arachidonic acid metabolites which help to regulate airway smooth muscle tone, epithelial mucus secretion, neurotransmitter release and, is also involved in inflammation (44-46).

EPITHELIAL RESPONSE TO INJURY Tissue repair is dependent on a structured progression of events that re-establish the integrity of the damaged tissue. The precise mechanisms involved in regeneration of the airway epithelium are still a matter of debate. Much of what is known about epithelium injury and repair (46) either comes from in vivo studies in which the epithelium is experimentally injured, such as by chemical (47), drugs (48), physical factor (49) and repair processes are followed histologically over time, or from cell


7

culture models (50). It is clear that the airway epithelium has a tremendous capacity to repair itself after injury. In a recent in vivo study, Erjefalt et al. showed that in as little as 15 min after an 800-µm wide wound was made in the tracheal epithelium of guinea pigs, epithelial cells on the damaged margin (including secretory and ciliated cells) began to dedifferentiate, flatten and migrate over the denuded area (51). In this study, the denuded area was completely covered by a thin layer of flattened undifferentiated epithelium. Proliferation of epithelial cells was not observed until about 30 hours after the initial stimuli. Within 5 days a fully differentiated epithelium was present again. At approximately the same time as cells begin to differentiate, a plasma exudate was observed which might be an indication of functional repair (52-55).

2.2. Airway epithelium injury during transplantation

Airway epithelium injury is frequently observed upon transplantation. Airway epithelium damage may be already induced before transplantation by the donor's brain death (56), and by ischemia-reperfusion (57-59) during the transplantation procedures. Evidence of inflammatory cytokines up-regulation such as interleukin (IL)-1, IL-2, IL-6, tumor necrotic factor alpha (TNF-α) and interferon gama (IFN-γ) is shown in peripheral organs including lung tissue from brain-dead donors shortly after harvesting (60). It is known that TNF-α and IL-1 are the “early response cytokines” produced by alveolar macrophages in acute lung injury through activation of the transcription factor nuclear factor-kappa B (NF-kB) (61). Upon transplantation, these cytokines induce inflammatory responses towards donor epithelial cells by attracting recipient macrophages and other inflammatory cells (62). This may contribute to the acute injury seen in donor tissue after transplantation. Furthermore, donor tissue that expresses allo MHC antigens induces cellular and humoral alloimmunity in the recipient. Human epithelial cells are known to continuously express MHC antigens in high density (63,64). A recent study shows that the binding of MHC alloantibodies to human epithelial cells induces apoptosis of these cells (65). Epithelial cells apoptosis was also observed in animal airway transplant OAD (66).

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Clinical pathologic features of OB suggest that in particular, the injury of epithelial cells by persistent inflammation in small airways hampers epithelium regeneration and stimulates fibroproliferation due to aberrant tissue repair. In animal tracheal transplants, airway epithelium is involved in the rejection process as one of the targeted tissue leading to OAD (67). The loss of airway epithelium by transplant rejection in allografts but also by enzymatic removal in isografts resulted in OAD (68;69). This stresses the importance of epithelium for the development of OB/OAD. Figure 1. Hypothesis of airway epithelium in OAD/OB occurence

In the illustration, airway allograft is transplanted and rejection occurs. There are two type of rejecting process: 1. Graft-independent process (left down arrows). It is caused by normal grafting surgical procedures and foreign body responses. It occurs to every type of grafting and is not dependdent on the type of grafts. Epithelium mostly experiences ischemia-reperfusion damage and could quickly recover. 2. Graft-dependent process (right down arrows). It is caused by alloimmune responses that recognize allo-MHC antigens expressed in allografts. It occurs only in the allgraft transplantation and is suggested to be the main cause of graft injury and dysfunction.


9

3. Hypothesis and experimental models

3.1. Hypothesis of OB/OAD Studies in patients and animal models indicate that donor airway epithelium injury is playing a role in the process of OB (6;70) and OAD (71). We hypothesize that transplant injury causes airway epithelium damage and that excessive and persistent loss of epithelium results in fibrosis that eventually leads to the occurrence of OB/OAD. Firstly, airway epithelium is one of the primary target tissues during alloimmune responses that are graft-dependent. It is also the target of graft-independent factors, such as general inflammatory response and transplant surgical injury. This type of injury is presented among all types of airway tissue (67) and is largely dominated by alloimmunity. Loss of epithelium during injury ‘denudes’ the transplanted airway, resulting in loss of the defense barrier and, generally, epithelium dysfunction. As a result, the submucosa tissue may directly encounter foreign pathogens that increase the risk of infection. Secondly, damaged airway epithelium may lose its own functional role in regulating the airway repair process during injury (72). In response to injury, airway epithelial cells are capable to dedifferentiate and to regenerate to replace the injured cells, as indicated above. In vitro (73;74)and vivo (75) studies have shown that airway epithelial cells also regulate fibroblast proliferation. Normal epithelial cells inhibit fibroblast proliferation in vitro probably by excretion of inhibitory cytokines (72;76). More specific investigation of the response of epithelium to transplantaton injury seems justified. At the moment, however, we have no good model directly focusing on role of airway epithelial cells in the development of OB/OAD.

3.2. Transgenic animal model for epithelium specific immune

response in OAD study Trachea transplantation is a convenient technique for investigation of epithelial responses. A drawback of studying OAD in an allogeneic tracheal transplant is that immune responses are directed against all cell types of the graft tissue. This is so, because the recipient’s immune system recognizes alloantigens on all cells expressing these

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antigens. So all cells in the trachea grafts are attacked and is impossible to investigate specifically the airway epithelium injury and its effects in OAD development after transplantation.

Transgenic animals expressing a neoantigen on a specific type of tissue may provide a new option to investigate the role of epithelium specific response in OAD. In a recent study, trachea transplants from HLA-class I transgenic mice were shown to induce alloreactive CD4 T cells and alloantibodies against the HLA-class I neoantigens (77). Although this is clearly an improved model to investigate the role of a single alloantigen (HLA-class I) in OAD, it still has a major drawback, since all types of tissue cells may express the neoantigen, making it not suitable for the study of single type of tissue, such as epithelium. To allow investigation of immune injury to be directed against an epithelium specific neoantigen in mouse tracheas, we decided to use the human epithelial glycoprotein-2 (hEGP-2) transgenic mice as donors. In these transgenic mice, hEGP-2 antigens are expressed exclusively on epithelial cells. Using hEGP-2 transgenic mice as donors, we were able to investigate whether the hEGP-2 antigen induces epithelium specific immune response after trachea transplantation and whether the response causes epithelial injury in the transgenic trachea transplants. This model also gave the opportunity to immunize recipients prospectively with the hEGP-2 antigen before transplantation. In this way, a pre-existing immunity directed exclusively to transplanted epithelium was induced.

3.3. Blockade of immune responses and airway epithelium protection

In clinical transplantation, immunosuppressive agents such as cyclosporine A (CsA), FK506 and rapamycin have been shown to effectively prolong graft survival (78). Using these drugs, alloreactive T cell responses are either reduced or blocked. Possible side effects, such as the CsA involvement in pro-fibroproliferation (79) or the pro-inflammatory effect of CsA and FK506 (11), are hampering the full exploration of these therapies. Another approach is the treatment by (monoclonal) antibodies against T cells, such as Anti-thymocyte Globulin (80-82), Anti-Cytotoxic T Lymphocyte Antigen-4 (83) or antibodies against CD2 and CD3 T cell common antigens (84). These antibodies have been used for depletion of T cells for prolong graft


11

survival. However, all these drugs are not specific for alloreactive T cells but modulate other normal T cells as well. This increases the recipient sensitivity to infection as the body defense system is suppressed.

Recently, a novel monotherapy using anti-CD45RB monoclonal antibodies (mAb) to deplete recipient T cells was investigated to block T cell responses and to prolong allograft survival (85). The CD45RB molecule belongs to a family of transmembrane protein tyrosine phosphatases that is expressed by leukocytes and play a critical role in regulating T-cell activation through modulating the activation status of the T cell receptor. In animal studies, mAb MB23G2 against CD45RB prolonged allograft survival through T cell depletion. These antibodies are T cell specific, easy in management and cause no severe side effect in animal models. Prolonged survival has been demonstrated after engraftment of islets (86), hearts and kidneys in MHC disparate mice after the treatment of recipients with anti-CD45RB mAb (87;88). Therefore, it seems worthwhile to investigate if a protective effect on transplant airway epithelium preventing OAD can be found in our mouse trachea transplant model.

4. Main issues addressed and the scope of this thesis

4.1. Epithelium dynamics during injury and its role in OAD

It is essential to know the behavior of airway epithelium upon transplantation injury and to establish its role in airway obliteration. We transplanted rat tracheas to observe airway changes post transplantation in a rat model (chapter 2). Injury was caused by rejection of the MHC fully mismatched tracheas and by enzymatic denudation of syngeneic transplants. Analyses were focused on epithelium integrity, the time point at which fibroproliferation and obliteration occurred. A series of experiments was carried out to stimulate repair of airway epithelium by isolation and reseeding of epithelial cells in trachea transplants.

4.2. Epithelium specific immune responses In chapter 3, we introduced the hEGP-2 transgenic mouse OAD model for the study of epithelium specific immune responses. The

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recipient immune responses against the transgenic grafts were studied in regards of both humoral and cellular reactivity. The effect of the immune responses on epithelium and its subsequent effect on OAD were analyzed. This is for the first time that the effect of a single type of tissue specific immune response is studied in a transplant setting.

4.3. OAD development under pre-existing immunity In chapter 4 the OAD development under pre-existing epithelial cell specific immunity was studied further. Recipient mice were immunized before transplantation to induce pre-existing immunity for hEGP-2 grafts. The reasoning is to mimic the immunity in allotransplantation where alloantibodies and T cells may directly recognize alloantigens. In our model, however, the pre-existing immunity was directed exclusively towards epithelium. This allowed us to analyse epithelium behavior and its effect on OAD development.

4.4. Anti-CD45RB antibody monotherapy to prevent OAD

The prevention of OAD by blocking allreactive T cells in an allogeneic trachea transplantation model is evaluated in chapter 5. To reduce the recipient T cells, a leukocytes antibody anti-CD45RB was administered in this model and the effect of this antibody on epithelium protection and OAD development was evaluated.


13

Reference 1. Taylor DO, Edwards LB, Boucek MM, Trulock EP, Keck BM, Hertz MI.

The Registry of the International Society for Heart and Lung Transplantation: twenty-first official adult lung transplant report--2004.J Heart Lung Transplant. 2004 Jul;23(7):796-803.

2. Heng D, Sharples LD, McNeil K, Stewart S, Wreghitt T, Wallwork J. Bronchiolitis obliterans syndrome: incidence, natural history, prognosis, and risk factors. J Heart Lung Transplant 1998:17: 1255-1263.

3. Boehler A, Kesten S, Weder W, Speich R. Bronchiolitis obliterans after lung transplantation: a review. Chest 1998:114: 1411-1426.

4. Estenne M, Hertz MI. Bronchiolitis obliterans after human lung transplantation. Am J Respir Crit Care Med 2002:166: 440-444.

5. Reynaud-Gaubert M. [Pathophysiology of obliterative bronchiolitis in lung transplants]. Rev Mal Respir 2003:20: 224-232.

6. Paradis I. Bronchiolitis obliterans: pathogenesis, prevention, and management. Am J Med Sci 1998:315: 161-178.

7. Sundaresan S, Trulock EP, Mohanakumar T, Cooper JD, Patterson GA. Prevalence and outcome of bronchiolitis obliterans syndrome after lung transplantation. Washington University Lung Transplant Group. Ann Thorac Surg 1995:60: 1341-1346.

8. Allen MD, Burke CM, McGregor CG, Baldwin JC, Jamieson SW, Theodore J. Steroid-responsive bronchiolitis after human heart-lung transplantation. J Thorac Cardiovasc Surg 1986:92: 449-451.

9. Bando K, Paradis IL, Similo S et al. Obliterative bronchiolitis after lung and heart-lung transplantation. An analysis of risk factors and management. J Thorac Cardiovasc Surg 1995:110: 4-13.

10. Boehler A, Estenne M. Obliterative bronchiolitis after lung transplantation. Curr Opin Pulm Med 2000:6: 133-139.

11. Borger P, Kauffman HF, Timmerman JA, Scholma J, van den Berg JW, Koeter GH. Cyclosporine, FK506, mycophenolate mofetil, and prednisolone differentially modulate cytokine gene expression in human airway-derived epithelial cells. Transplantation 2000:69: 1408-1413.

12. Sundaresan S. Bronchiolitis obliterans. Semin Thorac Cardiovasc Surg 1998:10: 221-226.

13. Prop J, Ehrie MG, Crapo JD, Nieuwenhuis P, Wildevuur CR. Reimplantation response in isografted rat lungs. Analysis of causal factors. J Thorac Cardiovasc Surg 1984:87: 702-711.

CHAPTER 1

14

14. Tazelaar HD, Prop J, Nieuwenhuis P, Billingham ME, Wildevuur CR. Airway pathology in the transplanted rat lung 128. Transplantation 1988:45: 864-869.

15. Winter JB, Gouw AS, Groen M, Wildevuur C, Prop J. Respiratory viral infections aggravate airway damage caused by chronic rejection in rat lung allografts. Transplantation 1994:57: 418-422.

16. Hertz MI, Jessurun J, King MB, Savik SK, Murray JJ. Reproduction of the obliterative bronchiolitis lesion after heterotopic transplantation of mouse airways. Am J Pathol 1993:142: 1945-1951.

17. Kawahara K, Hiratsuka M, Mikami K et al. Obliterative airway disease and graft stenting in pig-to-dog tracheal xenotransplantation. Jpn J Thorac Cardiovasc Surg 2001:49: 53-57.

18. Ikonen T, Uusitalo M, Taskinen E et al. Small airway obliteration in a new swine heterotopic lung and bronchial allograft model. J Heart Lung Transplant 1998:17: 945-953.

19. Salminen US, Ikonen T, Uusitalo M et al. Obliterative lesions in small airways in an immunosuppressed porcine heterotopic bronchial allograft model. Transpl Int 1998:11 Suppl 1: S515-S518.

20. Uusitalo MH, Salminen US, Ikonen TS et al. Alloimmune injury preceding airway obliteration in porcine heterotopic lung implants: a histologic and immunohistologic study. Transplantation 1999:68: 970-975.

21. Hausen B, Berry GJ, Dagum P et al. The histology of subcutaneously implanted donor bronchial rings correlates with rejection scores of lung allografts in a primate lung transplant model. J Heart Lung Transplant 1999:18: 714-724.

22. Hele DJ, Yacoub MH, Belvisi MG. The heterotopic tracheal allograft as an animal model of obliterative bronchiolitis. Respir Res 2001:2: 169-183.

23. Heng D, Sharples LD, McNeil K, Stewart S, Wreghitt T, Wallwork J. Bronchiolitis obliterans syndrome: incidence, natural history, prognosis, and risk factors. J Heart Lung Transplant 1998:17: 1255-1263.

24. Reznik SI, Jaramillo A, Zhang L, Patterson GA, Cooper JD, Mohanakumar T. Anti-HLA antibody binding to hla class I molecules induces proliferation of airway epithelial cells: a potential mechanism for bronchiolitis obliterans syndrome. J Thorac Cardiovasc Surg 2000:119: 39-45.

25. Jaramillo A, Smith MA, Phelan D et al. Development of ELISA-detected anti-HLA antibodies precedes the development of bronchiolitis obliterans


15

syndrome and correlates with progressive decline in pulmonary function after lung transplantation. Transplantation 1999:67: 1155-1161.

26. Neuringer IP, Mannon RB, Coffman TM et al. Immune cells in a mouse airway model of obliterative bronchiolitis. Am J Respir Cell Mol Biol 1998:19: 379-386.

27. Boehler A, Chamberlain D, Kesten S, Slutsky AS, Liu M, Keshavjee S. Lymphocytic airway infiltration as a precursor to fibrous obliteration in a rat model of bronchiolitis obliterans. Transplantation 1997:64: 311-317.

28. Qu N, De Haan A, Harmsen MC, Kroese FG, De Leij LF, Prop J. Specific immune responses against airway epithelial cells in a transgenic mouse-trachea transplantation model for obliterative airway disease. Transplantation 2003:76: 1022-1028.

29. Kelly KE, Hertz MI, Mueller DL. T-cell and major histocompatibility complex requirements for obliterative airway disease in heterotopically transplanted murine tracheas. Transplantation 1998:66: 764-771.

30. Hertz MI, Henke CA, Nakhleh RE et al. Obliterative bronchiolitis after lung transplantation: a fibroproliferative disorder associated with platelet-derived growth factor. Proc Natl Acad Sci U S A 1992:89: 10385-10389.

31. Smith MA, Jaramillo A, SivaSai KS et al. Indirect recognition and antibody production against a single mismatched HLA-A2-transgenic molecule precede the development of obliterative airway disease in murine heterotopic tracheal allografts. Transplantation 2002:73: 186-193.

32. Sherman LA, Chattopadhyay S. The molecular basis of allorecognition. Annu Rev Immunol 1993:11: 385-402.

33. Higuchi T, Jaramillo A, Kaleem Z, Patterson GA, Mohanakumar T. Different kinetics of obliterative airway disease development in heterotopic murine tracheal allografts induced by CD4+ and CD8+ T cells. Transplantation 2002:74: 646-651.

34. Reynaud-Gaubert M, Thomas P, Badier M, Cau P, Giudicelli R, Fuentes P. Early detection of airway involvement in obliterative bronchiolitis after lung transplantation. Functional and bronchoalveolar lavage cell findings. Am J Respir Crit Care Med 2000:161: 1924-1929.


36. Reznik SI, Jaramillo A, SivaSai KS et al. Indirect allorecognition of mismatched donor HLA class II peptides in lung transplant recipients with bronchiolitis obliterans syndrome. Am J Transplant 2001:1: 228-235.

CHAPTER 1

16

37. Richards DM, Dalheimer SL, Ehst BD et al. Indirect minor histocompatibility antigen presentation by allograft recipient cells in the draining lymph node leads to the activation and clonal expansion of CD4+ T cells that cause obliterative airways disease. J Immunol 2004:172: 3469-3479.



40. Gao GF, Jakobsen BK. Molecular interactions of coreceptor CD8 and MHC class I: the molecular basis for functional coordination with the T-cell receptor. Immunol Today 2000:21: 630-636.

41. Hong KU, Reynolds SD, Watkins S, Fuchs E, Stripp BR. Basal cells are a multipotent progenitor capable of renewing the bronchial epithelium. Am J Pathol 2004:164: 577-588.

42. Hong KU, Reynolds SD, Watkins S, Fuchs E, Stripp BR. In vivo differentiation potential of tracheal basal cells: evidence for multipotent and unipotent subpopulations. Am J Physiol Lung Cell Mol Physiol 2004:286: L643-L649.

43. Holgate ST, Lackie P, Wilson S, Roche W, Davies D. Bronchial epithelium as a key regulator of airway allergen sensitization and remodeling in asthma. Am J Respir Crit Care Med 2000:162: S113-S117.

44. Knight D. Epithelium-fibroblast interactions in response to airway inflammation. Immunol Cell Biol 2001:79: 160-164.

45. Knight DA, Holgate ST. The airway epithelium: structural and functional properties in health and disease. Respirology 2003:8: 432-446.

46. Holgate ST. Epithelial damage and response. Clin Exp Allergy 2000:30 Suppl 1: 37-41.

47. O'Brien DW, Morris MI, Ding J, Zayas JG, Tai S, King M. A mechanism of airway injury in an epithelial model of mucociliary clearance. Respir Res 2004:5: 10.

48. Adamson IY, Bowden DH. Bleomycin-induced injury and metaplasia of alveolar type 2 cells. Relationship of cellular responses to drug presence in the lung. Am J Pathol 1979:96: 531-544.


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49. Kay SS, Bilek AM, Dee KC, Gaver DP, III. Pressure gradient, not exposure duration, determines the extent of epithelial cell damage in a model of pulmonary airway reopening. J Appl Physiol 2004:97: 269-276.

50. Terzaghi-Howe M, Ford J. Effects of radiation on rat respiratory epithelial cells: critical target cell populations and the importance of cell-cell interactions. Adv Space Res 1994:14: 565-572.

51. Erjefalt JS, Erjefalt I, Sundler F, Persson CG. In vivo restitution of airway epithelium. Cell Tissue Res 1995:281: 305-316.

52. Erjefalt JS, Korsgren M, Nilsson MC, Sundler F, Persson CG. Prompt epithelial damage and restitution processes in allergen challenged guinea-pig trachea in vivo. Clin Exp Allergy 1997:27: 1458-1470.

53. Erjefalt JS, Sundler F, Persson CG. Epithelial barrier formation by airway basal cells. Thorax 1997:52: 213-217.

54. Persson CG. Epithelial cells: barrier functions and shedding-restitution mechanisms. Am J Respir Crit Care Med 1996:153: S9-10.

55. Erjefalt JS, Erjefalt I, Sundler F, Persson CG. Microcirculation-derived factors in airway epithelial repair in vivo. Microvasc Res 1994:48: 161-178.

56. Fisher AJ, Donnelly SC, Hirani N et al. Enhanced pulmonary inflammation in organ donors following fatal non-traumatic brain injury. Lancet 1999:353: 1412-1413.

57. Soria A, Vicente R, Ramos F, Lopez LM, Francia C, Montero R. [Lesion caused by ischemia-reperfusion in lung transplantation]. Rev Esp Anestesiol Reanim 2000:47: 380-385.

58. Clark SC, Sudarshan C, Khanna R, Roughan J, Flecknell PA, Dark JH. Controlled reperfusion and pentoxifylline modulate reperfusion injury after single lung transplantation. J Thorac Cardiovasc Surg 1998:115: 1335-1341.

59. Eppinger MJ, Deeb GM, Bolling SF, Ward PA. Mediators of ischemia-reperfusion injury of rat lung. Am J Pathol 1997:150: 1773-1784.

60. Takada M, Nadeau KC, Hancock WW et al. Effects of explosive brain death on cytokine activation of peripheral organs in the rat. Transplantation 1998:65: 1533-1542.

61. Lentsch AB, Ward PA. Regulation of experimental lung inflammation. Respir Physiol 2001:128: 17-22.

62. Rizzo M, SivaSai KS, Smith MA et al. Increased expression of inflammatory cytokines and adhesion molecules by alveolar macrophages of human lung allograft recipients with acute rejection:

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decline with resolution of rejection. J Heart Lung Transplant 2000:19: 858-865.

63. Cunningham AC, Milne DS, Wilkes J, Dark JH, Tetley TD, Kirby JA. Constitutive expression of MHC and adhesion molecules by alveolar epithelial cells (type II pneumocytes) isolated from human lung and comparison with immunocytochemical findings. J Cell Sci 1994:107 ( Pt 2): 443-449.

64. Cunningham AC, Zhang JG, Moy JV, Ali S, Kirby JA. A comparison of the antigen-presenting capabilities of class II MHC-expressing human lung epithelial and endothelial cells. Immunology 1997:91: 458-463.

65. Jaramillo A, Smith CR, Maruyama T, Zhang L, Patterson GA, Mohanakumar T. Anti-HLA class I antibody binding to airway epithelial cells induces production of fibrogenic growth factors and apoptotic cell death: a possible mechanism for bronchiolitis obliterans syndrome 84. Hum Immunol 2003:64: 521-529.

66. Alho HS, Salminen US, Maasilta PK, Paakko P, Harjula AL. Epithelial apoptosis in experimental obliterative airway disease after lung transplantation. J Heart Lung Transplant 2003:22: 1014-1022.

67. Fernandez FG, Jaramillo A, Chen C et al. Airway epithelium is the primary target of allograft rejection in murine obliterative airway disease 87. Am J Transplant 2004:4: 319-325.


69. Adams BF, Brazelton T, Berry GJ, Morris RE. The role of respiratory epithelium in a rat model of obliterative airway disease. Transplantation 2000:69: 661-664.

70. Mauck KA, Hosenpud JD. The bronchial epithelium: a potential allogeneic target for chronic rejection after lung transplantation. J Heart Lung Transplant 1996:15: 709-714.


72. Holgate ST. Epithelial damage and response. Clin Exp Allergy 2000:30 Suppl 1: 37-41.

73. Nakamura Y, Tate L, Ertl RF et al. Bronchial epithelial cells regulate fibroblast proliferation. Am J Physiol 1995:269: L377-L387.


19

74. Pan T, Mason RJ, Westcott JY, Shannon JM. Rat alveolar type II cells inhibit lung fibroblast proliferation in vitro. Am J Respir Cell Mol Biol 2001:25: 353-361.

