Ann Thorac Surg 2003;75:1123-1127
© 2003 The Society of Thoracic Surgeons
Original article: general thoracic
CTLA4Ig-gene transfection inhibits obliterative airway disease in rats
Yusuke Kita, MDa*,
Kazuya Suzuki, MDb,
Hiroshi Nogimura, MDa,
Tsuyoshi Takahashi, MDb,
Teruhisa Kazui, MDb
a Department of Thoracic Surgery, Haibara General Hospital, Shizuoka, Japan
b First Department of Surgery, Hamamatsu University School of Medicine, Hamamatsu, Japan
Accepted for publication October 25, 2002.
* Address reprint requests to Dr Kita, Department of Thoracic Surgery, Haibara General Hospital, 2887-1 Hosoe, Haibara-cho, Haibara-gun, Shizuoka-pref, Japan 421-0493
e-mail: kita21{at}aqua.ocn.ne.jp
 |
Abstract
|
|---|
BACKGROUND: Obliterative airway disease (OAD) is a major cause of long-term morbidity following lung transplantation. Its pathologic characteristics are small-airway inflammation and occlusion by fibrous tissue. However, the pathogenesis is uncertain and therapy is ineffective. This study presents the effects of CTLA4Ig-gene therapy on OAD in heterotopically transplanted rat tracheal allografts.
METHODS: Dark Agouti (DA, RT1a) allografts and Lewis (LEW, RT1l) isografts were transplanted into Lewis recipients. The tracheal graft was transplanted heterotopically into the subcutaneous pocket into the back. Adenoviral vectors (1.0x109 pfu) containing the CTLA4Ig-gene (AdCTLA4Ig) or the LacZ-gene (AdLacZ) were injected into the tail vein immediately after grafting. Grafts were harvested and examined after more than 35 days for mononuclear cell infiltration development and lumen occlusion with fibrosis.
RESULTS: Fully allogenic DA tracheas, treated with AdCTLA4Ig had significantly lower pathologic scores and infiltrating scores than the control allografts. The pathologic findings of the grafts, treated with AdCTLA4Ig, were very similar to those of the syngeneic grafts. The animals experienced no adverse events during follow-up. No evidence of vector-mediated tissue damage was seen in any graft.
CONCLUSIONS: Adenoviral vectors containing the CTLA4Ig-gene markedly inhibited the obliteration of the airway lumen. OAD may be associated with T-cell responses against graft tissue and alloimmune injury.
|
Introduction
|
|---|
Obliterative airway disease (OAD) is the most common complication after lung transplantation [1,2]. Despite contemporary immune suppression, some lung transplant recipients develop fatal chronic rejection. The pathologic OAD hallmark is fibrous small cartilaginous airways and variable peribronchiolar inflammatory infiltrate. This lesion differs from that of acute rejection, which is defined by perivascular mononuclear cell infiltration, possibly reflecting different immune targets or mechanisms [3]. Previous clinical studies support the concept that the OAD results from alloimmune-mediated injury [4–6] and graft function stabilization can be achieved with immune suppression [7, 8]. Graft rejection may lead to OAD [9, 10] and T cells play an important role in disease development [11].
CTLA4Ig is a soluble recombinant fusion protein, containing the CTLA4 extracellular domain and IgG1 Fc portion. CTLA4Ig strongly attaches to the B7 molecule to block CD28-mediated costimulatory signals, and inhibits the lymphocyte activation and immune responses [12–14]. CTLA4Ig administration to recipients with organ grafts has achieved prolonged graft survival in several rodent models [15–19]. In vivo gene transfer using adenoviral vectors achieves a high transfection rate into organ cells, which usually contain adenoviral receptors.
In this study, we investigated the effects of systemically administered adenoviral vectors containing CTLA4Ig gene (AdCTLA4Ig) for OAD, using the rat model of heterotopic tracheal transplantation.
|
Material and methods
|
|---|
Adenoviral vector
The recombinant adenovirus, AxCAhCTLA4Ig (AdCTLA4Ig) and AxCALacZ (AdLacZ), were provided by Dr. S. Hayashi (Nagoya University School of Medicine, Nagoya, Japan), Dr. H. Hamada (Japanese Foundation for Cancer Research, Tokyo, Japan), and Dr. I. Saito (Laboratory of Molecular Genetics, Institute of Medical Science, University of Tokyo, Tokyo, Japan). The adenovirus containing the expression cassette for human CTLA4Ig cDNA or Escherichia coli ß-galactosidase gene (LacZ) was constructed by homologous recombination between the expression cosmid cassette (pAdex/CAhCTLA4Ig) and the parental virus genome [20]. The recombinant viruses were subsequently propagated with 293 cells. The prepared vector solutions were stored at -80°C.