75. Moore BB, Peters-Golden M, Christensen PJ et al. Alveolar epithelial cell inhibition of fibroblast proliferation is regulated by MCP-1/CCR2 and mediated by PGE2. Am J Physiol Lung Cell Mol Physiol 2003:284: L342-L349.

76. Sacco O, Silvestri M, Sabatini F, Sale R, Defilippi AC, Rossi GA. Epithelial cells and fibroblasts: structural repair and remodelling in the airways. Paediatr Respir Rev 2004:5 Suppl A: S35-S40.


78. Garrity ER, Jr., Mehra MR. An update on clinical outcomes in heart and lung transplantation. Transplantation 2004:77: S68-S74.

79. Hostettler KE, Roth M, Burgess JK et al. Cyclosporine A mediates fibroproliferation through epithelial cells 85. Transplantation 2004:77: 1886-1893.

80. Brock MV, Borja MC, Ferber L et al. Induction therapy in lung transplantation: a prospective, controlled clinical trial comparing OKT3, anti-thymocyte globulin, and daclizumab. J Heart Lung Transplant 2001:20: 1282-1290.

81. Diamond DA, Michalski JM, Lynch JP, Trulock EP, III. Efficacy of total lymphoid irradiation for chronic allograft rejection following bilateral lung transplantation. Int J Radiat Oncol Biol Phys 1998:41: 795-800.

82. van Tiel FH, Rasmussen L, Merigan TC. Cytomegalovirus-specific cell-mediated immune responses in heart and heart-lung transplant recipients are not predictive for the occurrence of symptomatic CMV disease or tissue rejection. J Interferon Res 1991:11: 221-229.

83. Perico N, Imberti O, Bontempelli M, Remuzzi G. Toward novel antirejection strategies: in vivo immunosuppressive properties of CTLA4Ig. Kidney Int 1995:47: 241-246.

84. Chavin KD, Qin L, Lin J, Yagita H, Bromberg JS. Combined anti-CD2 and anti-CD3 receptor monoclonal antibodies induce donor-specific tolerance in a cardiac transplant model. J Immunol 1993:151: 7249-7259.

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85. Gao Z, Zhong R, Jiang J et al. Adoptively transferable tolerance induced by CD45RB monoclonal antibody. J Am Soc Nephrol 1999:10: 374-381.

86. Visser L, Poppema S, de Haan B et al. Prolonged survival of rat islet xenografts in mice after CD45RB monotherapy. Transplantation 2004:77: 386-391.

87. Zhang Z, Lazarovits A, Grant D, Garcia B, Stiller C, Zhong R. CD45RB monoclonal antibody induces tolerance in the mouse kidney graft, but fails to prevent small bowel graft rejection. Transplant Proc 1996:28: 2514.

88. Ko S, Jager MD, Tsui TY et al. Long-term allograft acceptance induced by single dose anti-leukocyte common antigen (RT7) antibody in the rat. Transplantation 2001:71: 1124-1131.

Chapter 2

Integrity of Airway Epithelium is Essential against Obliterative Airway Disease in

Transplanted Rat Tracheas

Ning Qu; Paul de Vos; Maaike Schelfhorst; Aalzen de Haan; Wim Timens; Jochum Prop

Journal of Heart and Lung Transplantation. 2005, Aug.

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Integrity of airway epithelium is essential

23

Abstract Background The pathogenesis of obliterative bronchiolitis following lung transplantation requires further elucidation. In this study we used the rat trachea transplantation to examine the role of epithelium in the progression of obliterative airway disease. Methods Normal and denuded (i.e., epithelium removed) trachea grafts from Lewis (LEW) and Brown Norway (BN) rats were transplanted subcutaneously into LEW rats. Viable trachea epithelial cells (to recover epithelium) were seeded into the lumen of some of the denuded tracheas. Grafts were removed at different time points between 2 days to 8 weeks after transplantation. Histological analysis was performed to evaluate the cellular infiltration of inflammatory cells, loss of epithelium, and obliteration of trachea lumen. Results Obliteration was found to occur in trachea transplants after loss of epithelium, caused by rejection in allografts or by enzymatic denudation in isografts. In these situations, fibroblasts started to proliferate and to migrate into the lumen in the second week after transplantation. Obliteration could be prevented when the epithelial integrity was restored by seeding epithelial cells; no obliteration occurred when denuded trachea isografts were seeded with epithelial cells, whereas non-seeded denuded tracheas were obliterated at day 6 after transplantation. Conclusions We conclude that integrity of airway epithelium is essential for rat trachea transplants to be safeguarded from obliterative airway disease. For clinical lung transplantation the results of our study suggest that protection of the integrity of airway epithelium may be important in prevention of the development of obliterative bronchiolitis.

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Introduction Clinical lung transplantation is associated with obliterative bronchiolitis (OB) in 50% of the transplanted patients, resulting in graft failure and high mortality of the recipients (1). Up to now, observations in human lung allograft recipients indicate that OB is an immune-mediated process characterized by infiltration of inflammatory cells, fibroproliferation, and in severe cases occlusion of airways, most notably of the bronchioles (2). The only therapy available for OB is augmentation of immunosuppression to arrest the functional decline of the transplanted lung (3-5). Unfortunately, when obliterative bronchiolitis is established augmented immunosuppression is ineffective in most cases (3,5). More insight into the pathogenesis of OB is required to design adequate treatment modalities.

The pathogenesis of human OB after lung transplantation has been investigated in experimental studies using the model of obliterative airway disease (OAD) in trachea grafts (6-11). These studies suggest that the integrity of airway epithelium is essential for the success of lung allografts. It was found that loss of epithelium as a result of rejection in allografted rat tracheal transplants preceded obliteration of the tracheal lumen (12,13). Also, it was found that exogenous injury of epithelial cells in tracheal isografts induced obliteration of the lumen that resembled OAD in rejected allografts (14,15). These findings suggest that the airway epithelium has a regulatory role in preventing obliteration of airways in lung and trachea transplants. This may well be accomplished by reducing fibroblasts growth and extracellular matrix production, since fibroblasts are mainly responsible for the obliteration of the airway lumen in OB and OAD (13,16).

In the present study a series of three experiments was undertaken in rats to test the hypothesis that the integrity of airway epithelium is essential to prevent OAD in trachea transplants. Firstly, we compared epithelial integrity and luminal obliteration in rejecting trachea allografts (without immune suppression) and in normal trachea isografts during a prolonged post-transplant period (from 4 days up to 8 weeks after transplantation). Then, we investigated if epithelial injury (induced by enzymatic denudation) accelerated the progression of OAD in allo- and isografted tracheas. Finally, we studied whether healthy epithelial cells seeded into denuded trachea isografts could prevent the development of OAD.


25

Material and methods Design of the study For the first set of experiments in the study, with the aim to correlate integrity of airway epithelium and luminal obliteration with inflammatory cell infiltration in OAD, trachea isografts and allografts were transplanted subcutaneously from Lewis (LEW) (n=19) and Brown Norway (BN) (n=20) donor rats, respectively into LEW recipient rats (n=39). Grafts were harvested at 4 days and 1,2,3,4, and 8 weeks after transplantation to assess the integrity of the epithelium, the luminal obliteration and the inflammatory infiltration. Next, the influence of loss of epithelial integrity on luminal obliteration was investigated in 32 LEW isografts and 31 BN allografts by removal of airway epithelial cells before transplantation of the tracheas into LEW recipient rats (i.e., denuded tracheas). Grafts (3 to 8 at each time point) were harvested at 2,4,6,10,14, and 21 days after transplantation for histological analysis.

Finally, to study whether epithelial cells can prevent the obliteration in denuded trachea transplants, we seeded epithelial cells into denuded rat tracheas which were subsequently transplanted isogenically into LEW rats. Grafts were harvested at 2,4, and 6 days (n=5 at each time point) after transplantation and histologically analyzed for the integrity of the epithelium and for luminal obliteration. Denuded trachea isografts (n=2 to 5 at the same time points) filled with medium instead of epithelial cells served as controls. Animals Inbred LEW and BN rats (male, specific-pathogen free) were obtained from Harlan (Harlan Netherlands, Horst, The Netherlands) and were used in the experiments at 12 weeks old. The rats were housed in the Central Animal Laboratory of Groningen University. All animals received care in compliance with the Dutch regulations and laws. Experimental protocols were approved by the institutional animal ethical review committee. Chemicals and antibodies Protease (Protease Streptomyces griseus, p8811), 4 units/mg solid powder and collagenase type IV were purchased from Sigma-Aldrich Chemie B.V. Zwijndrecht, Netherlands. The enzymes were diluted in Hank’s Balanced Salt

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26

Solution (HBSS) (GIBCO, Cat. No.14025-092) to obtain appropriate concentration.

For detection of specific inflammatory cells in the cellular infiltrates the following primary mouse anti rat antibodies were applied: anti-CD3 for T cells, anti-His 48 for granulocytes, anti-ED1 for macrophages and the antibodies for epithelial cells staining, keratin-18 specific monoclonal antibody-RGE53 (Cat.No.69861N) were purchased from BD Pharmingen Alphen aan den Rijn, The Netherlands. The anti-alpha-actin for myofibroblasts was purchased from Roche Molecular Biochemicals, Almere, The Netherlands. Trachea excision, denudation, epithelial cell seeding and transplantation Donor and recipient rats were anesthetized with halothane and N2O/O2 gas. Tracheas of donor rats were exposed by anterior midline incision and carefully dissected from the esophagus and other surrounding tissues. The tracheas were excised by transsection at the thyroid cartilage and the carina bifurcation and were immediately brought into cold saline, stored at 4 oC until further processing or transplantation. Denudation of tracheas and the isolation of epithelial cells were performed by enzymatic digestion of the extracellular matrix between the epithelial cell layer and the submucosa tissue in the trachea. The lumen was washed 5 times with 1 ml cold HBSS to remove blood and other contaminants, filled with enzyme using a 25 G needle, and ligated at both ends with a 3-0 polypropylene ligature. The tracheas were brought into HBSS for incubation. After incubation, the tracheas were cut open at both ends and the lumen was washed with HBSS to flush out the dissociated cells. For epithelial cells isolation, 1 ml cold Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco BRL, Grand Island, NY, USA) was used to flush. The collected cells were washed by centrifugation at 300g for 10 minutes and subsequently resuspended in 25 cm2 culture flask coated with Collagen I gel. Epithelial cells growth factor enriched culture medium DMEM/F12 (17) was added to allow only the epithelial cells to recover and grow. Before seeding, the epithelial cells were cultured for 24 hours. Cells viability was tested using trypan blue staining.

In a pilot study the procedures for denudation and for the isolation of epithelial cells were optimized. Therefore, different concentrations of enzymes (collagenease IV and protease), temperatures, and incubation times were tested (see Table 1). The efficacy of the denudation procedure was assessed


27

histologically by assessing the remaining epithelial cells lining the trachea lumen and scoring the integrity of the submucosal tissue. The applicability of the isolation procedure of epithelial cells was assessed based on the viability of the isolated epithelial cells after the enzymatic digestion. We found that the best procedure for denudation of the tracheas was incubation with 4 units of protease at 37 0C for 1 hour, since this procedure resulted in a complete removal of all epithelial cells from the trachea without significant damage of the submucosal tissue of the grafts. For the isolation of epithelial cells, the best procedure was incubation with 4 units of protease at 4 0C of 18 hours, yielding epithelial cells with a viability of 93%. (Table 1). Therefore, these procedures were applied in later experiments for isolation and subsequent seeding of epithelial cells. Table 1. Effect of denudation procedures of rat tracheas and isolation of trachea epithelial cells by enzymatic treatment.*

Procedures Results

n enzyme (unit/ml)

incubation time

incubation temperature

epithelium presence

submucosa presence

epithelial cells viability

8 Collagenase IV

100u 1h 37◦C ++ ++ <10%

7 Collagenase IV

500u 1h 37◦C + <10%

4 Collagenase IV

1000u 1h 37◦C - - <20%

6 Protease 4u 1h 37◦C - ++++ <40%

9 Protease 8u 1h 37◦C - + <35%

14

Protease 4u 18h 4◦C + + >93%

* Histology was scored on a scale from – to ++++ representing: no epithelium/submucosa (-) to intact epithelium/submucosa (++++) presence. Shown are the median values from the number of experiments given in the table. Cells viability is given as a percentage of total cells determined by trypan blue exclusion.

Seeding of epithelial cell into denuded tracheas was performed as follows. Tracheal epithelial cells, harvested and cultured as described above, were prepared at 2x106 cells/ml. Subsequently, the epithelial cells were

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28

injected into the lumen of denuded Lewis tracheas. Control denuded tracheas were filled with DMEM/F12 medium only. The tracheas were closed at both ends with surgical clips (Ligation clip 316l, Ethicon, Cincinnati, OH, USA) for transplantation.

For transplantation of the trachea, a small incision was made at the lateral side of the back of the recipient rat. By blunt dissection, a subcutaneous pouch was made and the clipped trachea was put into the pouche. The incision was closed by 2 single stitches (5-0 polypropylene suture). One trachea graft was implanted per recipient.

Histology and immunohistochemistry Tracheas from transplantation experiments were explanted by meticulous excision, and were cut into two segments. One segment was fixed in 10% formalin for paraffin embedding and Haematoxylin & Eosin (H&E) and Verhoeff's elastin staining while the second segment was snap frozen in liquid nitrogen for immunohistochemical staining. Tracheas for testing denudation procedures were cross cut into three segments (i.e., an upper, a middle, and a lower part) and fixed with 10% formalin for 24 hours. These tracheas were embedded in paraffin and were only H&E stained.

To examine the integrity of the epithelium, trachea cross sections stained by H&E were analyzed and epithelium was scored in five degrees categorized from 0 to 4 for low to high integrity. The degree of epithelial cells differentiation (the presence of well ciliated and pseudostratified epithelium indicates high differentiation representing normal epithelium, otherwise, the presence of single layer of flattened non-ciliated epithelium indicates poor differentiation representing abnormal epithelium), and the epithelium lining of the trachea lumen (in percentage) were evaluated.

To examine the degree of cellular infiltration in the H&E stained grafts we assessed the infiltration level on a semi-quantitative scale of 0 to 4 (18). The scale corresponds to the following infiltration grades: 0-no infiltration, 1-minimal infiltration: scattered or diffuse cells infiltrates, 2-mild infiltration: diffuse cell infiltrates with one area of dense infiltrates, 3-moderate infiltration: more than one area infiltrates, 4-severe infiltration: a thick layer of dense cell infiltrates.

The degree of luminal obliteration of the trachea was assessed by examining the thickness of the submucosa (i.e., between epithelium and cartilage) on a scale of 0 to 4 (18). The scores represent the following grades:


29

0-no thickening of submucosa, 1-minimal thickening: focal submucosa thickening, 2-mild thickening: submucosa thickening equal to the thickness of the cartilage ring, 3-moderate thickening, submucosa thickening resulting in a small lumen, 4-severe thickening: total lumen obliteration.

Immunohistochemistry was performed on frozen sections of tracheas. The tracheas were sectioned and processed as previously described (18). Sections were fixed in acetone and washed 3 times in 0.01M phosphate-buffered saline (PBS). Subsequently, we applied the appropriate primary antibodies in 1:50 dilutions. After 60 minutes incubation with the primary antibody the slides were washed with PBS. Next, we applied the peroxidase-conjugated secondary rabbit anti mouse antibody at 1:50 dilution (Dako, Glostrup, Denmark). After 60 minutes incubation, the slides were washed with PBS, and processed for staining, using amino-aethyl carbazole (AEC) (Sigma, 0.5mg in 3.75 ml DMF plus 70ml Acetate buffer, PH =4.9) together with H2O2, as a reagent giving a reddish-brown precipitate. Slides were counterstained with haematoxylin (Mayers’ haematoxylin 1:10). The slides were examined for number of positive cells from low to high numbers as 0 to grade 5 (six levels).

Epithelial cells were stained by keratin-18 specific monoclonal antibody (RGE53) and myofibroblasts were stained by anti-alpha-actin specific antibody. The staining procedure was identical to the protocol described above. All histology counting and scoring was done by two to three independent observers. Statistical analysis Results are expressed either as mean ± SEM or as median plus range (table). Statistical comparisons for difference between groups were evaluated by Mann-Whitney U test. A p-value < 0.05 was considered statistically significant. Results Transplantation induced OAD in allografts but not in isografts In trachea iso- and allografts we investigated how the integrity of the tracheal epithelium and the degree of luminal obliteration correlated with the inflammatory cell infiltration at 4 days and at 1,2,3,4,8 weeks after transplantation. Isografting was not associated with significant cellular infiltration or loss of integrity of the epithelium (Table 2A), despite the ischemic and surgical injury at the time of transplantation. We did observe a few

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30

granulocytes, macrophages in the isografts. In a minority of the tracheas, we observed some abnormal architecture of the epithelium as demonstrated by loss of cilia and the absence of pseudostratified epithelium. This was completely restored in the first week after transplantation. Figure 1.

0.5 1 2 3 4 8

0

1

2

3

4

5

weeks after transplantation

grad

ing

of in

filtra

tion,

epith

eliu

m a

nd m

yofib

robl

ast

foun

d in

the

graf

ts

Figure 1. Composition of cellular infiltrates and epithelium presence in rat trachea allografts. BN trachea allografts (n= 3 to 9) were analyzed by immunohistochemistry at each time point after subcutaneous transplantation into LEW rats. Open squares (□) represent the epithelium integrity; black triangles (▲) represent the macrophages; open triangles (∆) represent T cells; asterisks (*) represent myofibroblasts; black circles (●) represent granulocytes. A value of 0 represents absence of infiltrate while value 5 represents a severe infiltration of the graft. Values are medians of at least 3 experiments.

The pathology of rejecting allografts (tracheas transplanted from BN to

LEW rats) was very different from that of isografts: severe inflammatory cell infiltration was associated with loss of airway epithelium within the first weeks after transplantation, resulting in severe luminal obliteration at 3 weeks (Table 2B). Cellular infiltration during the first week after transplantation was most obvious in the tissue outside of the tracheal cartilage. The infiltration inside of the trachea started later, to reach its highest level at 2 weeks. Immunohistochemical analysis of the infiltrating cells showed that these were mainly macrophages, granulocytes and T cells (Figure 1), a pattern consistent with acute rejection. The intensity of the infiltration was associated with the


31

higher loss of integrity of the airway epithelium. In the first week after transplantation, the appearance of the epithelium was flattened and part of the epithelial cells was lost. The loss of epithelium was complete at 2 weeks after transplantation. Simultaneously, the lumen of the trachea became obliterated (Table 2B). At 1 week, the obliteration could be explained by thickening of the tracheal submucosa by infiltrating cells (Figure 1). As of the moment of disappearance of epithelial cells from the lumen (2 weeks post-transplantation), the obliteration was caused by large numbers of myofibroblasts and increase of connective tissue in the tracheas (Figure 1). Denudation of tracheal epithelial cells and OAD In order to investigate the relation between the integrity of epithelium and

luminal obliteration in more detail, we next studied the obliteration after

grafting of tracheas in the absence of epithelial cells (i.e., denuded tracheas).

Obliteration was assessed in normal and in denuded tracheas at short

intervals after transplantation (at 2,4,6,10,14, and 21 days, n=3 to 8). The

amount of epithelial cells profoundly decreased in control trachea isografts in

the first days after transplantation (Figure 2 A). This loss was compensated by

fast regeneration of the epithelial cells during the first week after

transplantation, resulting in complete recovery of the epithelial layer by day

10. In control allografts, the loss of epithelial cells showed a similar pattern of

recovery after transplantation (Figure 2 B). We found an initial loss of the

epithelium at day 2 with gradual recovery up to day 6. Then, the epithelial presence dropped sharply to disappear completely by day 10 after transplantation. These data are in agreement with the less detailed observations in the first experiment of this study. As expected, the denuded iso- and allografts showed virtually no epithelium in

the period immediately after transplantation (Figure 2 C, D, n=4 to 5) although

in spite of the enzymatic removal of epithelium, denuded isografts showed

some epithelial cells in the lumen at day 4 and day 6 (Figure 2 C). These

epithelial cells may be responsible for the complete recovery of the epithelial

layer seen at day 21 after implantation. There was no epithelium regeneration

observed in denuded allografts after transplantation (Figure 2 D).

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32

Figure 2. □ epithelium coverage ● obliteration X: days after transplantation


33

Figure 2. Integrity of the epithelium (open squares □) and degree of luminal obliteration (black circles ●) after transplantation of normal and denuded LEW iso- and BN allografts into LEW recipients. The integrity of the epithelium is expressed as the percentage of luminal area that is covered with epithelial cells. The obliteration level is expressed from low to high as 0-4, i.e. a value of 0 represents absence of obliteration while 4 represents complete luminal obliteration. Values represent medians and ranges of 3 to 9 experiments.

The essential role of the presence of epithelial cells in preventing obliteration is also demonstrated in Figure 2. With the grafting of denuded isografts and allografts, we found a pronounced and progressive obliteration of the lumen (Figure 2 C, D), whereas in normal isografts we found only minimal obliteration, disappearing after the complete recovery of the epithelium (Figure 2 A). In the control allografts, obliteration developed more slowly than in the denuded allografts (Figures 2 B, D). The best illustration of the role of luminal epithelial cells in preventing obliteration is given by the denuded isografts. Here obliteration developed quickly after transplantation, but this stopped by day 10 which coincided with the recovery of the epithelium (Figure 2 C).

We also evaluated those grafts in which the epithelium was not completely removed before transplantation (i.e., incompletely denuded group). The epithelium in these grafts started to recover immediately after transplantation at day 2, and was mostly restored within 6 days (Figure 2E, n=2 to 3). The obliteration of the lumen remained at minimal to mild level during the whole study period, similar to the control isografts.

Transplantation of epithelial cells into denuded trachea grafts to prevent OAD The experiments described above suggest a regulatory role of epithelial cells in preventing luminal obliteration in OAD. In order to confirm this role of airway epithelium in preventing obliteration, we studied whether seeding of isolated, viable epithelial cells could prevent the early obliteration in trachea isografts.

In the epithelial cell seeded, denuded isografts investigated at 2 days after transplantation, the lumen of the trachea was filled with proteinacous material containing loose cells, amongst which were many granulocytes and erythrocytes. At that moment no epithelium layer could be recognized, but already on day 4 after transplantation a thin layer of epithelial cells lined the partially open lumen of the treated trachea (Figure 3, seed d4). The epithelial layer was still irregular, at some places with flattened epithelial cells, at other

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34

places with cells in a multilayer up to 5 cells thick without nuclear polarity. The remainder of the trachea lumen was filled with similar material as seen on day 2, with decreasing numbers of granulocytes. At day 6, the epithelium was restored to normal as demonstrated by the presence of pseudostratified epithelium with ciliated epithelial cells; the lumen of the tracheas was open without significant fibrotic obliteration (Figure 3, seed d6). In the control denuded isografts without cell seeding, luminal obliteration at day 4 after transplantation (Figure 3, denu d4) progressed at day 6, with many cells in the lumen showing the morphology of fibroblasts (Figure 3, denu d6). In these tracheas no epithelium regeneration was observed at these early time points after transplantation. Table 2. Inflammation, integrity of epithelium, and obliteration in trachea iso- and allografts at 4 days, 1, 2, 3, 4, and 8 weeks after transplantation*. A. Isografts

Inflammation Epithelium integrity

Luminal obliteration

Time n Median(Range) Median(Range) Median(Range) D4 3 0 (0-0) 3 (3-4) 0 (0-0) 1w 3 0 (0-0) 4 (4-4) 0 (0-0) 2w 3 0 (0-0) 4 (4-4) 0 (0-0) 3w 3 0 (0-0) 4 (4-4) 0 (0-0) 4w 3 0 (0-0) 3 (0-4) 0 (0-0) 8w 4 0 (0-0) 3.5 (3-4) 0 (0-0) B. Allografts

Inflammation inside trachea

Inflammation outside trachea

Epithelium integrity

Luminal obliteration

Time n Median(Range) Median(Range) Median(Range) Median(Range) D4 3 0 (0-0) 3.5 (3-4) 2 (0-2) 0 (0-0) 1w 3 0 (0-4) 4 (4-4) 2 (2-2) 0 (0-4) 2w 3 3 (0-3) 4 (4-4) 0 (0-0) 0 (0-4) 3w 3 2 (2-2) 4 (4-4) 0 (0-0) 4 (2-4) 4w 3 3 (2-4) 4 (3-4) 0 (0-0) 4 (2-4) 8w 5 0 (0-2) 2 (0-3) 0 (0-0) 4 (2-4)

* Scores represent grading from low to high as 0-4.


35

Figure 3.

Figure 3. The histology of denuded isografts: non-seeding and seeded with epithelial cells after transplantation. In the denuded non-seeding graft at day 4, no epithelium was present and the lumen was partially obliterated by cellular debris. At day 6, epithelium remained absent, and the lumen was largely obliterated by fibrotic tissue. In the epithelial cell seeded group at day 4, a thin epithelium layer was lining the lumen (arrow). In the lumen was some cellular debris. On day 6, a totally restored epithelium was lining the lumen, while epithelial cells regained cilia and polarity. Histology slides were Verhoeff's elastin stained; pictures were taken at 40x magnification, the insets at 100x magnification.

Discussion From the three experiments in this study it can be concluded that development of OAD in trachea transplants correlates with injury and repair of the airway epithelium. Injury of the epithelium with subsequent luminal obliteration coincided with inflammatory responses in the trachea grafts. In this inflammation-induced injury to airway epithelium we distinguish two injury phases after transplantation of rat tracheas: the “surgical” injury phase (most obvious in isografts immediately after transplantation) and the rejection injury phase (most obvious in allografts from 10 days after transplantation).

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36

Furthermore, the progression of OAD is facilitated if repair of epithelium is prevented by denudation of trachea iso- and allografts. In contrast, development of OAD is prevented when the integrity of epithelium is restored quickly, in our experiments by seeding of epithelial cells into denuded trachea isografts. Taken together, these observations demonstrate that integrity of airway epithelium is essential for trachea transplants to be safeguarded from OAD.

How does epithelial injury in the two phases lead to OAD? In the surgical injury phase a non-specific inflammatory reaction with influx of granulocytes and macrophages develops in the tracheas immediately after transplantation. This inflammation is the response to ischemia in the period between removal of the graft from the donor and complete engraftment in the recipient and to reperfusion in the subsequent period. In this surgical injury phase epithelial cells are partially lost from the trachea transplants, irrespective of alloreactivity as its severity is similar in trachea isografts and allografts. In isografted tracheas, epithelium can recover from the surgical injury phase; it regrows quickly and covers the trachea lumen completely. The mild and transient obliteration of the lumen observed in some isografts in our experiments probably reflects transient swelling of the submucosa by the inflammatory reaction, without fibroblast proliferation. Even in allografts, the epithelium recovers from the surgical injury phase during the first postoperative week. These observations show that inflammation from the surgical injury phase has no lasting effect on trachea epithelium, provided no allograft-rejection related inflammation is involved.

In the rejection injury phase the airway epithelium is injured more severely by the inflammatory reaction as a result of acute rejection. Alloreactive, antigen-dependent T cells have been shown to play a role in the development of OAD (19,20). In agreement, a significant proportion of the inflammatory cells in our trachea allografts have the T cell phenotype. Remarkably, the inflammation starts in the periphery of the trachea allograft, to reach the epithelium between 1 and 2 week post-transplantation. It is exactly in this period that the epithelial cells are injured beyond repair and disappear from the trachea allografts. Simultaneously, myofibroblasts start to grow and obliterate the trachea lumen in a process typical of OAD as described by Boehler et al (21) and King et al (13). During the rejection injury phase, recovery of the trachea allograft is still possible to a certain degree. This was shown in a study of Brazelton (22) where OAD in allografts was prevented when the progression of rejection was stopped. They removed


37

trachea allografts from the recipients before getting irreversible epithelium damage (day 10) and transplanted the tracheas back to syngeneic recipients of the donor strain. In these retransplanted grafts the airway epithelium regenerated and the fibroproliferative obliteration of the lumen did not occur. These data are in line with the hypothesis that rejection-induced inflammation is the major factor causing epithelial injury that ultimately leads to OAD.

The importance of epithelium for the repair from injury is emphasized by our observations in two ways. On the one hand, when recovery of epithelium is hampered by denudation from trachea grafts, OAD develops both in isografts and in allografts. On the other hand, development of OAD is effectively prevented if epithelium is allowed to regrow quickly in isografts seeded with epithelial cells. These experiments show that early recovery of epithelium can prevent OAD even in situations with severe epithelial injury at the time of transplantation.

Intact epithelium is likely to play a regulatory role on fibroproliferative responses in the trachea transplants. We presume that under normal circumstances, like in the isografted tracheas, fibroblast growth is suppressed by the luminal epithelium layer, releasing regulating cytokines and growth factors. This is confirmed by studies showing that myofibroblasts start to proliferate only after damage or inadequate function of the airway epithelium, both in vivo (23) and in vitro (24-26). In vitro studies (27,28) have shown that cytokines produced by epithelial cells, such as prostaglandin E2 (29) and transforming growth factor beta (28), are successful inhibitors of fibroproliferation. In the absence of epithelial cells in the rejected or denuded tracheas, myofibroblasts will have the opportunity to grow into the lumen with unregulated growth and to obliterate the lumen as a consequence.

Our observation that seeding of viable epithelial cells into the lumen of tracheas can prevent OAD corroborates the findings of the group of Morris. They showed successful engraftment without OAD when epithelium was transplanted into trachea isografts from which the epithelium was partly digested with protease (15). In that study, however, it could not be excluded that the prevention of OAD was due to early regeneration of the epithelial cells in the trachea since the protease-treated tracheas showed epithelial cells at the time of transplantation. This is in accordance with our incomplete denudation group in which we found that the epithelium recovered quickly after grafting without significant obliteration. In an elegant model (30,31) using trachea allografts flanked with segments of trachea isografts, it was shown that the epithelium in the rejecting tracheas was regenerated by cells growing

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38

from the adjacent isografts and that the lumen remained patent. From these very different studies we conclude that the replacement of epithelium by well-functioning epithelial cells can help to prevent airway obliteration.

This trachea transplantation study may be of relevance for clinical lung transplantation, although the development of OB in patients is influenced by many more noxious factors such as infection (32) and HLA specific antibodies (34),. Yet, we think that also in patients the integrity of the airway epithelium is an essential component of adequate prevention of OB. The induction of OB is clearly linked to episodes of acute rejection (2) which was most detrimental for epithelial integrity in our study. Besides by rejection, OB can be induced by many more factors. From our experiments with denuded and epithelial-cell seeded trachea isografts it can be concluded that it is essential to preserve or repair the epithelial integrity in order to prevent OAD. At this moment it is hard to envision how seeding of epithelial cells could be performed in recipients of lung transplants, although the use of recipient stem cells might be a future perspective for development of a similar treatment. A more plausible approach at present to prevent OB development would be the protection of airway epithelial cells by activation of 'protective genes' like HO-1 (35), which might be capable to reduce local inflammation at the epithelial level in case of allograft reactivity. This might be achieved by exposure of donor lung to carbon monoxide, a treatment that was effective in a rat lung transplantation model (36). The efficacy and possible side effects of such an approach for prevention of OB after human lung transplantation have yet to be investigated.


39

Acknowledgement The authors want to thank Gineke Drok and Hans Vos for their help with histological staining, reading and scoring and Arjen Petersen for the help with the animal surgery.

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Reference

1. Hertz MI, Taylor DO, Trulock EP et al. The registry of the international society for heart and lung transplantation: nineteenth official report-2002. J Heart Lung Transplant 2002:21: 950-970.


3. Verleden GM. Bronchiolitis obliterans syndrome after lung transplantation: medical treatment. Monaldi Arch Chest Dis 2000:55: 140-145.






9. Takao M, Gu Y, Shimamoto A, Adachi K, Namikawa S, Yada I. Administration of exogenous interleukin-2 enhances obliterative airway disease in cyclosporine-treated rats following tracheal allografts. Transplant Proc 1999:31: 180-181.

10. Yamada A, Konishi K, Cruz GL et al. Blocking the CD28-B7 T-cell costimulatory pathway abrogates the development of obliterative bronchiolitis in a murine heterotopic airway model. Transplantation 2000:69: 743-749.

11. Yonan NA, Bishop P, el Gamel A, Hutchinson IV. Tracheal allograft transplantation in rats: the role of immunosuppressive agents in development of obliterative airway disease. Transplant Proc 1998:30: 2207-2209.

12. Adams BF, Berry GJ, Huang X, Shorthouse R, Brazelton T, Morris RE. Immunosuppressive therapies for the prevention and treatment of


41

obliterative airway disease in heterotopic rat trachea allografts. Transplantation 2000:69: 2260-2266.

13. King MB, Pedtke AC, Levrey-Hadden HL, Hertz MI. Obliterative airway disease progresses in heterotopic airway allografts without persistent alloimmune stimulus. Transplantation 2002:74: 557-562.

14. Haider Y, Yonan N, Mogulkoc N, Carroll KB, Egan JJ. Bronchiolitis obliterans syndrome in single lung transplant recipients--patients with emphysema versus patients with idiopathic pulmonary fibrosis. J Heart Lung Transplant 2002:21: 327-333.

15. Bui JD, Despotis GD, Trulock EP, Patterson GA, Goodnough LT. Fatal thrombosis after administration of activated prothrombin complex concentrates in a patient supported by extracorporeal membrane oxygenation who had received activated recombinant factor VII. J Thorac Cardiovasc Surg 2002:124: 852-854.

16. Hertz MI, Henke CA, Nakhleh RE et al. Obliterative bronchiolitis after lung transplantation: a fibroproliferative disorder associated with platelet-derived growth factor. Proc Natl Acad Sci U S A 1992:89: 10385-10389.

17. Kaartinen L, Nettesheim P, Adler KB, Randell SH. Rat tracheal epithelial cell differentiation in vitro. In Vitro Cell Dev Biol Anim 1993:29A: 481-492.




21. Aris RM, Walsh S, Chalermskulrat W, Hathwar V, Neuringer IP. Growth factor upregulation during obliterative bronchiolitis in the mouse model. Am J Respir Crit Care Med 2002:166: 417-422.

22. Brazelton TR, Adams BA, Cheung AC, Morris RE. Progression of obliterative airway disease occurs despite the removal of immune reactivity by retransplantation. Transplant Proc 1997:29: 2613.

23. Luce JM. Acute lung injury and the acute respiratory distress syndrome. Crit Care Med 1998:26: 369-376.

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24. Crouch E. Pathobiology of pulmonary fibrosis. Am J Physiol 1990:259: L159-L184.

25. Witschi H. Responses of the lung to toxic injury. Environ Health Perspect 1990:85: 5-13.

26. Dagle GE, Sanders CL. Radionuclide injury to the lung. Environ Health Perspect 1984:55: 129-137.

27. Young L, Adamson IY. Epithelial-fibroblast interactions in bleomycin-induced lung injury and repair. Environ Health Perspect 1993:101: 56-61.



30. Ikonen TS, Romanska HM, Bishop AE, Berry GJ, Polak JM, Morris RE. Alterations in inducible nitric oxide synthase (iNOS) and nitrotyrosine (NitroY) during re-epithelialization of heterotopic rat tracheal composite grafts. Transplant Proc 1999:31: 182.

31. Ikonen T, Briffa N, Brazelton T, Shorthous R, Berry G, Morris R. Prevention of Obliterative Airway Disease (OAD) in heterotopic rat tracheal allografts without immunosuppression: Anastomosis of recipient trachea to donor trachea enables re-epithelialization of allografts by recipient epithelium. J Heart Lung Transplantation 1998:17.

32. Carreno MC, Ussetti P, Varela A et al. [Infections in lung transplantation]. Arch Bronconeumol 1996:32: 442-446.

33. Husain AN, Siddiqui MT, Holmes EW et al. Analysis of risk factors for the development of bronchiolitis obliterans syndrome. Am J Respir Crit Care Med 1999:159: 829-833.

34. Jaramillo A, Smith MA, Phelan D et al. Development of ELISA-detected anti-HLA antibodies precedes the development of bronchiolitis obliterans syndrome and correlates with progressive decline in pulmonary function after lung transplantation. Transplantation 1999:67: 1155-1161.

35. Visner GA, Lu F, Zhou H, Latham C, Agarwal A, Zander DS. Graft protective effects of heme oxygenase 1 in mouse tracheal transplant-related obliterative bronchiolitis. Transplantation 2003:76: 650-656.

36. Song R, Kubo M, Morse D et al. Carbon monoxide induces cytoprotection in rat orthotopic lung transplantation via anti-inflammatory and anti-apoptotic effects. Am J Pathol 2003:163: 231-242.

Chapter 3

Specific Immune Responses against Airway Epithelial Cells in a Transgenic Mouse Trachea Transplantation Model

for Obliterative Airway Disease

Ning Qu; Aalzen de Haan; Martin C. Harmsen; Frans G.M. Kroese Lou F.M.H. de Leij; Jochum Prop

Transplantation. 2003 Oct 15;76(7):1022-8.

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44

Specific immune responses in transgenic mouse trachea transplantation

45

Abstract Background Immune injury to airway epithelium is suggested to play a central role in the pathogenesis of obliterative bronchiolitis (OB) after clinical lung transplantation. In several studies a rejection model of murine trachea transplants is used resulting in obliterative airway disease (OAD) with similarities to human OB. To focus on the role of an immune response specifically against airway epithelium, we transplanted tracheas from transgenic mice expressing human epithelial glycoprotein-2 (hEGP-2) on epithelial cells. We hypothesised that the immune response against the hEGP-2 antigen would result in OAD in the trachea transplants. Methods Tracheas from hEGP-2 transgenic and control non-transgenic FVB/N mice were heterotopically transplanted into FVB/N mice and harvested at week 1, 3, 6, and 9. Anti-hEGP-2 antibodies were determined in the recipient blood. The trachea grafts were analyzed for cellular infiltration, epithelial cell injury, and luminal obliteration. Results Recipients of transgenic tracheal grafts gradually developed anti-hEGP-2 antibodies. In the transgenic grafts, the submucosa was infiltrated predominantly by CD4+ T cells. Epithelial cells remained present but showed progressive abnormality. The tracheal lumen showed a mild degree of obliteration. All these changes were absent in non-transgenic FVB/N trachea transplants. Conclusions The hEGP-2 antigen on the epithelial cells of transgenic trachea transplants induces specific humoral and cellular immune responses leading to a mild form of OAD. It provides a suitable model for further investigation of the role of epithelial cells in the development of OAD in animals and OB in human lung transplantation.

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Introduction Obliterative bronchiolitis (OB), one of the most severe complications after lung transplantation, affects 50% of all lung transplant recipients and results in high mortality within 5 years (1,2). Several risk factors have been identified for the development of OB, amongst which episodes of acute rejection are the most prominent. Yet, the pathogenesis of OB remains unclear. As an animal model allowing investigation of human OB after lung transplantation, Hertz and colleagues heterotopically transplanted murine tracheas (3). They found that allogeneic tracheas implanted subcutaneously were rejected with massive cellular infiltration and epithelium loss as opposed to isografts which did not show these changes. By day 21, this lead to obliteration of the lumen of the transplanted trachea by fibroproliferation. This process is called the obliterative airway disease (OAD), and it shares similarities to human OB. Further studies showed that the rejection of tracheal allografts was mediated by CD4+ and CD8+ T lymphocytes, macrophages and granulocytes, preceding epithelium loss and complete luminal obliteration at day 21 (4-6). In addition to the T cell-driven responses, humoral responses were also found to be involved in OAD (2,7) and the blockade of the complement system could either prevent or inhibit the development of OAD (8). Clinical histopathologic features of OB suggest that in particular the injury of epithelial cells result from persistent inflammation in small airways leads to ineffective epithelial regeneration and excessive fibroproliferation due to aberrant tissue repair (9). Also, in animal tracheal transplants, the epithelium is involved in the rejection process leading to OAD (5,10). The effect of airway epithelium injury was investigated by the group of Morris in a rat trachea OAD model. They demonstrated that the loss of airway epithelium either by enzymic removal in isografts, or by transplant rejection in allografts resulted in OAD. The re-seeding of epithelial cells in the isografts (11) or re-growth in allografts (12) largely reduced the level of OAD. These findings suggested that the airway epithelium regulates fibroblast growth and thus protects against fibrotic obliteration of the tracheal transplants. Other studies showed strong sub-mucosal infiltration of immune cells (13), and loss of epithelium preceding the obliteration of the trachea grafts (10,14) supporting that the regulating role of the epithelial cells on fibroblast proliferation may be lost as the result of immune injury.

A limitation for the investigation of epithelium specific injury in all studies on tracheal allografts is that the alloreactive immune response is not


47

specifically directed against epithelial cells but against all cell types of the graft tissue, as all the cells express alloantigens. Therefore, it is possible that immune reactivity against cell types other than epithelial cells, for example fibroblasts, also contributes to tissue injury and subsequent fibroproliferation of the trachea. Transgenic animals expressing neoantigens on specific tissues or cell types may provide new options to investigate the role of epithelium in OAD. In this study, we used transgenic FVB/N mice as donors of tracheas for heterotopic transplantation into non-transgenic syngeneic mice. In these mice, human epithelial glycoprotein-2 (hEGP-2) is expressed specifically on epithelial cells, including trachea epithelial cells, driven by the endogenous specific promoter (15). We hypothesized that the epithelium specific hEGP-2 neoantigen induces an epithelial specific immune responses upon trachea transplantation and the responses cause epithelial injury and the subsequent development of OAD. Material and methods Experimental design Tracheas were transplanted from hEGP-2 transgenic FVB/N mice to non-transgenic FVB/N mice subcutaneously (s.c.) (n=27). As controls, tracheas from non-transgenic FVB/N mice were transplanted s.c. to non-transgenic FVB/N recipients (n=21). Also, as allogeneic controls, MHC fully mismatched C57BL/6 (H-2b) mice tracheas were s.c. transplanted to non-transgenic FVB/N (H-2q) recipients (n=18). About 3 to 11 grafts from each group at each time point were harvested at 1, 3, 6 and 9 weeks after transplantation and were studied for histology by haematoxylin and eosin (H&E) staining and for analyzing cellular infiltration by immunohistochemistry. Serum samples from recipient mice were collected and levels of antibodies against EGP-2 antigen and alloantigens were determined by ELISA and flow cytometry. Experimental animals Inbred FVB/N mice transgenic for the hEGP-2 were bred and housed in the Central Animal Facility of Groningen University according to the standard breeding rules for transgenic animals in a conventional condition. Normal FVB/N and C57BL/6 inbred mice were purchased from Harlan (Harlan Zeist,

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The Netherlands). Animals used in the experiments were between 8-10 weeks of lifetime. All animals received care in compliance with the Dutch regulations and laws. Experimental protocols were approved by the institutional animal ethical review committee. Heterotopic trachea transplantation

Donor mice (hEGP-2 transgenic and non-transgenic FVB/N or C57BL/6 mice) were anesthetized with halothane and N2O/O2 gas. The mice were then euthanized by abdominal aorta bleeding. Tracheas from these mice were exposed through the anterior midline incision and esophagus, vessels and other surrounding tissues were gently separated. After isolation, the tracheas were transected below the thyroid cartilage and above the bifurcation. Tracheas were immediately put into cold saline. The tracheal lumen was washed three times with 300µl cold saline by 1ml syringe to remove blood and other fluid inside the lumen. To avoid bending of the trachea after implantation, an orthodontic stainless steel wire (0.036", GAC, New York, USA) was prepared to the same length of the trachea. Both ends of the wire were folded back and made blunt. Tracheas were clipped along with the prepared wires by stainless steel surgical clips (Ligation Clip 316L, Ethicon Endo-Surg. Inc. Cincinnati, OH 45242-2839, USA) at both ends.

The non-transgenic FVB/N recipients were anesthetized in the same way as the donor mice. After shaving, a small incision was made at the lateral side of the back. By blunt dissection, a subcutaneous pouch was made, the clipped trachea was put into the pouch and the wound was closed by one stitch (6-0 Prolene suture). Each recipient received one trachea graft.

At designated time points the trachea grafts were explanted through an incision immediately outside the grafts tissue. After separating the wires and cutting off the clips, each trachea was cut into two segments: one half was fixed in 10% formalin for H&E staining and the other half was snap frozen in liquid nitrogen for immunohistochemical staining.

ELISA assay for anti-hEGP-2 antibody detection Whole blood samples were collected at different time points after transplantation. Serum hEGP-2 specific antibody levels were detected by ELISA. Briefly, 96-well ELISA plates


49

were coated overnight with 100 µl/well recombinant soluble hEGP-2 protein (300ng/ml). Serum samples (100µl/well from each sample) in two-fold dilutions incubated in the coated plates for 1.5h at 37oC. Purified mouse-anti-hEGP-2 mAb Moc31 (Purified from hybridoma culture supernatant) (15) at a concentration of 3.5µg/ml was used as the standard. Serum samples from non-transgenic graft recipients were used as negative control. Plates were then washed with PBS/Tween-20 solution and rabbit-anti-mouse IgG peroxidase labeled antibodies (Dako, UK) was applied and incubated for 1h at 37oC. After washing, TBM peroxidase substrate solution was added and plates were stained for 15 min on a shaking bed. The staining reaction was stopped by adding 1M H2SO4 100µl/well and the plates were read on a microplate reader (Emax, Molecular Device.) at 450 nm. Data were analyzed by reader’s software (SoftmaxPro version 1.2.0) and the EGP-2 specific antibodies level related to Moc31 level was calculated. Flow cytometry assay for alloantibody detection Alloantibodies directed against donor (C57BL/6) alloantigens were measured in serum from allograft recipients (FVB/N) by analyzing the amount of alloantibodies bound to donor derived spleen T cells by flow cytometry. Serum samples from non-transplanted control mice (normal FVB/N) served as negative control. Serum was de-complemented by incubation at 56oC for 30 min. Serum samples were serially diluted (10 fold) with PBS/FCS (PBS plus 1% FCS) from 1:50 till 1:500,000. Splenocytes from the donor strain (C57BL/6 ) that express alloantigens were harvested and stained with anti-CD3-PE (1:50, Human-anti-Mouse PE labeled, BD Pharmingen) to identify T cells. These cells lack Fc receptors, and as a consequence, do not bind antibodies non-specifically. Splenocytes were then incubated with the diluted serum samples for 20 min on ice, washed and stained with FITC labeled goat-anti-mouse IgG antibodies (1:100, BD Pharmingen) for 20 min on ice. After another wash, double stained cells (CD3-PE and FITC labeled cells with bound alloantibodies) were detected by flow cytometry (FacsCaliber flow cytometer, Becton Dickinson). After gating on CD3+ T cells, the geometric Mean Fluorescent Intensity (MFI) of the FITC signal was taken. Data was analyzed with WinList software (5.0 version, Verity Company). Light microscopy and analysis

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Formalin fixed trachea segments were paraffin embedded. Serial cross sections (3µm thick) were cut at 3 levels 50µm apart of each trachea segment, and 3 sections at each level were stained by normal H&E method and evaluated at 50 to 400 magnifications. To analyze the infiltration level of the trachea grafts, a semi-quantitive scoring system was used with the following five grades: 0-no infiltration, 1-minimal infiltration: scattered or diffuse cellular infiltrates, 2-mild infiltration: diffuse cellular infiltrates with one area dense infiltrate, 3-moderate infiltration: more than one area infiltrates, 4-severe infiltration: a thick layer of dense cellular infiltrates.

To analyze the obliteration degree of the trachea lumen, the thickness of submucosa (between epithelium and cartilage) was scored using following five grades: 0-no thickening of submucosa, 1-minimal thickening: focal submucosa thickening, 2-mild thickening: submucosa thickening equal to the thickness of the cartilage ring, 3-moderate thickening, submucosa thickening result in a small lumen, 4-severe thickening: total lumen obliteration.

The epithelium was categorized in two types: normal and abnormal based on morphological aspects of epithelial cells, i.e. the differentiation (the presence of ciliated epithelium) and the pseudo-stratification (the presence of pseudostratified epithelium layer). The coverage of the trachea by normal epithelium and abnormal epithelium were scored in percentage.

Immunohistochemistry Frozen tracheas were cut into 5µm thick cross sections, and were immunohistochemically stained by monoclonal antibodies for different markers (4). Briefly, the sections were fixed in acetone for 10 min and then washed 3 times in 0.01M phosphate-buffered saline (PBS). Primary rat antibodies specific for mouse CD5, CD4, CD8 T cells and macrophages [Primary antibodies directed against mouse CD5 (Ly-1), CD4 (L3T4), and CD11b (mac-1, for macrophages) were from hybridoma culture supernatants, and purified antibodies specific for mouse CD8α (Ly-2) were from BD Pharmingen, San Diego, USA] were applied and incubated for 1h (hybridomas in culture supernatants were not diluted, CD8α was diluted 1:50). The slides were then washed again with PBS. Secondary antibodies (rabbit-anti-rat peroxidase labeled, 1:50 dilution, Dako, UK) plus 3% normal FVB/N mouse serum were applied and incubated for another 1h. After washing in PBS, the slides were colored with AEC (Sigma, 0.5mg in 3.75 ml DMF plus 70ml acetate buffer, pH =4.9). Endogenous peroxidase was blocked by adding 0.03% H2O2 in the


51

AEC solution. Slides were counterstained with Mayer’s haemotoxilin (1:10). Additionally, epithelium was stained by keratin-specific antibody RGE53 (Eurogentec, Parc scientifique du Sart Tilman , 4102 Sering, BE) for epithelium lumen coverage evaluation on 3 selected trachea segments from each time point. The hEGP-2 expression in the transgenic grafts was examined by the anti-hEGP-2 specific monoclonal antibody Moc31 (15).

Trachea infiltrating cell phenotypes were analyzed both inside and outside trachea ring in three sections from each trachea segment (stained by monoclonal antibodies for CD5+, CD4+, CD8+, T cells and macrophage). The positive cells in the grafts were scored, using semi-quantification system with five grades: 0-no positive cells, 1-scattered positive cells, 2-small clusters or one area with dense positive cells, 3-more than one area or a large area plus clusters of positive cells. 4-whole tissue dense positive cells. Additionally, from 4 areas each section, CD4+ and CD8+ T cells and macrophages in the same tissue area per microscopic view were counted (400 magnifications) and CD4+/ CD8+ ratios were calculated. All the histology scoring and counting were done by two independent observers. Results Immunogenicity of hEGP-2 transgenic trachea grafts We used hEGP-2 transgenic trachea grafts as a model to study the role of epithelial injury in the development of OAD. We explored whether the transplantation of hEGP-2 transgenic grafts induce epithelial cell specific immune responses by analyzing anti-hEGP-2 antibodies responses and cellular infiltrations. Antibodies Anti-hEGP-2 antibodies became detectable in the recipients recieved hEGP-2 tracheas at 3 weeks after transplantation and levels increased sharply between 3 and 6 weeks. (Figure 1A). No anti-hEGP-2 antibodies were detected in the recipients received non-transgenic grafts. This indicated that a hEGP-2 antigen specific humoral response was induced by grafting the hEGP-2 transgenic tracheas. As control, we examined alloantibodies development in C57BL/6 allograft recipients, alloantibodies were present already at 3 weeks and continue to increase at 6 and 9 weeks. (Figure 1B).

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Figure 1. A. hEGP-2 Antibodies

control 3w 6w 9w0

2500

5000

7500

10000

week after transplantation

conc

entra

tionµ g

/ml

B. Alloantibodies

control 3w 6w 9w0

50

100

150

200


mea

n flu

ores

cent

inte

nsity

Figure 1. Antibody dynamics in recipients of hEGP-2 transgenic and C57BL/6 allogeneic trachea transplants. Anti-hEGP-2 antibodies were detectable in the recipients of hEGP-2 transgenic grafts at 3 weeks after transplantation, and reached high concentrations at 6 and 9 weeks (A). Alloantibodies were present at 3 weeks and continued to increase at 6 and 9 weeks (B). Values represent mean ± standard deviations of four to five animals.

Infiltration Cellular infiltrations were observed in the hEGP-2 tracheas mainly in the submucosa inside the trachea ring as demonstrated by H&E staining (Figure 2). The grading of the infiltration increased from minimal to mild cellular


53

infiltration inside trachea ring at 1 week to mild to moderate level at 3, 6, 9 week after transplantation (Table 1). The infiltration outside the trachea was minimal and there was no clear increase with time. Analysis of the cellular infiltration by immunohistochemistry showed that CD5+, CD4+ and CD8+ T cells appeared at 1 week post-transplantation reached a plateau at 3, 6 weeks and decreased at 9 weeks inside the trachea rings (Table 2). CD4+ T cells were dominant in numbers compared to CD8+ T cells at the same infiltrating tissue area, with CD4+/ CD8+ ratio between 2.7 to 3.1 at different time points (Table 3). Fewer T cells were observed outside the trachea ring and their numbers did not increase with time. This was accordant to the H&E stained findings. Macrophages appeared in the hEGP-2 grafts at 1 week, peaked at 3 and 6 weeks, started to decrease at 9 weeks (Table 3). In control non-transgenic FVB/N grafts, only minimal infiltration was found inside and outside

Figure 2.

Figure 2. Histology of hEGP-2 transgenic, non-transgenic FVB/N and allogeneic C57BL/6

tracheas at 3 weeks after transplantation. Transgenic grafts showed moderate cellular

infiltrations in the submucosa. Few infiltrating cells were observed outside the trachea

cartilage ring (A). The epithelium was partly flattened (D) and luminal obliteration was mild

(A). Non-transgenic grafts showed minimal cellular infiltrations inside and outside the tracheal

cartilage ring (B). Well differentiated normal epithelium was observed (E), and no obliteration

of the lumen (B). Allogeneic grafts showed minimal cellular infiltrations both inside and

outside trachea cartilage ring (C). No epithelium was left and the lumen was totally obliterated

(F,C).

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Table 1. Cellular infiltrationa inside and outside trachea cartilage rings (H&E)

EGP-2 grafts FVB/N grafts C57BL/6 grafts infiltration infiltration infiltration

n Median(Range)c n Median(Range)c n Median(Range)c Inb 1w 5 1.25 (1-2) 5 1 (0-2) 3 2 (1-2.5) 3w 11 2.5 (1-4) 9 1 (0-1) 8 1.1 (1-2.5) 6w 7 2.75 (1-4) 4 1 (1-1) 5 0.75 (0.5-1) 9w 3 2.5 (1.5-3) 2 1 (0.5-1.5) 2 0 (0-0) Outc 1w 5 1 (0-1) 5 1.5 (0-2) 3 2.5 (1-3) 3w 11 1 (0-2.5) 9 0 (0-0.5) 8 2 (1-2.5) 6w 6 0.5 (0-1) 4 0 (0-2) 5 0.9 (0.5-2.5) 9w 2 1 (0.5-1.5) 2 0 (0-0) 2 0.5 (0.5-0.5)

aSoring standard: 0-no infiltration, 1-minimal infiltration: scattered or diffuse cellular infiltrates, 2-mild infiltration: diffuse cellular infiltrates with one area dense infiltrate, 3-moderate infiltration: more than one area infiltrates, 4-severe infiltration: a thick layer of dense cellular infiltrates; binside trachea cartilage ring; coutside trachea cartilage ring; the trachea rings, and this did not increase with time (Table 1) supporting that the response against transgenic grafts was hEGP-2 specific and was not caused by transplantation injury.

In the allograft controls, an acute cellular infiltration was observed with a mild to moderate cellular infiltration at 1 week, decreased thereafter and disappeared by 9 weeks (Table 1). Interestingly, the localization of the infiltration in allografts differed from that in the hEGP-2 transgenic grafts: in the allografts, the infiltration was more observed outside trachea while in the hEGP-2 grafts the infiltration was mainly observed within the submucosa inside the trachea (Table 1). Also, CD5+, CD4+ and CD8+ cellular infiltration was predominantly localized outside of the trachea ring at 1, 3, 6 weeks (Table 2) in the allografts. In contrast to the transgenic grafts, CD4+ T cells in the allografts were outnumbered by CD8+ T cells at 1 and 3 weeks with a CD4+/CD8+ ratio <0.6 (Table 3). Macrophages were observed in high numbers at 1, 3, 6 weeks after transplantation (Table 3).


55

Table 2. Phenotypes of T cells and infiltration levelsa inside and outside trachea (IHC)

C57BL/6 grafts

CD5 CD4 CD8

n Median(Range) Median(Range) Median(Range)

1w 4 0.5 (0-1) 0.8 (0-1.5) 0 (0-1) 3w 5 2.25 (1-2.5) 1 (0-2) 1.5 (1-2) 6w 5 1 (0-1) 0.5 (0-2.5) 0 (0-1)

in

9w 2 0 (0-0) 0.5 (0-1) 0.8 (0.5-1) 1w 3 2 (2-3) 2.5 (1-2.5) 2 (1-2.5) 3w 5 1.5 (0-2.5) 1 (0-2.5) 1.5 (1-3) 6w 5 2 (1-2) 1.5 (0-2) 0.5 (0-1)

outc

9w 1 0 (-) - 0.5 (-)

aScoring standard: 0-no positive cells, 1-scattered positive cells, 2-small clusters or one area with dense positive cells, 3-more than one area or a large area plus clusters of positive cells. 4-whole tissue dense positive cells; binside trachea cartilage ring; coutside trachea cartilage ring; Table 3. CD4/CD8 ratios and macrophage numbers EGP-2 grafts (n=5), mean C57BL/6 grafts (n=4), mean week CD4 CD8 CD4/CD8a MΦ CD4 CD8 CD4/CD8a MΦ 1w 34.2 12.8 2.7 51.8 41 100.8 0.41 69.4 3w 186.7 61.75 3 191.5 28.25 47.3 0.60 158 6w 162 51.5 3.1 194.5 75 36 2.1 83 9w 39 13.5 2.8 74 - - - -

aThe ratios were calculated based on the positive cell numbers found in the same tissue area positive for CD4 and CD8 T cells infiltrations;

EGP-2 grafts CD5 CD4 CD8

n Median(Range) Median(Range) Median(Range)

1w 5 1.5 (0-2) 2 (1-3) 0 ( 0-1) 3w 6 2 (1-3) 3 (2-3.5) 1 (0.5-1) 6w 5 2 (0-3) 3 (1-3) 1 (0-1)

inb

9w 3 1 (1-2) 1.5 (1-2) 0.5 (0-1) 1w 5 1 (0-2) 0.5 (0.5-1) 0.5 (0-1) 3w 3 1 (0-2.5) 2 (1-2) 0.5 (0-1) 6w 5 1 (0-1) 0.5 (0-3) 0 (0-2.5)

outc

9w 2 1.5 (1-2) 2 (1-2) 0 (0-1.5)

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Epithelial cells abnormality and luminal obliteration

After transplantation, the epithelium of hEGP-2 transgenic grafts was present, but it was partially abnormal: a thin layer of flattened non-ciliated cells (Figure 3A). This remained unchanged up to 6 weeks. At 9 weeks the percentage of abnormal epithelial cells increased significantly while the percentage of epithelium coverage decreased slightly, indicating a progressing epithelial injury (Figure 3A). Expression of hEGP-2 in the transgenic graft epithelium was checked by hEGP-2 specific Moc31 antibodies and both normal and abnormal epithelium expressed hEGP-2 at all time points. The keratin specific staining indicated that the cells lining the lumen after transplantation in the grafts were of epithelial origin (data not shown). Obliteration grading showed a slight thickening of the submucosa from minimal at 1 week to mild level at 9 week (Figure 3B).

In non-transgenic control trachea grafts, the epithelium showed a well ciliated and pseudostratified appearance (Figure 3A). In parallel with the normal epithelium, no obliteration of the lumen was detectable in non-transgenic trachea grafts (Figure 3B). In the allograft controls, the loss of epithelium and significant epithelium abnormality were already observed at 1 week post-transplantation (Figure 3A). The epithelium was totally absent and the lumen was occluded from 3 week and onwards. (Figure 3A,B).


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Figure 3. A. Epithelium coverage and abnormality

1 3 6 9 1 3 6 9 1 3 6 90

50

100ep-coverage

abnormal

EGP2 C57BL/6FVB/N


trach

ea lu

men

epi

thel

ium

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B. Luminal obliteration

0

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5

1 3 6 9 1 3 6 9 1 3 6 9


EGP-2 FVB/N C57BL/6

lum

inal

obl

itera

tion

scor

e(0

-4)

Figure 3. Epithelium coverage, abnormality and luminal obliteration of trachea transplants. Epithelium was present almost 100% in hEGP-2 transgenic grafts. The appearance of the epithelium was less than 50% abnormal during the first 6 weeks. At 9 weeks, most of the epithelium has become abnormal (A). Obliteration grading in hEGP-2 grafts showed the thickening of the submucosa from minimal level at 1 week to mild level at 9 week (B). In the FVB/N trachea grafts, the epithelium showed normal, well ciliated and pseudostratified appearance (A) and no obvious obliteration of the lumen was observed (B). In the C57BL/6

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allografted tracheas, the loss of epithelium was already observed at 1 week (A) and the remaining epithelium was abnormal. The tracheal lumen was occluded within 3 week (B). Error bars in Figure 3A indicated the standard error of the mean of three to five experiments. Values in Figure 3B represent the median and range of three to five experiments. Discussion In this study we transplanted tracheas from transgenic FVB/N mice, in which expression of the hEGP-2 neoantigen is restricted to epithelial cells. The tracheas were transplanted to non-transgenic FVB/N mice to study the role of immune injury to airway epithelial cells in the development of OAD. Our data show a strong immune response against epithelial hEGP-2 antigens, as demonstrated by high levels of hEGP-2 specific antibodies and heavy cellular infiltrations, leading to epithelial cell abnormality and partial luminal obliteration in the transplantated tracheas.

The presence of anti-hEGP-2 antibodies prove that antigens expressed on tracheal epithelial cells can induce an epithelial specific immune response. The potential role of the antibodies in the development of OAD depends on their functional capabilities. Therefore, we performed a pilot study testing the cytotoxicity of hEGP-2 antibody positive serum in a complement-dependent cell cytotoxicity assay. We found that the hEGP-2 Ab positive serum (dilution 1: 10) was capable to lyse up to 50% of a hEGP-2 expressing cell line (data not shown). A control hEGP-2 negative cell line was not lysed under the same conditions supports that the antibodies induced by the transgenic trachea transplant have cytotoxic-capabilities. Comparing the antibody responses after transgenic and allogeneic trachea transplantation, we noticed that the antibody level increased more sharply in the hEGP-2 transgenic recipients during 3 to 6 weeks (Figure 1A,B) while alloantibody levels showed a steady increase. This may indicates a late-coming development of hEGP-2 antibodies. Despite a later appearance of anti-hEGP-2 antibodies, their cytotoxic characteristics may contribute to the graft injury, as in clinical studies where the development of alloantibodies correlated to the occurrence of OB (16-18).

In parallel, the pattern of cellular infiltration in the hEGP-2 transgenic tracheas is consistent with a specific immune response against epithelial cells: the infiltrates are mainly located within the submucosa of the trachea near the hEGP-2 antigen expressing epithelium. It can be excluded that this infiltration is caused by non-specific factors related to the transplantation procedure


59

because the infiltration was absent in the control non-transgenic trachea transplants (Table 1). Furthermore, the infiltration in allografts is more diffusely spread throughout the tracheal tissue, suggesting targeting towards the alloantigens expressed on all cells. It is remarkable that the proportion of CD8+ was low within the infiltrating cells in the transgenic transplants. This phenomenon was particularly obvious when comparing the CD4+/CD8+ ratios in the transgenic tracheas with those in the allogeneic tracheas, where our CD4+/CD8+ ratios were in agreement with values described in the literature (4). The low proportion of CD8+ T cells in the hEGP-2 transgenic tracheas may be due to the different pathway of recognition of the hEGP-2 neoantigen compared to alloantigens. In allograft rejection, it is well documented that significant numbers of alloreact CD4+ and CD8+ T cells pre-exist in recipients are able to directly recognize alloantigens (19). In our transgenic situation, such pre-existing antigen-reactive T cells directly recognizing the hEGP-2 antigen are lacking. Thus, CD4+ and CD8+ T cells in the transgenic situation need to be activated de novo via an indirect antigen recognition pathway, involving processing of exogenous and endogenous neoantigen and presentation of antigen-derived peptides in MHC class II and I molecules, respectively. MHC class II-restricted antigen presentation of exogenously derived antigen, such as hEGP-2 from necrotic or apoptotic epithelial cells most likely depends on presentation of the hEGP-2 by professional antigen presenting cells (APCs) and activate mainly CD4+ T cells. MHC class I-restricted antigen presentation of endogenous hEGP-2 is likely to depend on hEGP-2 expressing epithelial cells. Being 'non-professional' APC, these cells may not have the capacity of inducing an MHC class I-restricted CD8 T cell response. This may explain the predominance of CD4+ T cells amongst the infiltrating cells in the transgenic trachea transplants. Although it has also been shown that both CD4+ and CD8+ T cells could contribute to the development of OAD (20,21) the low proportion of CD8+ T cells in transgenic grafts may indicate that a cytotoxic T cell response is weak.

The development of OAD with epithelial injury and luminal obliteration in the transgenic transplants is in line with the moderate epithelial cells specific immune responses that we found in the tracheas. The epithelium of transgenic tracheas exhibited a significant degree of abnormality, but this became apparent as late as 9 weeks after transplantation (Figure 3A). In addition, the epithelial cells were still of donor origin as they continued to express the hEGP-2 antigen after transplantation. This is in contrast to the allogeneic tracheas, where epithelial cells were largely abnormal at 1 week

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and were virtually absent by 3 weeks after transplantation. The obliteration of the tracheal lumen inversely correlated with the presence of epithelium: only mild obliteration in the transgenic tracheas and complete obliteration in the allogeneic tracheas. Several studies have shown that airway epithelial cell can inhibit proliferation of fibroblast, both in vitro (22,23) and in vivo (24,25). In rat trachea transplants, epithelial cells were capable to inhibit luminal obliteration after transplantation. This was shown in trachea transplants that were made devoid of epithelium by exogenous methods (11). Upon transplantation these tracheas obliterated quickly, unless epithelium was allowed to re-grow either from adjacent recipient tissue (12) or from co-transplanted donor tissue (11). The mild obliteration in our transgenic tracheas, however, does not seem to result mainly from fibroproliferation but rather reflect the volume of infiltrating cells in the submucosa. This is consistent with the possible inhibiting role of the presence of partial abnormal epithelium in the lumen on fibroproliferation.

We have shown that transplantation of hEGP-2 transgenic tracheas induces an epithelial cell specific immune response composed of both antibodies and T cell reactivity. This response causes epithelium abnormality and partial airway obliteration. In future studies the epithelial cell specific immune response can be intensified, because hEGP-2 antibodies appeared late and CD8+ T cells proportion remained low in the submucosal infiltrates and the enhancement could be done by pre-transplant induction of anti-hEGP-2 antibodies and hEGP-2 specific cytotoxic T cells through immunization with hEGP-2. We conclude that the transplantation of hEGP-2 transgenic tracheas provides a suitable model for further investigation of the role of epithelial cells in the development of OAD in animals and OB in lung transplanted patients. Acknowledgement The authors thank Henk van de Molen for the assistance in the animal experiments; Dr. Wijnand Helfrich for supplying the purified hEGP-2 protein and Dr. Yijin Ren, from Orthodontic Department of University Medical Center of Nijmegen, The Netherlands for helping with the orthodontic wires.


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Reference 1 Hertz MI, Taylor DO, Trulock EP et al. The registry of the international

society for heart and lung transplantation: nineteenth official report-2002. J Heart Lung Transplant 2002:21: 950-970.

2 Estenne M, Hertz MI. Bronchiolitis obliterans after human lung transplantation. Am J Respir Crit Care Med 2002:166: 440-444.

3 Hertz MI, Jessurun J, King MB, Savik SK, Murray JJ. Reproduction of the obliterative bronchiolitis lesion after heterotopic transplantation of mouse airways. Am J Pathol 1993:142: 1945-1951.

4 Neuringer IP, Mannon RB, Coffman TM et al. Immune cells in a mouse airway model of obliterative bronchiolitis. Am J Respir Cell Mol Biol 1998:19: 379-386.

5 Hele DJ, Yacoub MH, Belvisi MG. The heterotopic tracheal allograft as an animal model of obliterative bronchiolitis. Respir Res 2001:2: 169-183.

6 Neuringer IP, Walsh SP, Mannon RB, Gabriel S, Aris RM. Enhanced T cell cytokine gene expression in mouse airway obliterative bronchiolitis. Transplantation 2000:69: 399-405.

7 Palmer SM, Davis RD, Hadjiliadis D et al. Development of an antibodies specific to major histocompatibility antigens detectable by flow cytometry after lung transplant is associated with bronchiolitis obliterans syndrome. Transplantation 2002:74: 799-804.

8 Kallio EA, Lemstrom KB, Hayry PJ, Ryan US, Koskinen PK. Blockade of complement inhibits obliterative bronchiolitis in rat tracheal allografts. Am J Respir Crit Care Med 2000:161: 1332-1339.

9 Yousem SA, Berry GJ, Cagle PT et al. Revision of the 1990 working formulation for the classification of pulmonary allograft rejection: Lung Rejection Study Group. J Heart Lung Transplant 1996:15: 1-15.

10 King MB, Pedtke AC, Levrey-Hadden HL, Hertz MI. Obliterative airway disease progresses in heterotopic airway allografts without persistent alloimmune stimulus. Transplantation 2002:74: 557-562.

11 Adams BF, Brazelton T, Berry GJ, Morris RE. The role of respiratory epithelium in a rat model of obliterative airway disease. Transplantation 2000:69: 661-664.

12 Ikonen TS, Brazelton TR, Berry GJ, Shorthouse RS, Morris RE. Epithelial re-growth is associated with inhibition of obliterative airway

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disease in orthotopic tracheal allografts in non- immunosuppressed rats. Transplantation 2000:70: 857-863.

13 Boehler A, Chamberlain D, Kesten S, Slutsky AS, Liu M, Keshavjee S. Lymphocytic airway infiltration as a precursor to fibrous obliteration in a rat model of bronchiolitis obliterans. Transplantation 1997:64: 311-317.

14 Uusitalo MH, Salminen US, Ikonen TS et al. Alloimmune injury preceding airway obliteration in porcine heterotopic lung implants: a histologic and immunohistologic study. Transplantation 1999:68: 970-975.

15 McLaughlin PM, Harmsen MC, Dokter WH et al. The epithelial glycoprotein 2 (EGP-2) promoter-driven epithelial-specific expression of EGP-2 in transgenic mice: a new model to study carcinoma-directed immunotherapy. Cancer Res 2001:61: 4105-4111.

16 Jaramillo A, Smith MA, Phelan D et al. Temporal relationship between the development of anti-HLA antibodies and the development of bronchiolitis obliterans syndrome after lung transplantation. Transplant Proc 1999:31: 185-186.

17 Jaramillo A, Naziruddin B, Zhang L et al. Activation of human airway epithelial cells by non-HLA antibodies developed after lung transplantation: a potential etiological factor for bronchiolitis obliterans syndrome. Transplantation 2001:71: 966-976.

18 Reznik SI, Jaramillo A, SivaSai KS et al. Indirect allorecognition of mismatched donor HLA class II peptides in lung transplant recipients with bronchiolitis obliterans syndrome. Am J Transplant 2001:1: 228-235.

19 Smith MA, Jaramillo A, SivaSai KS et al. Indirect recognition and antibodies production against a single mismatched HLA-A2-transgenic molecule precede the development of obliterative airway disease in murine heterotopic tracheal allografts. Transplantation 2002:73: 186-193.

20 Smith MA, Jaramillo A, SivaSai KS et al. Indirect recognition and antibodies production against a single mismatched HLA-A2-transgenic molecule precede the development of obliterative airway disease in murine heterotopic tracheal allografts. Transplantation 2002:73: 186-193.

21 Szeto WY, Krasinskas AM, Kreisel D, Popma SH, Rosengard BR. Donor antigen-presenting cells are important in the development of obliterative airway disease. J Thorac Cardiovasc Surg 2000:120: 1070-1077.

22 Pan T, Mason RJ, Westcott JY, Shannon JM. Rat alveolar type II cells inhibit lung fibroblast proliferation in vitro. Am J Respir Cell Mol Biol 2001:25: 353-361.


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23 Fan ZJ, Wei HR, Wang A. Inhibition of fibroblast proliferation by human iris pigment epithelial cells in vitro: preliminary results. Graefes Arch Clin Exp Ophthalmol 1996:234: 64-66.

24 Neuringer IP, Aris RM, Burns KA, Bartolotta TL, Chalermskulrat W, Randell SH. Epithelial kinetics in mouse heterotopic tracheal allografts. Am J Transplant 2002:2: 410-419.

25 Romaniuk A, Prop J, Petersen AH, Nieuwenhuis P, Wildevuur CR. Class II antigen expression on bronchial epithelium in rat lung allografts is prevented by cyclosporine treatment. Transplant Proc 1987:19: 218-219.

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Chapter 4

Obliterative Airway Disease Development in Mouse Trachea Transplants

under Pre-existing Immunity

Ning Qu; Martin C.Harmsen; Lou M.F.H. de Leij; Jochum Prop; Aalzen de Haan

Submitted

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Abstract Background Animal trachea transplantation models have suggested that airway epithelium plays a central role in the pathogenesis of obliterative airway disease OAD, a proces that shares similarities with human Obliterative Bronchiolitis after lung transplantation. Here we investigated, in a murine transgenic trachea transplantation model with human epithelial glycoprotein-2 (hEGP-2) expressed exclusively on epithelial cells, if pre-existing immunity to hEGP-2 induces damage of airway epithelial cells and if this contributes to the development of OAD. Methods Immunity in recipient FVB/N mice was induced by immunization and tested by screening levels of serum hEGP-2-specific antibodies in vivo and their complement fixing capacity in vitro, as well as cellular immunity by in vivo delayed type hypersensitivity testing (DTH). Tracheas from hEGP-2 transgenic FVB/N mice were then transplanted into hEGP-2 immune or non-immune FVB/N recipients. Grafts were harvested at week 1, 3, 6 after transplantation and were analyzed for cellular infiltration, epithelial coverage and morphology, and the degree of obliteration. Results Immunization resulted in hEGP-2-specific cellular immunity as demonstrated by positive DTH tests and serum hEGP-2-specific antibodies with the capacity to induce complement –dependent lysis of a hEGP-2+ cell line in vitro. Upon transplantation of hEGP-2 trachea graft in hEGP-2 immune recipients, the grafts showed an increased infiltration and a higher degree of epithelium damage and abnormality compared to grafts transplanted in non-immune recipients. Despite this, levels of luminal obliteration of grafs in immune vs non-immune mice were similar. Conclusions Pre-existing immunity targeting exclusively towards tracheal graft epithelial cells induces a high degree of epithelium abnormality but this does not lead to full obliteration. Therefore, the development of OAD may additionally require extensive epithelial cells loss and targeting and damage of other graft cell types, e.g. fibroblasts, by the graft-reactive immune responses.

Obliterative airway disease development in mouse trachea transplants

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Introduction In clinical lung transplantation, obliterative bronchiolitis (OB) affects 50% of all lung transplant recipients and results in high mortality within 5 years (1,2). Histopathologic features of OB suggest that the airway epithelial cells injury result from persistent inflammation in small airways leading to ineffective epithelial regeneration and excessive fibroproliferation due to aberrant tissue

repair (3). In animal tracheal transplants, rejection leads to obliterative airway disease (OAD) (4,5), a process that shares similarities to human OB. The role of airway epithelium injury in trachea OAD was investigated by the group of Morris in rats (6). They demonstrated that the loss of airway epithelium, either by enzymatic removal in isografts or by transplant rejection in allografts, resulted in OAD. Re-seeding of epithelial cells in the isografts (7) or re-growth in allografts (8) largely reduced the level of OAD. These findings suggested that the airway epithelial cells regulate fibroblasts growth and thus protect the airway against fibrotic obliteration. Other studies showed strong submucosa infiltration of immune cells (9), and loss of epithelium preceding the obliteration of the trachea grafts (5,10) supporting that the regulating role of the epithelial cells on fibroblast proliferation may be lost as the result of immune injury.

In a previous study (11) we hypothesised that OAD in trachea transplants was primarily caused by epithelial injury resulting in loss or dysfunction of the epithelial cells; fibroblast growth in OAD would be secondary to this epithelial injury. To investigate this role of epithelium in OAD we used transgenic animals expressing neoantigens on airway epithelial cells. (11) In these mice, human epithelial glycoprotein-2 (hEGP-2) is expressed specifically on epithelial cells, including trachea epithelial cells, driven by the endogenous specific promoter (12). Upon transplantation of tracheas from hEGP-2 transgenic FVB/N mice donors into non-transgenic FVB/N mice, humoral immune responses against the hEGP-2 antigen on epithelial cells developed leading to epithelium abnormality and partial airway obliteration. In contrast to allogeneic trachea transplantation, the epithelium was not totally destroyed and total obliteration did not occur in this model. We postulated that the weaker OAD in transgenic tracheas could be explained by the absence of graft reactive immunity at the time of transplantation. In allogeneic transplantation, alloreactivity exists as the result of cross reactivity of pre-existing alloantigen specific antibodies and memory T cells towards the alloantigens, creating a strong and immediate immune attack against the

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graft. In transgenic trachea transplantation, this pre-existing immunity does not exist and epithelial cell specific immune responses have to evolve after transplantation. In the present study, we therefore investigate if pre-existing immunity to the epithelial hEGP-2 neoantigen augments injury of airway epithelial cells and if this contributes to the development of OAD in hEGP-2 transgenic trachea transplants. Material and methods Experimental design Immunity to hEGP-2 was induced in nontransgenic FVB/N recipient mice by immunization with syngeneic hEGP-2 antigen expressing cells. Immunity to hEGP-2 prior to transplantation was analysed by ELISA and the cytotoxic potential of these antibodies was analysed by an antibody mediated complement-dependent cytotoxicity assay. Cellular immunity to hEGP-2 induced by the immunization was analysed by a delayed type hypersensitivity (DTH) testing. Tracheas from hEGP-2 transgenic mice (n=38) were transplanted to the immunized (n=11) and non-immunized (n=27) FVB/N mice subcutaneously (s.c.). As controls, tracheas from nontransgenic FVB/N mice were transplanted s.c. to immunized (n=7) and non-immunized (n=21) non-transgenic FVB/N recipients. Trachea grafts were harvested at 1, 3, 6 weeks after transplantation and were studied for epithelial integrity and abnormality by haematoxylin and eosin (H&E) staining and for cellular infiltration by immunohistochemistry. Experimental animals Inbred FVB/N mice transgenic for the hEGP-2 were bred and housed in the Central Animal Facility of Groningen University according to the standard breeding rules for transgenic animals in a conventional condition. Normal FVB/N inbred mice were purchased from Harlan (Harlan Horst, The Netherlands). Animals used in the experiments were between 8-12 weeks old. All animals received care in compliance with the Dutch regulations and laws. Experimental protocols were approved by the institutional animal ethical review committee.


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Immunization of recipient mice Non-transgenic FVB/N mice were immunized by injection of syngeneic hEGP-2 expressing cells. These cells were harvested as primary cell isolates from hEGP-2 transgenic mice and were used for the primary immunization and for the booster. For primary cell isolates, transgenic mice were sacrificed and part of the intestine was harvested. The lumen of intestine was washed with cold PBS plus 2% Gentamycin. A 12 cm long piece of intestinal tract was then opened longitudinally and the epithelium was scraped off using a surgical scalpel. The collected epithelium was washed twice with ice cold HBSS and the protein content was determined using a BIO-RAD protein assay (BIO-RAD, München, Germany). Complete Freund’s adjuvant (CFA) (for primary immunization) or incomplete Freund’s adjuvant (IFA) (for booster) was mixed with amount of cells containing 750 µg of protein at volume ratio 1:1 and was injected s.c. in the neck of each mouse. The primary immunization was followed by a booster six weeks later. As controls of immunization, non-transgenic mice were injected with solvent plus CFA or IFA in the same way.

ELISA assay for anti-hEGP-2 antibodies To analyse humoral immunity towards hEGP-2, serum hEGP-2 specific antibody levels were analysed after immunization. Whole blood samples were collected from the orbital sinus at 2 time points: before immunization and 4 weeks after the booster (the day before transplantation). Serum hEGP-2 specific antibody levels were detected by ELISA as previously described (11). Briefly, ELISA plates were coated overnight with recombinant soluble hEGP-2 protein. Samples in two-fold dilutions were incubated in the coated plates for 1.5h at 37oC. Purified mouse-anti-hEGP-2 monoclonal antibody (mAb) (Moc31; (12)) was used as standard. Serum samples from non-transgenic FVB/N mice were used as negative control. Rabbit-anti-mouse IgG (subclass-specific) peroxidase labelled antibodies (Dako, Bucks, UK) were applied and incubated for 1h at 37oC. After washing, TBM peroxidase substrate solution was added and plates were stained and read on a microplate reader (Emax, Molecular Device, Santa Fe Springs, CA, USA) at 450 nm. Data were analysed by the reader’s software (SoftmaxPro version 1.2.0) and the hEGP-2 specific antibodies level related to the Moc31 level was calculated.

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Antibody mediated complement-dependent cytotoxicity assay To evaluate the contribution of hEGP-2 specific antibodies to the lysis of hEGP-2 expressing cells, we performed a complement-dependent cytotoxicity assay. First, a hEGP-2 expressing cell line was produced. This was done by infection of the MLE-12 cell line (FVB/N origin, purchased from ATCC, Manassas, VA, USA) with retroviral particles encoding both hEGP2 and enhanced green fluorescent protein (EGFP), as reported previously. (13) Briefly, hEGP2 cDNA was cloned into a retroviral vector derivative of LZRS-pBMNlacZ35. After transfection into the amphotrophic packaging cell line Phoenix, transfected cells were selected by culturing in the presence of 1 µg/ml puromycin, 300 µg/ml hygromycin and 1 µg/ml diphtheria toxin (BD Biosciences Clontech, Palo Alto, CA, USA). Viral particle-containing supernatant was harvested after 3 days and used for transduction of the hEGP-2-EGFP genes into the MLE-12 cell line. Transduced cells were selected both for EGFP fluorescence and hEGP2 expression (mAb Moc31-PE) using the MoFlo high-speed cell sorter (Cytomation, Fort Collins, CO, USA). These cells and non-transfected cells, as a control, were then used in a complement-dependent cytotoxicity assay. This test was set-up similar to standard tissue typing complement-dependent cytotoxicity techniques. In brief, MLE-12 cells with and without hEGP-2 expression were added to Terasaki plates (4000 cells/well). Serum at a 1:10 dilution was added to the cells. As a positive control, a complement fixing hEGP-2 specific mAb, Moc181 (1:10 diluted hybridoma culture supernatant) (a gift from Dr. Bart-Jan Kroese, Medical Biology Section, Department of Pathology and Laboratory Medicine, Groningen University, The Netherlands) was added. Plates were then incubated for 30 min at room temperature after which rabbit complement (Sanquin, Amsterdam, The Netherlands) was added. After 1 hour, a cocktail of ethidium bromide/acridine orange was added for visualisation of dead (red) or living (green) cells with the use of a fluorescence microscope. A semiquantitative analysis was then performed by grading cell death in a scale of 0-5 (0, <10%; 1, 10-20%; 2, 20-40%; 3, 40-60%; 4, 60-80%; 5, 80-100% dead cells).


71

Delayed type hypersensitivity test To analyse the induction of cellular immunity by the immunization, a DTH test was performed. The test was performed 10 days after the booster immunization. Using a Hamilton syringe (100µl) 25 µL of purified recombinant soluble hEGP-2 protein (300ng/ml) was injected into the left hind footpad of immunized mouse (n=5). Non-immunized animals (n=5) served as a control. Additionally, as a control, the same volume of solvent (sterile PBS) was injected into the right hind footpad of each mouse. Footpad thickness was measured before and 24, 48, and 72h after injection with an engineering calliper (Micromez, Hanover, Germany) and the incremental thickness was scored.

Heterotopic trachea transplantation Mouse tracheas were heterotopically transplanted as previously described (11). Briefly, donor mice were anaesthetised with halothane and N2O/O2 gas and killed by abdominal aorta bleeding. Tracheas from these mice were exposed through an anterior midline incision and were transected below the thyroid cartilage and above the bifurcation. The lumen was washed with cold HBSS and the trachea was immediately put into cold saline. The recipient mice were anaesthetised in the same way as the donor mice. A small incision was made at the lateral side of the back and a subcutaneous pouch was made, the trachea was clipped with surgical stainless clips at both ends and put into the pouch. Each recipient received one trachea. At designated time points the trachea grafts were explanted through an incision immediately outside the grafts tissue. Each trachea was cut into two segments: one half was fixed in 10% formalin for H&E staining and the other half was snap frozen in liquid nitrogen for immunohistochemical staining. Histological analysis

As previously described (11), formalin fixed trachea segments were paraffin embedded. Serial cross sections (3µm thick) were cut at 3 levels 50µm apart of each trachea segment, and 3 sections at each level were stained by normal H&E method and evaluated at 50 to 400 magnifications. To analyse the infiltration level of the trachea grafts, a semi-quantitative scoring system was used with the following five grades: 0-no infiltration, 1-minimal infiltration: scattered or diffuse cellular infiltrates, 2-mild infiltration: diffuse cellular

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infiltrates with one area dense infiltrate, 3-moderate infiltration: more than one area infiltrates, 4-severe infiltration: a thick layer of dense cellular infiltrates (11).

To analyse the obliteration degree of the trachea lumen, the thickness of submucosa (between epithelium and cartilage) was scored using following five grades: 0-no thickening of submucosa, 1-minimal thickening: focal submucosa thickening, 2-mild thickening: submucosa thickening equal to the thickness of the cartilage ring, 3-moderate thickening, submucosa thickening result in a small lumen, 4-severe thickening: total lumen obliteration.

The epithelium was categorised in two types: normal and abnormal based on morphological aspects of epithelial cells, i.e. the differentiation (the presence of ciliated epithelium) and the pseudo-stratification (the presence of pseudostratified epithelium layer). The coverage of the trachea luminal ring by normal and abnormal epithelium was scored in percentage. Immunohistochemistry Frozen tracheas were cut into 5µm thick cross sections, and were immunohistochemically stained as previously described (11). Briefly, primary rat antibodies specific for mouse CD5 (pan-T cells), CD4 (T-helper cells), CD8 (T-Cytotoxic cells) T cells or macrophages were applied and incubated for 1h (hybridomas in culture supernatants were not diluted, CD8α was diluted 1:50). Primary antibodies directed against mouse CD5 (Ly-1), CD4 (L3T4), and CD11b (mac-1, for macrophages) were from hybridoma culture supernatants, and purified antibodies specific for mouse CD8α (Ly-2) were from BD Pharmingen, San Diego, USA. The slides were then washed again with PBS. Secondary antibodies (rabbit-anti-rat peroxidase labelled, 1:50 dilution, Dako, Bucks, UK) plus 3% normal FVB/N mouse serum were applied and incubated for another 1h. After washing, the slides were coloured with AEC (Sigma, 0.5mg in 3.75 ml DMF plus 70ml acetate buffer, pH4.9). Slides were counterstained with Mayer’s haemotoxilin (1:10).

Trachea infiltrating cell phenotypes were analysed inside the trachea ring in three sections from each trachea segment. The positive cells in the grafts were scored, using a semi-quantitative scoring system with five grades: 0-no positive cells, 1-scattered positive cells, 2-small clusters or one area with dense positive cells, 3-more than one area or a large area plus clusters of positive cells, 4-whole tissue dense positive cells. All the histology scoring and counting were done by three independent observers (11).


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Results Immunization induces cytotoxic anti-hEGP-2 antibodies To determine whether immunized mice had developed anti-hEGP-2 immunity, hEGP-2 specific antibody levels and subclasses were determined before transplantation. hEGP-2 specific antibodies were detected in all immunized mice before transplantation (mean level of 126 ± 51 µg/ml). Control animals did not show detectable hEGP-2-specific antibody levels. The induced antibodies were mainly of the IgG1 subclass, with lower but detectable levels of IgG2a and lgG2b, but not IgG3 (data not shown). Anti-hEGP-2 sera were tested in complement-dependent cytotoxicity assay to determine their cytotoxic capacity towards hEGP-2 expressing cells in vitro. Significant cell Figure 1.

hEGP-2 cells non-hEGP-2 cells0

1

2

3

4

positive control Abimmune serumcontrol serum

complement onlymedium only

****

target cells

cell l

ysis

gra

de

Figure 1. Cell lysis of hEGP-2 positive target cells and control cells in a complement-dependent cytotoxicity assay. Serum from hEGP-2 immune recipients induced significant higher levels of cell lysis of hEGP-2 positive target cells than serum from non-immune recipients (control serum; asterisks indicates p<0.01). hEGP-2 immune serum-induced cell lysis was significant higher with hEGP-2 positive cells compared with control cells lacking hEGP-2 expression (asterisks indicates p<0.01). Columns are the mean value of 6 wells and error bars indicate the S.E.M. lysis was observed when sera from immunized animals were used, comparable with that seen with the complement-fixing monoclonal antibody

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Moc181 as a positive control (Figure 1). Control cells without hEGP-2 expression were lysed only at a minimal level which was comparable with the level seen with sera from non immunized animals (Figure 1). The results demonstrate that the antibodies in the immunized mice had cytotoxic capacity against hEGP-2 expressing target cells. Immunization induces hEGP-2 specific cellular immune responses To analyse if the immunization resulted in cellular immune responses in vivo, mice were challenged with purified hEGP-2 protein and tested for DTH. A significant swelling of footpad thickness, peaking at 24 hours after challenge, was measured in hEGP-2-immunized recipients, indicating a positive DTH response which gradually decreased after 72 hours (Figure 2). A positive DTH response was not found in the control animals (Figure 2). This indicates that a hEGP-2 specific cellular immune response was induced in immunized recipients. Figure 2.

24 48 72

-0.05

0.00

0.05

0.10

0.15immunized,hEGP2immunized,PBSnon-immunized,hEGP2non-immunized,PBS

*

hours after challenge

thic

knes

s in

crea

se(c

m)

Figure 2. Footpad swelling in DTH testing of immune and non-immune recipients. hEGP-2 immune recipients demonstrated a significant increased swelling at 24 h after hEGP-2 injection compared to non-immune recipients or injection of PBS alone (asterisk indicates P < 0.05). The tissue swelling developed quickly after challenge and peaked at 24hours, decreased to minimal level after 72 hours. Data are shown as the mean value of 5 mice. Error bars indicate the S.E.M.


75

Pre-existing hEGP-2 specific immunity enhances immune cells infiltration in transplants Upon transplantation of hEGP-2 tracheas, cellular infiltration was found in the submucosa of all grafts at all time-points. The infiltration of hEGP-2 grafts in immunized recipients ranged from a mild level at 1 week to a severe level at 3 and 6 weeks (Table 1). hEGP-2 grafts in non-immunized recipients were less infiltrated (ranging from minimal level at 1 week to mild level at 3 weeks and 6 weeks; Table 1). Few infiltrating cells were found in non-transgenic control FVB/N grafts from both immunized and non-immunized recipients at all time points (data not shown). The cellular infiltrates were mostly focused within the submucosa and no obvious infiltration was found outside the trachea rings. Immunohistochemical staining showed that the cells infiltrating the trachea grafts were mainly T cells (CD5+) and macrophages (data of macrophages not shown). A dominant CD4+ T cell infiltration was found in all grafts from both immunized and non-immunized group (Table 2). CD4+ cells infiltration levels increased from a mild level at 1 week to a moderated level at 3 weeks in both groups. The infiltration level of CD8+ cells was from minimal at 1 week to mild at 3 weeks in the immunized group and zero to minimal in the non-immunized group (Table 2). Table 1. Cellular infiltration in trachea submucosa* (H&E)

hEGP-2 grafts in non-immune recipients

hEGP-2 grafts in immune recipients

n infiltration in submucosa n

infiltration in submucosa

1w

3w

6w

5

11 7

1 (1-2) 2.5 (1-4) 2.5 (1-4)

3 5 4

1.5 (1-4) 4 (4-4) 4 (4-4)

*Infiltration in the trachea submucosa was scored in five grades: 0-no infiltration, 1-minimal infiltration: scattered or diffuse cellular infiltrates, 2-mild infiltration: diffuse cellular infiltrates with one area dense infiltrate, 3-moderate infiltration: more than one area infiltrates, 4-severe infiltration: a thick layer of dense cellular infiltrates. Shown are median values (and range).

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Table 2: T cell infiltration in transplanted tracheas (immunohistochemistry)

*The positive cells in the submucosa of the trachea grafts were scored in five grades: 0-no positive cells, 1-minimal infiltration: scattered positive cells, 2-mild infiltration: small clusters or one area with dense positive cells, 3-moderate infiltration: more than one area or a large area plus clusters of positive cells. 4-severe infiltration: whole tissue dense positive cells. Shown are median values (and range). Pre-existing hEGP-2 specific immunity enhances the epithelium abnormality but not the luminal obliteration Grafts in hEGP-2-immunized recipients showed almost completely abnormal epithelium at all time points. (Figure 3A) In contrast, grafts from non-immunized mice had only partly abnormal epithelium (Figure 3A). At 3 weeks after transplantation, hEGP-2 tracheas in immunized animals showed some degree of epithelial cell denudation, which was not observed in the non-immunized animals. Despite a higher degree of epithelial damage, the luminal obliteration in the hEGP-2 grafts transplanted to immunized animals did not significantly differ from the obliteration observed in grafts transplanted to non-immunized animals (Figure 3B). The submucosa in most tracheas was mildly increased in thickness, leaving the lumen largely free from fibrosis.

hEGP-2 grafts in non-immune recipients*

hEGP-2 grafts in immune recipients*

n CD5+ CD4+ CD8+ n CD5+ CD4+ CD8+

1w

3w

5

6

1.5 (0-2) 2 (1-3)

2 (1-3) 3 (2-3.5)

0 (0-1) 1 (0.5-1)

3 5

1.5 (0.5-4) 3 (1-4)

1.5 (0.5-4) 3 (0.5-4)

1 (0-2) 1.5 (0-2)


77

Figure 3.

1 3 6 1 3 60

50

100 total epithliumabnormal epithelium

non-immune immune

A

*


trach

ea lu

men

epi

thel

ium

cove

rage

(%)

0

1

2

3

4

1 3 6 1 3 6

B

non-immune immune


lum

inal

obl

itera

tion

grad

e

Figure 3. Epithelial coverage, abnormality and luminal obliteration in trachea grafts. Figure 3A shows a significantly lower coverage at 3 weeks, but not at 1 or 6 weeks, in grafts in the immune recipients compared to grafts in the non-immune recipients (p<0.05). Epithelial cell abnormality (dark columns) and indicates the fraction of abnormal epithelium relative to the total epithelium (open columns). At all time points the level of abnormal epithelium is higher in immune recipients than in non-immune recipients (Figure 3A; p< 0.05). The luminal obliteration does not differ significantly between immunized and non-immunized groups (figure 3B). Columns show mean values of 4 to 9 trachea grafts and error bars indicate S.E.M.

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Discussion In this study, we investigated if pre-existing immunity to epithelial antigens augments injury of airway epithelial cells and if this contributes to the development of OAD. This was analysed in a transgenic trachea transplantation model, where epithelium of the donor trachea carried the hEGP-2 neoantigen. Our data shows that the immunization induced a hEGP-2 specific pre-existing immunity composed of both humoral and cellular responses in recipient mice, as demonstrated by the induction of cytotoxic anti-hEGP-2 antibodies and a DTH response. Upon transplantation in immunized mice, the transplanted hEGP-2 tracheas revealed an enhanced level of infiltration and epithelium abnormality when compared to hEGP-2 tracheas transplanted to non-immunized mice, however, without a significant increase of the OAD level.

We previously hypothesised that OAD in trachea transplants is primarily caused by injury to epithelial cells resulting in epithelial loss or dysfunction, while fibroblast overgrowth is secondary to this epithelial injury (11). This is not fully supported by the results of the present study: despite evident increase of epithelial injury of trachea grafts in immunized recipients with persistent abnormal appearance of the epithelium, it was not associated with the development of OAD. These findings could illustrate a high degree of flexibility of airway epithelial cells to cope and respond to stress such as immune attack. It remains to be answered what factors could be involved in these processes. Recent studies on epithelial cell injury and repair showed that the expression of ‘protective genes’ such as HO-1 (14) contributes to the epithelial cell survival during injury. Under these circumstances epithelial cells undergo proliferation as well as dedifferentiation to avoid further damage (15). Similar mechanisms might operate in the dedifferentiation and survival of tracheal hEGP-2 expressing epithelial cells exposed to hEGP-2 specific immune attack. These dedifferentiated epithelial cells persisting in hEGP-2 trachea grafts, although injured and abnormal in appearance, may have maintained sufficient function to be capable to inhibit fibroblast proliferation.

One might speculate that even in our pre-immunized recipients the immune response was insufficient to cause severe epithelial injury leading to OAD. However, the immunization induced hEGP-2 specific antibodies with antibody mediated complement-dependent cytotoxicity in vitro. In a previous study, antibody-mediated complement activation (classical pathway) has been suggested to play a role in epithelial injury and development of OAD in rat


79

tracheal allografts. (16) There, an increase in intragraft complement components C3 and C5b-9 (membrane attack complex) as well as IgM and IgG deposits were found during the progressive loss of epithelium and airway fibroocclusion in nontreated allografts. Additionally, the blockade of the complement activation system effectively prevented OAD. It is possible that in the rats the complement mediated damage was caused by graft-reactive IgM rather than IgG. In the immunized mice, we found the hEGP-2 specific antibodies to be mainly of the IgG1 subclass and to a lower extend of the IgG2a subclass. The latter subclass is known to bind and activate complement. The induced IgG antibodies, although capable of complement-dependent cytotoxicity in vitro may have been less efficient of complement-dependent cytotoxicity in vivo. Besides the humoral responses, we also found cellular immunity with in vivo anti-hEGP-2 DTH responses in the immunized mice, indicating that the immunization procedure was effective. Despite the induced immune response against the epithelial hEGP-2 antigen, the loss of epithelium was only mild and transient and was not associated with the full airway obliteration as seen in OAD.

So far, we explained the absence of severe OAD in our study by inhibition of fibroproliferation by the tracheal epithelium. Alternatively, it is conceivable that fibroproliferation of the lumen failed to occur in the transgenic tracheas, because fibroblasts lacked certain stimuli that cause their activation and proliferation during OAD in allogeneic trachea transplantation. In support of this idea is the study of Smith et al., who described a different model of transgene-expressing trachea transplantation where OAD with complete obliteration developed rapidly upon transplantation. (17) Their transgenic mice express the human HLA-A2 as a neoantigen throughout the trachea on all types of tissue cells, including fibroblasts, whereas in our hEGP-2 transgenic mice the expression is restricted to epithelial cells. Thus, the main components of the immune response, being neoantigen-specific antibodies and immune cells in both the HLA-A2 transgenic and hEGP-2 transgenic model, target to either all type of cells (HLA-A2 transgenic) or epithelial cells only (hEGP-2 transgenic). As a consequence, the induced neoantigen-specific antibodies bind to all cell types including fibroblasts in the HLA-A2 transgenic model. Antibodies have been shown to induce tyrosine phosphorylation in targeted cells resulting in activation and proliferation (18,19). In targeted fibroblasts, such activation may lead to fibrosis. Similarly, neoantigen-specific CD4+ T cells target to all cells in the HLA-A2 transgenic graft provided that the cells are capable to express MHC class II and to indirectly present HLA-

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A2 antigenic epitopes. The activated T cells secrete profibrotic cytokines such as TGF-beta (20) that stimulate fibroblasts to proliferate. In our hEGP-2 model, immune responses target exclusively to the epithelial cells, resulting in injured and abnormal epithelium, but not in activation of fibroblasts. The absence of stimuli for fibroproliferation may explain why OAD with complete obliteration was not observed in our EGP-2 transgenic trachea transplants.

In conclusion, our data demonstrate that pre-existing immunity directed exclusively towards airway epithelial cells augments submucosal inflammation and epithelium abnormality in trachea transplants, but does not result in severe OAD. Although massive damage to epithelial cells has been shown to result in severe OAD, as for example in artificial denudation of epithelial cells from syngeneic trachea grafts, (21) a less severe injury to epithelial cells with persistent dedifferentiation does not cause significant airway obliteration. This study suggests that OAD only develops if graft-reactive immune responses affect both epithelial cells and fibroblasts in trachea transplants. Acknowledgement The authors thank Arjen H. Petersen and Henk van de Molen for the assistance in the animal experiments; Dr. Wijnand Helfrich and Edwin Bremer for supplying the purified hEGP-2 protein and hEGP-2 retroviral vector; Mrs. Greetje Groen for the help of immunohistochemical staining and Dr. Caroline Roosendaal for the help in antibody mediated complement-dependent cytotoxicity assay.


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Reference 1. Knollmann FD, Ewert R, Wundrich T, Hetzer R, Felix R. Bronchiolitis

obliterans syndrome in lung transplant recipients: use of spirometrically gated CT. Radiology 2002:225: 655-662.

2. Lu KC, Jaramillo A, Lecha RL et al. Interleukin-6 and interferon-gamma gene polymorphisms in the development of bronchiolitis obliterans syndrome after lung transplantation. Transplantation 2002:74: 1297-1302.

3. Yousem SA, Berry GJ, Cagle PT et al. Revision of the 1990 working formulation for the classification of pulmonary allograft rejection: Lung Rejection Study Group. J Heart Lung Transplant 1996:15: 1-15.

4. Nord M, Schubert K, Cassel TN, Andersson O, Riise GC. Decreased serum and bronchoalveolar lavage levels of Clara cell secretory protein (CC16) is associated with bronchiolitis obliterans syndrome and airway neutrophilia in lung transplant recipients. Transplantation 2002:73: 1264-1269.



7. Bui JD, Despotis GD, Trulock EP, Patterson GA, Goodnough LT. Fatal thrombosis after administration of activated prothrombin complex concentrates in a patient supported by extracorporeal membrane oxygenation who had received activated recombinant factor VII. J Thorac Cardiovasc Surg 2002:124: 852-854.

8. Ikonen TS, Brazelton TR, Berry GJ, Shorthouse RS, Morris RE. Epithelial re-growth is associated with inhibition of obliterative airway disease in orthotopic tracheal allografts in non- immunosuppressed rats. Transplantation 2000:70: 857-863.

9. Aris RM, Walsh S, Chalermskulrat W, Hathwar V, Neuringer IP. Growth factor upregulation during obliterative bronchiolitis in the mouse model. Am J Respir Crit Care Med 2002:166: 417-422.

10. Henke JA, Golden JA, Yelin EH, Keith FA, Blanc PD. Persistent increases of BAL neutrophils as a predictor of mortality following lung transplant. Chest 1999:115: 403-409.

11. Qu N, De Haan A, Harmsen MC, Kroese FG, De Leij LF, Prop J. Specific immune responses against airway epithelial cells in a transgenic mouse-

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trachea transplantation model for obliterative airway disease. Transplantation 2003:76: 1022-1028.

12. Reznik SI, Jaramillo A, SivaSai KS et al. Indirect allorecognition of mismatched donor HLA class II peptides in lung transplant recipients with bronchiolitis obliterans syndrome. Am J Transplant 2001:1: 228-235.

13. Bremer E, Kuijlen J, Samplonius D, Walczak H, de Leij L, Helfrich W. Target cell-restricted and -enhanced apoptosis induction by a scFv:sTRAIL fusion protein with specificity for the pancarcinoma-associated antigen EGP2. Int J Cancer 2004:109: 281-290.

14. Yamada N, Yamaya M, Okinaga S et al. Protective effects of heme oxygenase-1 against oxidant-induced injury in the cultured human tracheal epithelium. Am J Respir Cell Mol Biol 1999:21: 428-435.

15. Tesfaigzi Y. Processes involved in the repair of injured airway epithelia. Arch Immunol Ther Exp (Warsz ) 2003:51: 283-288.

16. Kallio EA, Lemstrom KB, Hayry PJ, Ryan US, Koskinen PK. Blockade of complement inhibits obliterative bronchiolitis in rat tracheal allografts. Am J Respir Crit Care Med 2000:161: 1332-1339.


18. Bian H, Harris PE, Mulder A, Reed EF. Anti-HLA antibody ligation to HLA class I molecules expressed by endothelial cells stimulates tyrosine phosphorylation, inositol phosphate generation, and proliferation. Hum Immunol 1997:53: 90-97.

19. Harris PE, Bian H, Reed EF. Induction of high affinity fibroblast growth factor receptor expression and proliferation in human endothelial cells by anti-HLA antibodies: a possible mechanism for transplant atherosclerosis. J Immunol 1997:159: 5697-5704.

20. Ihn H. Pathogenesis of fibrosis: role of TGF-beta and CTGF. Curr Opin Rheumatol 2002:14: 681-685.


Chapter 5

Anti-CD45RB Antibody Monotherapy

Protects Mouse Trachea

Allograft Epithelium

Ning Qu; Lydia Visser; Sibrand Poppema; Jochum Prop; Aalzen de Haan

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84

Anti-CD45RB antibody monotherapy protects airway epithelium

85

Abstract

Background Obliterative airway disease occurs upon experimental transplantation of trachea allografts. Its main histological features are graft airway epithelium injury and luminal fibrosis. Airway epithelium plays an important role in the pathogenesis of obliterative airway disease. Alloimmune responses, especially T cell responses, are thought to be mainly responsible for epithelium injury. Immunosuppressive therapy has been used to diminish T cell responses and prolong graft survival. To avoid the side-effect of classical therapy, new approaches including tolerance inducing antibodies may improve the success of airway (lung) transplantation. In the present study we investigated the effect of anti-CD45RB treatment on the protection of airway epithelium and its effect on obliterative airway disease. Methods Allogeneic tracheas were transplanted to recipient mice that were treated either with anti-CD45RB monoclonal antibodies or with solvent daily from day -1 to day 7. Grafts were explanted at 3 weeks after transplantation, and were analyzed histologically and immunohistochemically. Results In the anti-CD45RB treated group, T cell infiltration was minimal. Most of the trachea epithelium was preserved, although its morphology was abnormal. Luminal obliteration and obliterative airway disease was inhibited by the treatment. In the solvent treated group, higher level of T cell infiltration and severe level of epithelium loss were observed. The trachea lumen was partially occluded and obliterative airway disease occurred. Conclusions This study shows that anti-CD45RB monotherapy diminishes the T cell response towards trachea allografts, prevents severe trachea airway epithelium loss, and reduces the occurrence of obliterative airway disease. Combination of the anti-CD45RB antibody treatment with other immunosuppressive drugs may further improve its protective effect.

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Introduction Animal models of obliterative airway disease (OAD) using trachea grafts have been used to study the mechanism of obliterative bronchiolitis (OB), a hallmark of chronic rejection after human lung transplantation. (1-3). These models have shown that both cellular and humoral immune responses against donor tissue, in particular the airway epithelium, can play a role in the development of OAD (4,5). Recent studies on trachea transplantation in CD4 and CD8 T cell knockout mice have shown that the absence of T cells strongly reduced the development of OAD (6,7). Therefore, therapy aiming specifically at T cells may prevent airway epithelial damage and, consequently, reduce the development of OAD.

Classical therapies to interfere with T cell activity are based on lifelong generalized immunosuppression, such as continuous administration of calcineurin inhibitors cyclosporine A (CsA) or FK506. Novel therapies based on immunomodulation of T cells through monoclonal antibodies (mAb) carry the promise not to require lifelong administration and may, following a short period of dosing post-transplantation, lead to prolonged allograft survival or even tolerance (8). For example, a novel therapy using anti-CD45RB mAb for modulation of T cell response has been shown to lead to prolonged allograft survival in kidney, heart and islet transplantations (8-10). CD45 is a family of highly glycosylated transmembrane protein tyrosine phosphatases that are expressed by leukocytes and that are required for effective antigenic stimulation of B and T cells. CD45RB, a differentially expressed exon, is a potent immunomodulatory target that can be found in different quantities on all lymphocytes. The mechanism by which anti-CD45RB mAb induces tolerance is not completely clear, but the depletion of CD45RBhigh cells from the periphery plays an important role (11). In the grafts a population of CD45RBlow, IL-4 and IL-10-producing T cells (12) is found that have been reported to have immunoregulatory capacities (13-15). Although a beneficial effect of anti-CD45RB mAb treatment on graft rejection has been shown in a number of different transplantation models, the effect on the development of OAD in trachea grafts has not been studied before.

To investigate whether OAD development in trachea grafts could be prevented by anti-CD45RB mAb monotherapy, we performed allogeneic trachea transplantation in antiCD45RB, i.e. MB23G2, mAb-treated or untreated mice and subsequently analyzed cellular infiltration level, epithelium integrity and the obliteration level of the trachea transplants.


87

Material and methods Experimental design MHC fully mismatched mouse strains were used for allografting. C57B/6 mice (MHC: H2d) were used as trachea donors and FVB/N mice (MHC: H2q) were used as trachea recipients. Anti-CD45RB mAb MB23G2 (100µg in 100µl saline) was injected intravenously (tail vein) in recipient mice (n=7). A same volume of saline was injected intravenously in control mice (n=6). The mAb was administered daily from day -1 to day 7 of transplantation. Recipient mice were killed on day 21 after transplantation and the trachea grafts were explanted. These grafts were analyzed for cellular infiltration, epithelium integrity and lumen obliteration level.

Experimental animals FVB/N and C57BL/6 inbred mice were purchased from Harlan (Harlan, Horst, The Netherlands). Animals used in the experiments were between 8 and 10 weeks of age. All animals received care in compliance with the Dutch regulations and laws. Experimental animals were handled according to the approved protocol and animal welfare regulations by the institutional animal ethical review committee. Trachea transplantation Mouse tracheas were heterotopically transplanted as previously described (16). Briefly, donor mice were anesthetized and killed by abdominal aorta bleeding. Tracheas from these mice were exposed through the anterior midline incision and were transected below the thyroid cartilage and above the bifurcation. Tracheas were immediately put into cold saline and the lumen was washed. The recipient mice were anesthetized in the same way as the donor mice. A small incision was made at the lateral side of the back and a subcutaneous pouch was made, the trachea was clipped with surgical stainless clips at both ends and put into the pouch. Each recipient received one trachea graft. At day 21 the trachea grafts were explanted through an incision immediately outside the grafts tissue. Each trachea was cut into two segments: one half was fixed in 10% formalin for H&E staining and the other half was snap frozen in liquid nitrogen for immunohistochemical staining.

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Histological analysis Formalin fixed trachea segments were paraffin embedded. Serial cross sections at 3µm thick were cut and stained with H&E. To analyze the infiltration level of the trachea grafts, a semi-quantitative scoring system was used with the following five grades: 0, no infiltration; 1, minimal infiltration, scattered or diffuse cellular infiltrates; 2, mild infiltration, diffuse cellular infiltrates with one area of dense infiltrate; 3, moderate infiltration, more than one area of dense infiltrate; and 4, severe infiltration, a thick layer of dense cellular infiltrates. To analyze the obliteration degree of the trachea lumen, the thickness of submucosa (between epithelium and cartilage) was scored using the following five grades: 0, no thickening of submucosa; 1, minimal thickening, focal submucosa thickening; 2, mild thickening, submucosa thickening equal to the thickening of the cartilage ring; 3, moderate thickening, submucosa thickening resulting in a small lumen; and 4, severe thickening, total lumen obliteration. The epithelium was categorized in two types: normal and abnormal based on morphological aspects of epithelial cells, i.e. the differentiation (the presence of ciliated epithelium) and the pseudo-stratification (the presence of pseudostratified epithelium layer). The coverage of the trachea by normal epithelium and abnormal epithelium was scored in percentage.

Immunohistochemistry Frozen tracheas were cut into 5µm thick cross sections, and were immunohistochemically stained. The primary monoclonal antibodies were rat antibodies specific for mouse CD5+, CD4+, CD8+, CD45RB+ T cells and macrophages as described in previous chapters and anti-CD45RB mAb (MB4G4, purchased from ATCC). The secondary antibody was rabbit-anti-rat peroxidase labeled (Jackson), and 3-amino 9-ethylcarbazole (Sigma) was used as an immunoperoxidase substrate. Slides were counterstained with Mayer’s haematoxylin. Trachea infiltrating cell phenotypes were analyzed only inside the trachea ring in three sections from each trachea segment. A semi-quantitative scoring system was used as previously described (16) with five grades: 0, no positive cells; 1, scattered positive cells; 2, small clusters or one dense area with positive cells; 3, more than one area or a large area plus clusters of positive cells; and 4, whole tissue dense positive cells.


89

Statistical analysis Results are expressed either as mean ± SEM or as median plus range. Statistical comparisons for difference between groups were evaluated by Mann-Whitney U test. A p-value < 0.05 was considered statistically significant.

Results

Anti-CD45RB antibodies inhibit cellular infiltration in transplanted trachea allografts Trachea grafts from the untreated group revealed high cellular infiltration levels (median value 2; both inside and outside of the cartilage) indicating an active cellular response (Table 1). The infiltrating cells were mostly CD4+ and CD8+ T cells and macrophages (Table 2). In contrast, grafts from anti-CD45RB mAb treated animals showed a significantly lower level of cellular infiltration inside the trachea cartilage (Table 1, P=0.011) which was due to a lower level of CD4+ and CD8+ T cell infiltration (Table 2). Macrophage infiltration however was similar in grafts from untreated or treated animals (Table 2). Table 1. Cellular infiltrations inside/outside trachea ringa (H&E)

Mouse number 1 2 3 4 5 6 7 median

Ab treated grafts 0/2 1/1 0/1 1/1 1/2 1/1 1/1.5 1/1

Control grafts 2/2 2/2 2/2 1/2 2/3 1/1 - 2b/2c

aScoring standard: 0-no infiltration, 1-minimal infiltration: scattered or diffuse cellular infiltrates, 2-mild infiltration: diffuse cellular infiltrates with one area dense infiltrate, 3-moderate infiltration: more than one area infiltrates, 4-severe infiltration: a thick layer of dense cellular infiltrates; b p=0.011 c p=0.0507

The infiltrating cells in grafts from the untreated group showed a high level of CD45RB expression (Figure 1A). This is in accordance with the high level of cellular infiltration found in H&E stained sections and with the level of T cell infiltration demonstrated by the immunohistochemical staining. In

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contrast, grafts from the mAb treated group showed only a few dim CD45RB expressing T cells (Figure 1B). Table 2. Phenotypes of the infiltrating cells inside the transplanted trachea ringa

Infiltrating cell phenotypes, median (range)

CD4+ CD8+ CD5+ MΦ+ Ab treated grafts

(n=7) 0.5

(0-1) 0.5

(0-0.5) 0.5

(0.5-1.5) 2.5

(1.5-2.5) Control grafts

(n=5) 1.5

(1-1.5) 1.5

(1.5-2) 1.5

(1.5-2) 2

(1.5-2) aScoring standard: 0-no positive cells, 1-scattered positive cells, 2-small clusters or one area with dense positive cells, 3-more than one area or a large area plus clusters of positive cells. 4-whole tissue dense positive cells; Figure 1. A. Graft in non-treated group B. Graft in Ab treated group

Figure 1. Immunohistochemical staining for CD45RB Anti-CD45RB staining showed many clearly positive cells in trachea grafts in Ab non-treated group (A). (Arrow indicates example of positive cell). In contrast, there were only sporadic positive cells in Ab treated group (B). The staining was at 3 weeks after transplantation. (High power light microscope at 100x magnification).


91

Anti-CD45RB antibodies protect airway epithelium and inhibit OAD In grafts from the untreated group, severe epithelium loss had occurred (less than 25% epithelial coverage) while the remaining epithelium was totally abnormal (Figure 2A). The latter was demonstrated by the presence of flattened epithelium (i.e. epithelium without cilia and with less pseudostratification). These grafts also revealed a high level of submucosa thickening (scoring 2.75, Figure 2B) which resulted into moderate luminal obliteration. In grafts from the mAb-treated group however, little epithelium loss was observed, although most of the remaining epithelium appeared abnormal (abnormality range 70-85%). The total epithelium coverage remained high in the trachea lumen (approximately 80% epithelial coverage remaining, Figure 2A). Only a minimal level of submucosa tissue thickening was observed in most of the grafts from the mAb-treated group. Furthermore, the grafts did not show severe luminal obliteration (average score of 1.25, Figure 2B). Figure 2. A

. Ab treated non-treated0

25

50

75

100total coverageabnormal epith.

epith

eliu

m c

over

age

%

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B

non-treated Ab treated0

1

2

3

4lu

min

al o

blite

ratio

n

Figure 2. Epithelium coverage, abnormality and luminal obliteration of trachea transplants. Almost 80% of the epithelium was preserved in Ab treated grafts. However, the appearance of the epithelium was more than 55% abnormal (A). The non-treated group showed severe epithelium loss up to about 80% and absence of normal epithelium (A). Obliteration grading showed the thickening of the submucosa at a mild to moderate level in non-treated grafts (B). In the Ab treated group, submucosa thickening was from minimal to mild level and no obvious obliteration of the lumen was observed (B). Error bars in Figure 2A indicate the standard error of the mean and the values in Figure 2B represent the median and the range.

Discussion

In this study we demonstrate a protective effect of anti-CD45RB mAb treatment on donor airway epithelium with a prominent reduction in the development of OAD. Treatment of trachea recipients with anti-CD45RB mAb diminished the T cell infiltration level, improved the luminal epithelium coverage and inhibited luminal obliteration. The development of abnormal epithelium however was not prevented by anti-CD45RB mAb treatment.

The development of epithelium abnormality and only partial epithelium loss, as observed in the anti-CD45RB mAb treated group, resembled the outcome of previous trachea transplantation experiments (16). In that study, we transplanted tracheas from transgenic mice, expressing human epithelial glycoprotein-2 (hEGP-2) exclusively on epithelial cells, to syngeneic non–transgenic mice. After transplantation a hEGP-2-directed immune response was induced and this response was restricted to the grafted airway epithelium. Immunological analysis indicated that this was a predominantly antibody


93

mediated response. These epithelium specific immune responses resulted in abnormal epithelium and mild luminal obliteration. Although we did not analyze the development of alloantibodies in anti-CD45RB treated mice, the epithelium injury, i.e. development of abnormal epithelium, is most likely the result of a humoral response. Although the presence of abnormal epithelium does not appear to lead to fibroproliferation in the short term, it may be less favorable for graft survival in the long-term. CD4-knockout mice showed a delayed development of partial OAD on days 60 and 90 and of complete OAD by day 180 (6). It would be interesting to perform a follow-up study in animals for a longer period than 21 days in our present treatment protocol, to see if the development of OAD is delayed or if tolerance is achieved.

The diminished infiltration of CD4 and CD8 cells after treatment with anti-CD45RB is the most likely reason for the improvement in epithelial coverage and the inhibition of lumen obliteration. In kidney and islet transplantation models the total amount of infiltrating lymphocytes in the grafts was similar in anti-CD45RB treated and untreated mice (11,17). In those models the numbers of CD4 cells were approximately similar but there was a definite decline in the amount of infiltrating CD8 cells. In an islet xenograft model with anti-CD45RB monotherapy a delay in the infiltration of lymphocytes in the grafts prolonged the survival of the islets but could not induce tolerance (10). These experiments show the necessity of the presence of CD4 cells in the process of tolerance. In our model we find less infiltration of T cells but no shift to mostly CD4 cells; long-term experiments have to be performed to show if tolerance in this model is achieved.

The difference in CD45RB expression in treated and untreated grafts may reflect another mechanism of CD45RB antibodies, as has been shown in a pancreas allograft transplantation model in SCID mice (14). In this model SCID mice were repopulated with either CD4CD45RBhigh or CD4CD45RBlow cells, and only the CD45RBhigh expressing cells could reject the grafts. When CD45RBlow expressing cells were mixed in with the CD45RBhigh expressing cells the grafts were protected against rejection. The CD45RBhigh population contains naive and Th1 cell populations and the CD45RBlow population contains predominantly Th2 cells, but it is becoming clear that the regulatory T cell population is also contained in the CD45RBlow population. The low number of infiltrating lymphocytes in the treated trachea grafts is an indication for a milder, more tolerance inducing environment.

As indicated above, the incomplete protection of the epithelium by the anti-CD45RB monotherapy stresses the necessity for additional

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immunosuppression to prevent graft damage. In this respect it is important to note that regular doses of immunosuppressive calcineurin inhibitors, such as CsA, have been shown to abrogate the protective effects of anti-CD45RB mAb on long term graft survival (18), but lower doses may be beneficial (9,19). Additional treatment with rapamycin may be particularly useful since this drug, in addition to its T cell inhibitory effects, effectively reduces the formation of alloantibodies (20). When tested in combination with anti-CD45RB antibodies in a vascularized mouse allograft cardiac model rapamycin indeed acted synergistically (19). Another very effective combination with anti-CD45RB is the blockade of costimulation through CD154 as tested in several modes (21-24). In the mouse cardiac allograft model the combination of anti-CD45RB and anti-CD154 effectively prevented chronic allograft vasculopathy (19).

In conclusion, anti-CD45RB mAb monotherapy diminished the T cell response towards trachea allografts and prevented severe trachea airway epithelium loss and, consequently, strongly reduced the occurrence of OAD. Acknowledgement The authors thank Marja van Luyn, Greetje Groen and Linda Brouwer for the help with the histology analysis and Pieter Klok for the help with antibody injections. This work was partly supported by an Ubbo Emmius Scholarship of the University of Groningen to Ning Qu.


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Reference



3. Kawahara K, Hiratsuka M, Mikami K et al. Obliterative airway disease and graft stenting in pig-to-dog tracheal xenotransplantation. Jpn J Thorac Cardiovasc Surg 2001:49: 53-57.




7. Richards DM, Dalheimer SL, Hertz MI, Mueller DL. Trachea allograft class I molecules directly activate and retain CD8+ T cells that cause obliterative airways disease. J Immunol 2003:171: 6919-6928.


9. Parry N, Lazarovits AI, Wang J et al. Cyclosporine inhibits long-term survival in cardiac allografts treated with monoclonal antibody against CD45RB. J Heart Lung Transplant 1999:18: 441-447.

10. Visser L, Poppema S, de Haan B et al. Prolonged survival of rat islet xenografts in mice after CD45RB monotherapy. Transplantation 2004:77: 386-391.

11. Lazarovits AI, Visser L, Asfar S et al. Mechanisms of induction of renal allograft tolerance in CD45RB-treated mice. Kidney Int 1999:55: 1303-1310.

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12. Zhong RZ, Lazarovits AI. Monoclonal antibody against CD45RB for the therapy of rejection and autoimmune diseases. J Mol Med 1998:76: 572-580.

13. Bottomly K, Luqman M, Greenbaum L et al. A monoclonal antibody to murine CD45R distinguishes CD4 T cell populations that produce different cytokines. Eur J Immunol 1989:19: 617-623.

14. Davies JD, O'Connor E, Hall D, Krahl T, Trotter J, Sarvetnick N. CD4+ CD45RB low-density cells from untreated mice prevent acute allograft rejection. J Immunol 1999:163: 5353-5357.

15. Powrie F, Correa-Oliveira R, Mauze S, Coffman RL. Regulatory interactions between CD45RBhigh and CD45RBlow CD4+ T cells are important for the balance between protective and pathogenic cell-mediated immunity. J Exp Med 1994:179: 589-600.


17. Basadonna GP, Auersvald L, Khuong CQ et al. Antibody-mediated targeting of CD45 isoforms: a novel immunotherapeutic strategy. Proc Natl Acad Sci U S A 1998:95: 3821-3826.

18. Fecteau S, Basadonna GP, Freitas A, Ariyan C, Sayegh MH, Rothstein DM. CTLA-4 up-regulation plays a role in tolerance mediated by CD45. Nat Immunol 2001:2: 58-63.

19. Sho M, Harada H, Rothstein DM, Sayegh MH. CD45RB-targeting strategies for promoting long-term allograft survival and preventingchronic allograft vasculopathy. Transplantation 2003:75: 1142-1146.

20. Schmidbauer G, Hancock WW, Wasowska B, Badger AM, Kupiec-Weglinski JW. Abrogation by rapamycin of accelerated rejection in sensitized rats by inhibition of alloantibody responses and selective suppression of intragraft mononuclear and endothelial cell activation, cytokine production, and cell adhesion. Transplantation 1994:57: 933-941.

21. Sutherland RM, McKenzie BS, Zhan Y et al. Anti-CD45RB antibody deters xenograft rejection by modulating T cell priming and homing. Int Immunol 2002:14: 953-962.


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22. Rayat GR, Gill RG. Indefinite Survival of Neonatal Porcine Islet Xenografts by Simultaneous Targeting of LFA-1 and CD154 or CD45RB. Diabetes 2005:54: 443-451.

23. Camirand G, Rousseau J, Ducharme ME, Rothstein DM, Tremblay JP. Novel duchenne muscular dystrophy treatment through myoblast transplantation tolerance with anti-CD45RB, anti-CD154 and mixed chimerism. Am J Transplant 2004:4: 1255-1265.

24. Lee EN, Kim EY, Lee J et al. Changes in expression of T-cell activation-related molecules and cytokines during tolerance induction in an allogeneic skin transplantation murine model. Transplant Proc 2004:36: 2425-2428.

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Chapter 6

General Discussion

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Heterotopic tracheal allografts in small rodents have been shown to share histopathological characteristics with human OB (1) and therefore provide a suitable animal model for the study of OB (2;3) , although the pathogenesis may differ. Allografts in small rodents develop obliterative airway disease (OAD) within 3-4 weeks after transplantation (4-6). Histopathological analysis of these grafts reveals an early epithelium loss followed by fibroblast overgrowth; the overgrowing fibroblasts occlude the airway lumen. Human OB and animal OAD are both characterized histologically by airway epithelium damage and fibrotic luminal obliteration.

To study the airway epithelium and its behavior in OAD after transplantation, we used different animal models. In one part of the study the trachea transplantation model was used in rats (chapter 2), in other parts in mice (chapters 3, 4, and 5). These trachea transplant models facilitate the examination of the pathogenesis of the disease and the elucidation of the cellular and molecular mechanisms involved in its development. The model provides a less technically demanding alternative to whole lung transplantation in small rodents and may lead to quicker identification of new treatments that might prevent the development of post-transplantation OB in patients. The experiments described in this thesis address several aspects of the role of airway epithelial cells and its behavior in OAD. These aspects are 1) the role of epithelium injury in OAD occurrence during the post-transplant period (chapter 2), 2) the role of epithelium specific immune responses in this injury (chapters 3 and 4) and 3) a possible therapeutic option through the application of C45RB antibodies for OAD prevention (chapter 5). Airway epithelium plays important role inhibiting OAD Our work supports the idea that airway epithelium is the main target tissue during alloimmune rejection and plays a crucial role in development of OAD (7-10). In chapter 2, results are presented upon transplantation of allogenic trachea grafts, which are in line with other studies of animal OAD models (3;6;11). Severe loss of epithelium was found to start at the first week after transplantation with total loss within 3 weeks. Full development of OAD was present at 4 weeks. It is interesting to note that the fibroproliferation started at week 2 after transplantation, secondary to airway epithelium injury and ultimate loss (chapter 2) as a result of acute rejection in our rat trachea

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transplants. Furthermore, a direct effect of epithelial cells on fibroproliferation was demonstrated by grafting tracheas without epithelium, so-called denuded tracheas (chapter 2). In denuded allografts, immediate fibroproliferation was observed after grafting and fibroproliferation started within 6 days. Also in denuded isografts, which are not exposed to acute rejection, immediate fibroproliferation was observed after grafting. Interestingly, after partial denudation of isografts, the fibroproliferation stopped at the time point when epithelium started to recover (day 10). In addition, early fibroproliferation could be inhibited in denuded isografts by growth of seeded syngeneic epithelial cells. These findings indicate that the absence of epithelium results in fibroproliferation and that OAD is not the result from transplant rejection only, but could also result from other epithelium-targeted tissue injuries. It is likely that this is a reflection of a general tissue ‘injury-repair’ process in which epithelial cells are the injured targets and fibroblasts are the repair substitutes if epithelial cells are not able to regenerate and cope with the damage (12;13). The interaction between epithelial cells and fibroblasts has been investigated in several studies (14). These indicate that epithelial cells release various cytokines which are involved in ‘epithelial cell-fibroblast’ interaction, such as fibrogenic growth factor (FGF) (15), prostaglandin E2 (PGE2) and transforming growth factor (TGF) beta (14;16). The FGF is known to induce apoptosis in epithelial cells and to stimulate fibroproliferation (15), whereas PGE2 and TGF-beta are the inhibitor and stimulator of fibroproliferation, respectively (14;17-19).

During the process of epithelial cell injury, for example during transplant rejection or chemical denudation, epithelial cells start to release FGF before they get into apoptosis (15). As this process continues, decreasing levels of PGE2 and increasing level of TGF-beta are released by the epithelial cells, giving fibroblasts the opportunity to grow. As a result, fibroblasts will replace the lost epithelium and, in addition, will grow into the lumen in an unregulated manner and finally obliterate the lumen as a consequence. From such a concept it can be put forward that epithelial cells are the main mediator in maintaining the airway architecture after transplantation. Presence of epithelial cells largely inhibits the fibroproliferation while OAD occurs as a result of an unbalanced situation. Thus, epithelium loss as a result of transplant rejection will initiate fibroproliferation and will eventually result into luminal obliteration. Immune responses are a major component in the transplant rejection process. It is well documented that alloantigen specific antibodies (IgM and IgG), and

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more importantly, immune cells (mainly CD4+, CD8+ T cells) contribute to the initiation and execution of destruction in lung transplantation (20-22) and trachea transplantation (23-25) Antibody responses in OAD Alloantibodies against donor tissue may be directly involved in OB and OAD. Clinical investigations in lung transplantation show that both anti-HLA class I and II antibodies are associated with epithelium damage in patients with OB (20;26-28). In an animal study antibody-mediated complement activation has been shown to induce epithelium injury and to contribute to the development of OAD in rat tracheal allografts. (29). These results are in line with our findings in chapter 3, that alloantibodies appear as early as one week after transplantation in recipients of fully MHC mismatched tracheas. In these studies immune responses towards tissues other than epithelium could not be excluded to play a role in airway obliteration. In our transgenic mice model, antibodies are directed exclusively against an epithelium-specific antigen. Although cytotoxic in vitro, these antibodies didn't cause severe loss of epithelium (chapter 3). In immunized mice with higher levels of epithelium-specific antibodies before transplantation (chapter 4) the epithelium injury was significantly increased. Yet, no severe OAD developed in the transplanted tracheas at 1, 3 and 6 weeks after transplantation

Our results show that the presence of cytotoxic antibodies is insufficient to cause severe epithelial damage with subsequent OAD. Therefore it seems logical to assume that in trachea transplant models T cell responses are also involved in the development of OAD. Cellular responses in OAD It has been shown that both CD4+ and CD8+ T cells play an important role in obliteration of trachea transplants (11;30). In allograft recipients, significant numbers of pre-existing CD4+ and CD8+ T cells are able to directly recognize alloantigens (11). Indirect recognition through MHC Class II pathway mediated by CD4+ T cells and direct recognition through MHC Class I pathway mediated by CD8+ T cells are suggested to be the main mechanisms of cellular immune response (chapter 3, (31). Particularly, CD8+ T cells, namely the cytotoxic T cells (CTL) are the effector cells directly activated by class I

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molecules and are shown to be mainly responsible for inducing OAD in murine trachea transplant models (32;33). This has been confirmed by T cell-adoptive transfer model (32), where transferred alloreactive CD8+ T cells could efficiently induce epithelium loss leading to the occurrence of OAD.

In our rat and mouse trachea allografts, cellular infiltration by CD4+ and CD8+ T cells started from 10 days after transplantation, indicating an acute rejection of the tracheas (chapters 2 and 3). In the hEGP-2 transgenic grafts low numbers of CD8+ T cells were induced: the CD8+/CD4+ ratio was lower than in the allografts. This may indicate that the CTL response was weak in our transgenic model compared to allotransplantation, and was not capable of inducing severe epithelium damage. This weak cellular response (in combination with the antibody response) resulted in a mild form of OAD without total obliteration of the lumen (chapters 3 and 4).

Further indication of the role of cellular responses in the development of OAD is the effect of the blocking of T cell responses by anti-CD45RB antibodies (chapter 5). This treatment reduced the level of epithelium injury from heavy loss (allografts without antibody treatment) to only abnormality (flattened epithelium in antibody treated grafts) and thus prevented OAD developed (chapter 5). Other studies diminishing T cell responses by immunosuppressive agents (34;35) or T cell depletion show the same effect on protecting epithelium and preventing OAD. These data suggest that the T cell responses contribute to epithelium injury, inducing the development of OAD.

We conclude that antibody responses together with cellular responses cause OAD in trachea transplants. Our transgenic transplantation model allows for the first time to isolate the immune responses against airway epithelium. The studies with this model show that the immune responses may make the epithelium abnormal but that this is not enough to cause OAD. OAD is a result from epithelium loss We think that OAD is only found in trachea transplants after complete loss of epithelium, as shown in our studies (chapter 2, 3) and other studies (8-10). In isografts, epithelium is mildly injured by the transplantation procedure without loss and epithelium abnormality persists for only a short term (chapter 2, 5). These isografts usually recover without obliteration. A similar process is found in the hEGP-2 transgenic mice transplantation model: mild injury and abnormality with minimal loss of epithelium, resulting in minimal obliteration in

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the grafts (chapter 3, 4). In fact, this minimal obliteration was caused by submucosa thickening as a result of massive immune cell infiltrates rather than by fibroproliferation. This is in sharp contrast to the severe OAD as seen in allotransplants, where severe loss of epithelium correlates with fibroproliferation and obliteration of the lumen. Furthermore, OAD is also found in the absence of rejection after removal of epithelium (denudation) from trachea isografts (chapter 2(8)). This indicates that epithelium loss is the primary cause of fibroproliferation. Both in isografts and allografts, epithelium injury induces a general process in which injury and repair are balanced to a certain point. When the injury causes only a mild degree of epithelium injury (as we observed in the isografts and hEGP-2 grafts) the epithelium regeneration will be the dominant process. Thus, the integrity of luminal epithelium coverage can be maintained and fibroproliferation is not activated. Once the injury causes severe epithelium damage (as we observed in allografts), the epithelium regeneration process can not sufficiently maintain the epithelium integrity, thus fibroproliferation as alternative repairing process is activated. This will eventually manifest as OAD. In this concept we think that obliteration of airways can be prevented as long as protection of the epithelium is sufficient to avoid severe loss of epithelial cells. Epithelium protection and regeneration Different approaches to prevent OB/OAD have been tried in different centers. As airway epithelium injury is a result from alloimmune responses in lung transplantation, immunosuppressive agents such as Cyclosporin A and FK-506 are widely used to suppress immune responses and prolong graft survival (20;36-40). The principles of using these drugs are quite similar: suppressing cellular immunity. Common side effects of these drugs are nephrotoxicity and non-specific suppression of the immune system. In this way, the risk for infections and tumors increases.

For Cyclosporin A it has been proposed that it has an effect on epithelial cells: In vitro studies indicate that Cyclosporin A stimulates epithelial cells to produce cytokines that enhance fibroproliferation. (38;41). Therefore, new agents or methods to sufficiently suppress immune responses but avoid side effects are needed. In chapter 5 we used anti-CD45RB monoclonal antibodies (mAb) to modulate T cell response (42), protect airway epithelium, and prevent OAD. In this study we demonstrated the protective effect of anti-CD45RB mAb treatment on donor airway epithelium and its subsequent effect

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in OAD prevention. Treatment of trachea recipients with anti-CD45RB mAb resulted in diminished T cell infiltration, a preserved luminal epithelium coverage and inhibition of luminal obliteration. Using the trachea transplantation model, we showed an alternative way for airway epithelium protection with specific T cells suppression for OAD prevention.

Another approach to prevent development of OAD would be the protection of airway epithelial cells by activation of 'protective genes' such as HO-1 (43) known to be capable to reduce local inflammation at the epithelial level in case of allograft reactivity. This might be achieved by exposure of donor lung to carbon monoxide, a treatment that was effective in a rat lung transplantation model (43). This treatment is worth further investigation on how applicable it is in clinical situation to protect epithelium. Apart from protection, restoration of the epithelium after loss of its integrity has been shown to be effective in OAD prevention. The seeding of viable epithelial cells in denuded airways has successfully regenerated the epithelium and has prevented OAD (chapter 2) (8). Also, the reparation of damaged epithelium by recipient cells has been shown to be capable of OAD inhibition in transplanted allograft.

Although it is not evidential (44), stem cells may play a role in epithelium repair. In kidney transplantation model, stem cells were reported to re-build the kidney epithelial cells (45). A pilot study about bone-marrow derived stem cells in lung injury model suggested that bone-marrow stem cells could differentiate into epithelial cells repairing lung function. (46). The use of donor or recipient stem cells might be a future perspective for development of epithelium regenerative treatment.

To prevent injury during the clinical lung transplantation procedure, extra precautions in the clinical handling techniques, ranging from donor procedures to surgical transplantation, should be taken regarding the epithelium protection. During the period of brain death of the lung donor, inflammatory cytokines are released which are harmful to epithelial cells and cause injury (47). Inflation of the donor lung with oxygen before storing into preservation should be strictly according to the protocol; over-inflation should be avoided because high pressure may damage epithelium integrity. Furthermore, the airway should be kept clean and free from any toxic chemical fluids that may cause epithelium injury.

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Remarks on animal OAD study: advantages and limitations All animal OAD study just as in other animal studies, share the benefit of short investigation time, large in experimental animal numbers and cheap in cost. In transplant models, further benefits such as inbred strain availability for immune system study, transgenic possibility and tissue engineering, allow insight research for certain single aspect in a set up without encountering the clinical complexity. Especially for OB study, clinical OB is defined mainly by symptoms and confirmed by biopsies (20;21;36;48-54) which means every patient is different from another. It is difficult to find a group of similar OB patient that share the cause and stage in OB progress for a study. Also, the patient-tailored clinical treatment against OB might complicate the situation. These difficulties could be avoided in animal OAD models. Studies may be available in animal OAD models, in which a treatment can be standardized in a way that is impossible to test in patients. For example, to investigate the effect of immunosuppressive agents, treated groups can be compared with untreated groups. In transgenic animal models, the influence of a specific gene can be investigated for its behavior under certain circumstances (chapter 3, 21). Without animal models, we could not develop our scientific knowledge of the mechanism of human diseases.

The limitations of using animals for human disease study are also obvious. Simplest to say, they are not human. Animals and humans may have much similarity in body structure, physiology and pathology, yet, the differences between species in pathogenesis and pathophysiology may result in discordant processes. A limitation of the animal OAD model is that its pathogenesis seems to differ from that of OB. The process of the development of OB, for instance, may take several years in human lung transplants while in murine OAD models it takes only weeks or months. The use of such a model in animal experiments may lead to incorrect conclusions and to controversy in understanding of human diseases.

Another critical difference is that animals do not ‘complain’ as human does. Patients can describe the disease history and symptoms, which helps clinicians to make diagnose. In the clinical situation, OB develops gradually and the complaints of patients may lead to an early diagnosis. In an animal OAD model, the investigation time points are assigned arbitrarily and may introduce error in judgment. Thus the conclusions and the understandings based on animal model should be carefully interpreted into humans.

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Possible implications from this study for clinical OB management Our animal research is aimed to benefit clinical treatment. In general, the work in this thesis shows that airway epithelium is playing an important role in animal OAD and that protection of airway epithelium is crucial in preventing OAD. Based on the findings of the four studies in this thesis, the following suggestions might be useful for the management of lung transplantation patients.

Before and after transplantation, the induction of self-protective gene expression (HO-1, (43)) may help to preserve the integrity of the donor lung epithelium. This may be done by inhalation of a safe dose of CO, as has been discussed in chapters 2 and 3. It has been successfully used in animal model for airway protection (55)

The specific blocking of CTL response using anti-CD45RB, as described in chapter 5, may be applicable in clinical OB treatment. It is an increasingly used antibody in other organ transplantations (56). It could be also used combining other immunosuppressive drugs to reduce their dose and non-specific toxicity towards normal immune cells.

Although we did not show a severe destructive effect of antibodies towards donor epithelium, we believe it is still helpful to block its function, such as by blocking the complement binding pathway as discussed in chapter 4.

Further prospective is based on the epithelial cells seeding study. As it is difficult to envision how this is applicable in clinical situation now, but the stem cells transplant research widened the possibility for reconstruction/regeneration of the injured epithelium. Investigations on this direction may be promising on rebuilding the injured epithelium after transplantation or even already before transplantation.

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Reference 1. Hertz MI, Jessurun J, King MB, Savik SK, Murray JJ. Reproduction of the

obliterative bronchiolitis lesion after heterotopic transplantation of mouse airways. Am J Pathol 1993:142: 1945-1951.

2. McKane BW, Fernandez F, Narayanan K et al. Pirfenidone inhibits obliterative airway disease in a murine heterotopic tracheal transplant model. Transplantation 2004:77: 664-669.



5. Reichenspurner H, Soni V, Nitschke M et al. Obliterative airway disease after heterotopic tracheal xenotransplantation: pathogenesis and prevention using new immunosuppressive agents. Transplantation 1997:64: 373-383.

6. Yonan NA, Bishop P, el Gamel A, Hutchinson IV. Tracheal allograft transplantation in rats: the role of immunosuppressive agents in development of obliterative airway disease. Transplant Proc 1998:30: 2207-2209.



9. Fernandez FG, Jaramillo A, Chen C et al. Airway epithelium is the primary target of allograft rejection in murine obliterative airway disease. Am J Transplant 2004:4: 319-325.

10. Ikonen TS, Brazelton TR, Berry GJ, Shorthouse RS, Morris RE. Epithelial re-growth is associated with inhibition of obliterative airway disease in orthotopic tracheal allografts in non-immunosuppressed rats. Transplantation 2000:70: 857-863.

CHAPTER 6

110


12. Tesfaigzi Y. Processes involved in the repair of injured airway epithelia. Arch Immunol Ther Exp (Warsz ) 2003:51: 283-288.

13. Crouch E. Pathobiology of pulmonary fibrosis. Am J Physiol 1990:259: L159-L184.


15. Jaramillo A, Smith CR, Maruyama T, Zhang L, Patterson GA, Mohanakumar T. Anti-HLA class I antibody binding to airway epithelial cells induces production of fibrogenic growth factors and apoptotic cell death: a possible mechanism for bronchiolitis obliterans syndrome. Hum Immunol 2003:64: 521-529.

16. Ihn H. Pathogenesis of fibrosis: role of TGF-beta and CTGF. Curr Opin Rheumatol 2002:14: 681-685.

17. Vaillant P, Menard O, Vignaud JM, Martinet N, Martinet Y. The role of cytokines in human lung fibrosis. Monaldi Arch Chest Dis 1996:51: 145-152.

18. Martinet Y, Menard O, Vaillant P, Vignaud JM, Martinet N. Cytokines in human lung fibrosis. Arch Toxicol Suppl 1996:18: 127-139.




22. Etienne B, Mornex JF. [Immunological aspects of lung transplantation]. Rev Mal Respir 1996:13: S15-S22.



25. Smith MA, Jaramillo A, SivaSai KS et al. Indirect recognition and antibody production against a single mismatched HLA-A2-transgenic

General discussion

111

molecule precede the development of obliterative airway disease in murine heterotopic tracheal allografts. Transplantation 2002:73: 186-193.

26. Reznik SI, Jaramillo A, Zhang L, Patterson GA, Cooper JD, Mohanakumar T. Anti-HLA antibody binding to hla class I molecules induces proliferation of airway epithelial cells: a potential mechanism for bronchiolitis obliterans syndrome. J Thorac Cardiovasc Surg 2000:119: 39-45.

27. Jaramillo A, Smith MA, Phelan D et al. Development of ELISA-detected anti-HLA antibodies precedes the development of bronchiolitis obliterans syndrome and correlates with progressive decline in pulmonary function after lung transplantation. Transplantation 1999:67: 1155-1161.

28. Reinsmoen NL, Nelson K, Zeevi A. Anti-HLA antibody analysis and crossmatching in heart and lung transplantation. Transpl Immunol 2004:13: 63-71.



31. Smith CR, Jaramillo A, Duffy BF, Mohanakumar T. Airway epithelial cell damage mediated by antigen-specific T cells: implications in lung allograft rejection. Hum Immunol 2000:61: 985-992.

32. Richards DM, Dalheimer SL, Hertz MI, Mueller DL. Trachea allograft class I molecules directly activate and retain CD8+ T cells that cause obliterative airways disease. J Immunol 2003:171: 6919-6928.


34. King MB, Jessurun J, Savik SK, Murray JJ, Hertz MI. Cyclosporine reduces development of obliterative bronchiolitis in a murine heterotopic airway model. Transplantation 1997:63: 528-532.

35. Adams BF, Berry GJ, Huang X, Shorthouse R, Brazelton T, Morris RE. Immunosuppressive therapies for the prevention and treatment of obliterative airway disease in heterotopic rat trachea allografts. Transplantation 2000:69: 2260-2266.

CHAPTER 6

112

36. Boehler A, Kesten S, Weder W, Speich R. Bronchiolitis obliterans after lung transplantation: a review. Chest 1998:114: 1411-1426.


38. Borger P, Kauffman HF, Timmerman JA, Scholma J, van den Berg JW, Koeter GH. Cyclosporine, FK506, mycophenolate mofetil, and prednisolone differentially modulate cytokine gene expression in human airway-derived epithelial cells. Transplantation 2000:69: 1408-1413.

39. Glanville AR, Baldwin JC, Burke CM, Theodore J, Robin ED. Obliterative bronchiolitis after heart-lung transplantation: apparent arrest by augmented immunosuppression. Ann Intern Med 1987:107: 300-304.

40. Kur F, Reichenspurner H, Meiser BM et al. Tacrolimus (FK506) as primary immunosuppressant after lung transplantation. Thorac Cardiovasc Surg 1999:47: 174-178.

41. Hostettler KE, Roth M, Burgess JK et al. Cyclosporine A mediates fibroproliferation through epithelial cells. Transplantation 2004:77: 1886-1893.


43. Visner GA, Lu F, Zhou H, Latham C, Agarwal A, Zander DS. Graft protective effects of heme oxygenase 1 in mouse tracheal transplant-related obliterative bronchiolitis. Transplantation 2003:76: 650-656.

44. Emura M. Stem cells of the respiratory tract. Paediatr Respir Rev 2002:3: 36-40.

45. Oliver JA. Adult renal stem cells and renal repair. Curr Opin Nephrol Hypertens 2004:13: 17-22.

46. Yamada M, Kubo H, Kobayashi S et al. Bone marrow-derived progenitor cells are important for lung repair after lipopolysaccharide-induced lung injury. J Immunol 2004:172: 1266-1272.

47. Avlonitis VS, Fisher AJ, Kirby JA, Dark JH. Pulmonary transplantation: the role of brain death in donor lung injury. Transplantation 2003:75: 1928-1933.

48. Fournier M, Groussard O, Sleiman C, Mal H, Darne C, Pariente R. [Bronchiolitis obliterans after lung transplantation]. Presse Med 1992:21: 816-820.

General discussion

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49. Rajagopalan N, Maurer J, Kesten S. Bronchodilator response at low lung volumes predicts bronchiolitis obliterans in lung transplant recipients. Chest 1996:109: 405-407.

50. Estenne M, Van Muylem A, Knoop C, Antoine M. Detection of obliterative bronchiolitis after lung transplantation by indexes of ventilation distribution. Am J Respir Crit Care Med 2000:162: 1047-1051.

51. Lentz D, Bergin CJ, Berry GJ, Stoehr C, Theodore J. Diagnosis of bronchiolitis obliterans in heart-lung transplantation patients: importance of bronchial dilatation on CT. AJR Am J Roentgenol 1992:159: 463-467.

52. Aboyoun CL, Tamm M, Chhajed PN et al. Diagnostic value of follow-up transbronchial lung biopsy after lung rejection. Am J Respir Crit Care Med 2001:164: 460-463.

53. Baz MA, Layish DT, Govert JA et al. Diagnostic yield of bronchoscopies after isolated lung transplantation. Chest 1996:110: 84-88.

54. Reynaud-Gaubert M, Thomas P, Badier M, Cau P, Giudicelli R, Fuentes P. Early detection of airway involvement in obliterative bronchiolitis after lung transplantation. Functional and bronchoalveolar lavage cell findings. Am J Respir Crit Care Med 2000:161: 1924-1929.

55. Ameredes BT, Otterbein LE, Kohut LK, Gligonic AL, Calhoun WJ, Choi AM. Low-dose carbon monoxide reduces airway hyperresponsiveness in mice. Am J Physiol Lung Cell Mol Physiol 2003:285: L1270-L1276.

56. Sho M, Harada H, Rothstein DM, Sayegh MH. CD45RB-targeting strategies for promoting long-term allograft survival and preventingchronic allograft vasculopathy. Transplantation 2003:75: 1142-1146.

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Chapter 7

Summary

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Summary

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Summary

Obliterative bronchiolitis (OB) is one of the most severe complications after lung transplantation and affects about 35-50% of recipients within 5 years after transplantation. Features of OB include airway lymphocytic inflammation, epithelium damage and fibrotic occlusion of the airway's lumen. Animal models for studying OB in animal airway transplants, referred to as obliterative airway disease (OAD), suggest that airway epithelium plays a role in the pathogenesis of OAD.

Chapter 1 describes the background of this thesis, introduces a general hypothesis, and lists specific questions for the experimental investigations. Based on a review of the relevant literature, the author points at the importance of airway epithelium in OB/OAD occurrence: normal airway epithelium inhibits fibroproliferation; upon allotransplantation, transplant injury of airway epithelium may cause severe epithelium injury; the loss of epithelium integrity results in fibroproliferation; and the overgrowth of the fibroblasts eventually results in OB/OAD. This leads to the general hypothesis for the mechanism of OAD/OB: that transplant injury causes airway epithelium damage and that excessive, persistent loss of epithelium results in fibrosis that eventually leads to the occurrence of OB/OAD.

To investigate the role of airway epithelium injury in OAD, it was

decided to use rat and mouse trachea transplantation models. The investigations were focused on the epithelial cell’s integrity after transplantation under immune responses, such as the airway epithelium injury/repair and its effect on OAD, the possible immunological cause of epithelium injury and the prevention of OAD through epithelium protection.

In chapter 2, the response to injury of airway epithelium was investigated using trachea transplants in rats. Injury was caused by rejection in allogeneic fully MHC-mismatched tracheas and by enzymatic denudation of epithelium in syngeneic tracheas. To stimulate repair processes and rebuild the integrity of airway epithelium, viable epithelial cells were seeded into the lumen of denuded tracheas. The conclusion from this study is that integrity of airway epithelium is essential for trachea transplants to be safeguarded from OAD.

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In chapter 3 the hEGP-2 transgenic mouse was introduced in a new

model of OAD. The hEGP-2 antigen expressed on airway epithelial cells allowed investigation of epithelium specific immune responses. In this study, the recipient's humoral and cellular immune responses against the hEGP-2 antigen on the grafts were analyzed, as well as the subsequent development of OAD. It was found that hEGP-2 antigen on the epithelial cells of transgenic trachea transplants induced specific humoral and cellular immune responses towards epithelium. Yet, these specific responses did not cause severe loss of epithelium, and only resulted in mild OAD. This is the first time that the effect of a tissue-specific immune response is studied in a transplant setting. It allows for further investigation of epithelium-specific immune responses on epithelium injury in OAD model.

Chapter 4 is focused on the development of OAD under pre-existing epithelium-specific immunity, mimicking the immunity in allotransplantation where alloantibodies and T cells can directly recognize alloantigens in donor airway epithelial cells. Based on the transgenic model described in chapter 3, recipient mice were immunized before transplantation to induce pre-existing immunity against hEGP-2 antigens. Successful immunization was demonstrated by the detection of a high antibody levels against hEGP-2 antigen in blood (humoral) and a positive DTH test in the footpad (cellular) of immunized mice. The pre-existing immunity augmented the degree of epithelial injury in trachea transplants. However, it didn't aggravate the degree of OAD. It was concluded that more extensive epithelial cell loss with stimulation of other graft cells, such as fibroblasts, may be required for full development of OAD.

In chapter 5, it was investigated if OAD in allogeneic trachea transplants could be prevented by anti-CD45RB mAb monotherapy, blocking alloreactive T cells. Mouse trachea allografts were analyzed for cellular infiltration, epithelium integrity and obliteration at three weeks after transplantation. The anti-CD45RB Ab monotherapy was found to diminish the T cell response towards trachea allografts, to improve the epithelium integrity, and to prevent OAD in mice trachea allotransplant.

Chapter 6 is the general discussion of the main findings in this thesis and gives implications for the clinical management of OB. Our experimental

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studies are consistent with the following observations: airway epithelium injury plays an important role in the OAD pathogenesis; the loss of epithelium causes fibroproliferation and results in OAD. We conclude that OAD only develops after complete loss of epithelium in trachea transplants. In case of mild injury of the epithelium (as observed in the isografts and hEGP-2 grafts), the epithelium has the potency to regenerate and restore its integrity, thus suppressing fibroproliferation to obliterate the lumen. In this concept, obliteration of airways can be prevented as long as protection of the epithelium is sufficient to avoid severe loss of epithelial cells.

For clinical lung transplantation the results of the study may indicate that protection of the integrity of airway epithelium is important in prevention of the development of OB. Posttransplant injury of epithelial cells by inflammation may be avoided by careful surgical procedures and induction of self-protective gene expression. Rejection injury should be prevented by optimal immunosuppressive regimens, possibly with anti-CD45RB monoclonal antibodies to block CTL responses. And finally, stem cell transplantation may be a future option to regenerate the integrity of severely injured epithelium.

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Chapter 8

Summary in Dutch

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Samenvatting

Bronchiolitis obliterans (BO) is een van de ernstigste complicaties voor

patiënten na longtransplantatie. Ongeveer 35 tot 50% van de patiënten krijgt

hier binnen 5 jaar na de transplantatie last van. Histologische kenmerken van

BO zijn onder andere lymfocytaire infiltratie, schade aan het epitheel en

fibrotische vernauwing van het lumen. De pathogenese van BO kan worden

onderzocht bij proefdieren met subcutaan getransplanteerde luchtwegen,

meestal trachea's. Hierin ontstaat obliterative airway disease (OAD): een

vernauwing van de getransplanteerde trachea. Bij dit proces lijkt het luchtweg-

epitheel een centrale rol te spelen.

In hoofdstuk 1 wordt de achtergrond van dit proefschrift beschreven,

wordt een algemene hypothese gegeven en worden de onderzoeksvragen

geformuleerd voor de experimentele studies. Een overzicht van de

beschikbare literatuur wijst erop dat luchtwegepitheel belangrijk is bij het

ontstaan van BO/OAD: normaal luchtwegepitheel remt de groei van

fibroblasten; bij transplantatie kan het epitheel sterk beschadigd raken, vooral

als gevolg van rejectie; als de epitheliale bedekking verloren gaat treedt

fibroblastengroei op; door de overmatige groei van fibroblasten ontstaat

BO/OAD. Dit mondt uit in de algemene hypothese voor het mechanisme van

BO/OAD: door transplantatieschade gaat de epitheliale bedekking dermate

verloren dat fibroblastgroei de kans krijgt om het lumen te vernauwen.

De rol van schade aan luchtwegepitheel bij het ontstaan van OAD wordt in dit

proefschrift bestudeerd in trachea's die subcutaan getransplanteerd worden

bij ratten en muizen. De verschillende studies richten zich op schade aan de

epitheliale bedekking als gevolg van immuunreacties na de transplantatie, de

mogelijke immunologische mechanismen van de epitheelschade en de

bescherming van epitheel tegen immuunreacties ter voorkoming van OAD.

In hoofstuk 2 werd de reactie op schade van luchtwegepitheel

onderzocht in tracheatransplantaten bij ratten. De epitheelschade werd

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veroorzaakt door rejectie van allogene (d.w.z. genetisch verschillende)

trachea's en door enzymatische verwijdering van de epitheliale bedekking in

syngene (d.w.z. genetisch gelijke) trachea's. Om na te gaan of herstel van de

epitheelbedekking bespoedigd kon worden, werden levende epitheelcellen

gezaaid in het lumen van de syngene epitheel-loze trachea's. De conclusie

van dit hoofdstuk is dat herstel van de epitheliale bedekking essentieel is om

OAD in de tracheatransplantaten te voorkomen.

In hoofdstuk 3 vormt de transgene hEGP-2 muis de basis voor een nieuw

onderzoeksmodel voor OAD. De hEGP-2 antigenen op het epitheel van

tracheatransplantaten uit deze muis zouden het doel van een specifieke

immuunreactie kunnen zijn. De immuunreactie van niet-transgene ontvangers

werd geanalyseerd op humorale en cellulaire aspecten, met de schadelijke

invloed die de reactie had op het tracheaepitheel. Inderdaad konden humorale

en cellulaire immuunreacties tegen het hEGP-2 epitheel worden aangetoond.

Hoewel de immuunreactie specifiek gericht was tegen het tracheaepitheel,

ontstond er weinig epitheelschade door en ontwikkelde zich geen duidelijke

OAD. Dit unieke model met een specifieke immuunreactie tegen het epitheel

biedt verdere mogelijkheden om de rol van epitheel-specifieke schade bij

OAD te onderzoeken.

In hoofdstuk 4 wordt dit transplantatiemodel gebruikt om de vraag te

onderzoeken of OAD in tracheatransplantaten van hEGP-2 transgene

donoren zich sterker ontwikkelt wanneer de ontvangers van tevoren al

gesensibiliseerd zijn tegen het hEGP-2 antigeen. Dit komt beter overeen met

de situatie van allotransplantatie, waar aanwezige antistoffen en T-cellen

direct alloantigenen op het luchtwegepitheel kunnen herkennen. Dat muizen

goed konden worden gesensibiliseerd bleek uit hoge titers antistoffen tegen

het hEGP-2 antigeen in het bloed en uit een positieve vertraagd-type

overgevoeligheidsreactie tegen hEGP-2 eiwit ingespoten in de voetzool. In de

gesensibiliseerde ontvangers nam ook de epitheelschade in de

tracheatransplantaten toe. Toch versterkte dit niet de ernst van de OAD.

Hieruit wordt geconcludeerd dat OAD zich pas volledig ontwikkelt als de

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epitheelbedekking sterk is aangetast en er daarbij ook andere cellen in het

transplantaat, b.v. fibroblasten, worden gestimuleerd.

In hoofdstuk 5 wordt onderzocht of het ontstaan van OAD in muizen te

voorkomen is door behandeling van de ontvangers met een antistof tegen het

CD45RB antigeen op (geactiveerde) T-cellen. Allogene trachea's werden drie

weken na transplantatie onderzocht op cellulaire infiltratie, schade aan de

epitheelbedekking en vernauwing van het lumen. De anti-CD45RB antistoffen

bleken infiltratie door T-cellen te verminderen, het epitheel te beschermen en

de ontwikkkeling van OAD te vertragen.

In hoofdstuk 6 worden de resultaten uit dit proefschrift bediscussieerd en

worden mogelijke consequenties voor de behandeling van patiënten

aangegeven. Onze experimentele studies bevestigen de geformuleerde

hypothese: schade aan het luchtwegepitheel speelt een belangrijke rol in het

ontwikkelingsproces van OAD; het verlies van epitheelbedekking geeft ruimte

voor groei van fibroblasten en mondt daardoor uit in OAD. We stellen vast dat

OAD zich alleen ontwikkelt als de epitheelbedekking in de trachea volledig

verloren is gegaan. Bij een beperkte schade (zoals in de syngene en hEGP-2

transgene transplantaten) heeft het epitheel een sterke herstelcapaciteit,

waardoor fibroblasten niet de gelegenheid krijgen door hun groei het lumen af

te sluiten. Dit geeft aan dat vernauwing van de luchtwegen in transplantaten

kan worden voorkomen door ervoor te zorgen dat de bedekking met epitheel

intact blijft.

Voor de klinische behandeling van patiënten na longtransplantatie kan deze

studie suggesties voor verbetering geven. Net als in de trachea's, moet de

epitheelbedekking in longtransplantaten behouden blijven om BO te

voorkomen. Bij de transplantatie zou daarom schade aan epitheelcellen

voorkomen kunnen worden door zorgvuldige chirurgische procedures en door

de activiteit van zelf-beschermende genen te stimuleren. Schade aan epitheel

door rejectie zou voorkomen kunnen worden door de immunosuppressieve

behandelschema's verder te optimaliseren, mogelijk in combinatie met anti-

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CD45RB antistoffen tegen geactiveerde T-cellen. Voor de toekomst, tenslotte,

zou in situaties met ernstig beschadigd luchtwegepitheel de transplantatie van

stamcellen het herstel van de epitheelbedekking kunnen bespoedigen.

Chapter 9

Summary in Chinese

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论著摘要

第一章介绍了论文研究的背景知识以及对当前肺移植后阻塞性肺

炎研究不足的批评性讨论。通过系统性文献回顾，作者列出了本论文要研究的

重点问题，提出了呼吸道上皮在该症的发病机理中作用的假设及验证方法。

即：Ａ.常呼吸道上皮细胞对呼吸道上皮内的成纤维化过程起抑制作用；Ｂ. 移

植后免疫反应对呼吸道上皮具损伤作用；Ｃ.吸道上皮细胞损伤促进成纤维化过

程；Ｄ.异常的成纤维化过程造成呼吸道纤维增生从而导致移植后阻塞性肺炎的

发生。为了研究验证以上假设，作者选用了大鼠及小鼠的气管移植为模型。研

究的重点集中在呼吸道上皮细胞损伤／修复对移植后阻塞性肺炎发生过程的影

响及如何通过保护上皮细胞来预防肺移植后阻塞性肺炎。

第二章通过大鼠气管移植模型研究了气管上皮组织在移植后的病

理表现。在呼吸道上皮组织损伤模型中，呼吸道上皮损伤由同种异体移植免疫

反应或活性蛋白酶的酶解作用产生。而损伤后的气管上皮修复则由再植入的活

性上皮细胞完成。结果显示，在同种异体气管移植中，移植后免疫反应造成了

严重的呼吸道上皮损伤，诱发了呼吸道成纤维细胞增生，从而造成了移植后阻

塞性肺炎的发生。在同种同基因气管移植中，没有移植后免疫反应，未发生移

植后阻塞性肺炎。在同种同基因-但移植气管呼吸道上皮缺失的移植模型中，损

伤的呼吸道上皮未能修复，呼吸道成纤维细胞大量增生，最终导致移植后阻塞

性肺炎。而同种同基因-呼吸道上皮缺失-但活性上皮细胞植入的气管模型中，

移植的上皮细胞修复了缺失的呼吸道上皮并抑制了成纤维细胞增生从而也抑制

了移植后阻塞性肺炎的发生。通过该组实验作者得出了如下结论，即：呼吸道

上皮细胞完整性决定了移植后阻塞性肺炎的发生。

第三章重点介绍了 hEGP-2 转基因小鼠气管移植后阻塞性肺炎模

型。在这一模型中，移植后免疫反应被引导直接针对表达 hEGP-2 异种蛋白的

呼吸道上皮细胞，而其他组织细胞因不表达这一蛋白而不受免疫反应影响。所

以，由体液免疫反应及细胞免疫反应引起的免疫损伤机制只作用于表达一种蛋

白的呼吸道上皮细胞。实验结果显示，表达异种蛋白的呼吸道上皮细胞在受体

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中引导产生了大量的针对该上皮细胞异种蛋白的单克隆抗体以及 T 细胞。而这

些免疫反应产生了对上皮组织的损伤作用并最终造成了较轻程度的移植后阻塞

性肺炎。这是第一次利用转基因动物模型在移植研究领域获得了仅针对单一组

织的免疫反应。

第四章本章实验是基于第三章介绍的转基因小鼠气管移植后阻塞

性肺炎模型的延伸研究。主要阐述了在类似同种异体移植条件下，即在有大量

的抗原特异性抗体和抗原特异性免疫细胞存在的条件下移植后阻塞性肺炎的发

展进程。首先，在 hEGP-2 转基因鼠的移植后阻塞性肺炎模型中，受体鼠在移

植前先进行针对 hEGP-2 抗原的特异性免疫增强刺激。免疫增强结果经检验显

示，受体中产生了大量的针对 hEGP-2 的单克隆抗体和免疫细胞。但移植后，

经过增强的免疫反应仅造成呼吸道上皮细胞的进一步损伤（与第三章结果比

较）并未引发更显著程度的移植后阻塞性肺炎。这一结果说明了仅有针对上皮

细胞的免疫反应不足以产生移植后上皮细胞严重损伤及阻塞性肺炎。而其他组

织细胞的参与，如成纤维细胞的参与可能是产生移植后阻塞性肺炎的必要条

件。

第五章研究了如何通过注射抗 T 细胞抗体预防移植后阻塞性肺炎

的发生。本章实验应用了同种异体小鼠移植后阻塞性肺炎模型。在移植前和移

植后对受体鼠进行了 CD45RB 的单克隆抗体注射以阻断受体的 T 细胞的免疫反

应。实验结果显示，CD45RB 的单克隆抗体注射可以有效地阻断 T 细胞的免疫

反应。移植后气管上皮表现为轻度损伤，未发生严重缺损。无呼吸道成纤维细

胞增生。从而基本上预防了移植后阻塞性肺炎的发生。

第六章本章对全著进行了总结和延伸性讨论。并对本论文的结果

在临床上的应用提出了作者的见解：本论著中的实验结果证明呼吸道上皮细胞

在移植后阻塞性肺炎的发生机理中起重要作用。呼吸道上皮的严重损伤将引起

呼吸道内成纤维细胞增生，呼吸道纤维化，最终形成移植后阻塞性肺炎。实验

结果还显示，在呼吸道上皮轻微损伤（如 hEGP-2 模型）或严重损伤但能够修

复 (如第二章活性细胞植入) 时不发生移植后阻塞性肺炎。所以，呼吸道上皮

不可复性严重损伤是产生移植后阻塞性肺炎的原因。在呼吸道上皮损伤机制的

讨论中，免疫反应能够造成一定程度呼吸道上皮损伤，但没有诱发成纤维增

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生。这说明上皮细胞轻微损伤未造成其失去抑制成纤维增生的功能。也说明，

没有成纤维细胞的增生就不会发生移植后阻塞性肺炎。

在临床上，本论文结果显示，对呼吸道上皮细胞的保护将能在很大程度

上抑制移植后阻塞性肺炎的发生。如通过更加严格控制在临床肺移植过程中的

感染及炎症反应，以及通过特定方式刺激上皮细胞自我保护基因的表达等可在

一定程度上预防或延缓移植后阻塞性肺炎的发生。

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133

Acknowledgement I would like to express sincere thanks to my supervisor Dr. Jochum Prop, with whom I have worked everyday, by whom I have been inspired and instructed, and from whom I have learned so much that goes far beyond surgery and science; to my promoter Prof. dr. de Leij, who has given me the most support and encouragement, who has made me feel confident at research with my clinician background. I would also like to thank my colleague and supervisor Dr. Aalzen de Haan, who has been working with me intensively on several studies. He is so kind and eager to help and always positively consider my work. My special thank to Prof. dr. Boonstra, who supported me after my experimental work to facilitate me with every need to finish my thesis. He supported me both professionally and sentimentally, with the most generous recommendation and encouraging expectation.

Dr. Paul de Vos, thank you for your valuable advice to my research and your help on my thesis. Your point of view always broadens my thinking and enlightens me.

Arjen Petersen, thank you for helping me on my animal surgery and all the other works you’ve done to let my research be organized. It was your help that allowed my data to be smoothly collected and analysed.

Greetje Groen, working with you has been a pleasure and enjoyable process. Thanks for your help on my histology stainings. Henk van der Molen, Henk Moorlag thanks for your help. Diny and Herriëtte, thank you for your help in the past years to organize my document and even my Dutch letters. But isn’t that fun for you to learn some Chinese now? As China is getting not ‘red’ but ‘hot’.

Anita Niemarkt, Susan Jacobs, Henk Moes, Douwe Samplonius, Jelleke Dokter, Monica Dondorff, Baart de Haan, Linda Brouwer and Hans de Vos, it was so mice to work with you, friendly smiles.

Noelle Zweers, as my research roommate, it has never been as good as we hoped, we had a lot of fun together, hadn’t we? Jasper, Monika, Daniel, thank you for sharing a lot of memories and experience with me. I am glad that we’re all finally approaching to a harvest season!

Marco, Ingrid, Lydia (Pathology), Frans (Cell Biology), Bart-Jan, Bouke, Marja and Wijnand, thanks for your help on my scientific thinking, brainstorm and precious antibodies, EGP-2 protein for my research.

134

I’m not forgetting you my roommates now, Dirt-Jan and Yvonne, thank you for your patience on my noisy computer and non-stop typing. Now it will be quiet for some time.

Tjark, it was you signed the invatation letter in 1998 and open the door for me to come to this samll but great country. Thank you for providing me the opportunity to develop my future career in combination of patient care and medical science.

Prof. dr. Chengxin, Gao and Prof. dr. Owlin Huang (Shanghai Chest Hospital) thank you for leading me into this fascinating throacic surgery and organ transplantation wolrd. I know you will always back me up.

Finally, I would like to thank all the members of the Medical Biology and colleagues in the Department of Cardiothoracic Surgery of the University Medical Centre Groningen for their friendship and hospitality to me in the past years.

My dear wife Yijin, my parents, my brother Yang, thank you for your love and care in all these years. My friends Weidong, Xiaoyan, Shan, Peter, Yanji, Feiyan and dear Yinqi, I’m in debted to you for your kindness and care, this book is dedicated to all of you.

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Curriculum vitae

The author Ning Qu was born on January 08, 1968, Liaoyang, China. He received his medical education at China Medical University from 1986 to 1991. Afterwards he went for cardiothoracic surgery training in Shanghai Chest Hosptial from 1991 to 1997 and registerred in the specialty in 1997. He worked as a staff surgeon at thoracic surgery department in Shanghai Chest Hospital in 1998 before he came to Holland in November the same year. He started as a research fellow in Thoraxcentrum of Groningen University Medical Center and later on in 1999 received a grant from J.K. de Cock Foundation and an Ubbo Emmius Scholarship of Groningen University to support his PhD research project on lung transplantation. He finished the experimental work of research in 2003 and is working as practitioner under the supervision of Prof.dr. P.W.Boonstra in cardiothoracic surgery. He is also active in social activities and was elected as vice chairman of the 8th board of ACSSNL (Dutch Chinese scholars association). He is also the manager of the Chinese literature online-journal TULIP. He is member of several professional associations in medicine and cardiothoracic surgery, and has been invited to give lectures in several medical centers in Shenyang, Beijing and Shanghai, China.

university of groningen airway epithelium in obliterative

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