Experimental animals
All laboratory animals received humane care in compliance with the Principles of Laboratory Animal Care formulated by the Institute of Laboratory Animal Resources and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Science and published by the National Institutes of Health (National Institutes of Health, publication No. 86-23, revised 1985). The experimental protocols were approved by the Institutional Animal Care Use Committee, National Children’s Medical Research Center (Tokyo, Japan). The animals were purchased from Japan SLC Co. (Shizuoka, Japan) and maintained under standard conditions.
Heterotopic tracheal transplantation
Adult male Dark Agouti (DA, RT-la) rats and Lewis (LEW, RT-ll) rats were used as donors and recipients, respectively. Under ether anesthesia, the neck and upper chest of donor rats were incised and bluntly dissected. The trachea was resected from the larynx inferior border to the carina and placed in ice-cold phosphate-buffered saline (PBS) with penicillin (100 U/ml), streptomycin sulfate (100 µg/ml), and amphotericin B (0.25 g/ml) (Sigma, St. Louis, MO). The isolated donor tracheas were transplanted into subcutaneous pockets in the recipient back, essentially as described [9]. In brief, after the recipient’s back skin was prepared, about 1-cm skin incisions were made and subcutaneous pockets were formed by blunt dissection. The trachea grafts were planted heterotopically into the pockets without primary vascularization, and the wounds were closed with 3-0 silk sutures.
Experimental groups
The recipients were divided into the following groups: group 1 comprised DA-to-LEW rats injected with 1x109 plaque-forming units (p.f.u.) of control vector, AdLacZ; group 2 comprised DA-to-LEW rats administered 1x109 p.f.u. of AdCTLA4Ig; and group 3 comprised syngeneic grafts (LEW-to-LEW) without any treatment. The vectors were administered through the recipient tail vein immediately after grafting. The day of grafting was regarded as day 0 and the grafts were harvested weekly and examined over the course of 35 days.
Histologic studies
For histologic observation, the grafts were removed on 3, 5, 7, 14, 21, 28, and 35 days after transplantation and divided into four pieces. One of the middle pieces was fixed in formalin and paraffin-embedded for hematoxylin-eosin staining, the other was snap-frozen in tetrafluoroethane and stored at -80°C for immunohistochemical staining. Graft infiltrating cells in the thin cryocut section (6 µm) were stained with the monoclonal antibodies, CD2 (a mixture of MRC OX-54 and 55; Serotec, Oxford, UK). Color development was performed with nickel-cobalt-diaminobenzidine product (DAB; 049-22833 [Wako, Osaka, Japan]).
Scoring
Slides of grafts were examined two blinded reviewers and given numeric T-cell infiltration scores (on a scale of 0 to 4) based on the degree of CD2 positive mononuclear cell infiltration (Table 1).
Hematoxylin-eosin staining sections were examined by the same, blinded reviewers and given numeric pathologic scores (on a scale of 0 to 4) based on the presence or absence of mononuclear cell infiltrate, epithelial injury, and fibrosis (Table 1).
Determination of serum CTLA4Ig levels
To determine the time course of the serum CTLA4Ig levels, we injected 1x109 p.f.u. of AdCTLA4Ig into the recipient rats. Blood samples were collected on day 0 before injection and 1, 3, 5, 7, 10, 14, 21, and 35 days after injection. The serum concentration of CTLA4Ig was assayed by enzyme-linked immunosorbent assay, as described previously [21].
|
Results
|
|---|
To examine the lymphocyte role in OAD development, tracheal grafts were transplanted into recipients and removed after grafting for histologic study. The control grafts (group 1) revealed that the epithelial squamous metaplasia or subepithelial thickening were accerelated from day 5 to 7. Severe epithelial abnormalities became prominent until day 14, and a marked fibrous proliferation and lumenal obstruction were complete before day 28, whereas few epithelial abnormalities and no fibrous change were observed in group 2 and group 3 by hematoxylin-eosin staining (Fig 1).
We compared these differences by pathologic scoring criteria (Table 1) [11]. We compared an average of six grafts in each group. DA tracheas treated with AdCTLA4Ig (group 2), harvested on day 5, 7, 14, 21, and 28 had significantly lower pathologic scores than the control, group 1 (day 5; p < 0.05, day 7 to 28; p < 0.01, unpaired Student’s t-test; Fig 2).
The pathologic findings of group 2 were very similar to those of the syngeneic grafts (group 3). CD2 immunohistologic staining was performed to distinguish lymphocytes from other infiltrating monocytes. CD2 was expressed on both resting and activated T cells. To examine the lymphocyte infiltration, we used the infiltrating scoring criteria (Table 1) [11]. DA tracheas treated with AdCTLA4Ig (group 2), harvested on day 3, 5, and 7 had significantly lower scores than the control, group 1 (p < 0.01, unpaired t-test; Fig 3).
However, after day 14, the scores of group 2 were not significantly different from those of group 1. The infiltrating scores of group 3 were also significantly lower than those of group 1 from day 3 to 7, whereas the scores of group 3 were not significantly different from those of group 2 on any day investigated (Fig 3). The animals experienced no adverse events during follow-up. No pathologic changes appeared at autopsy or in postmortem analysis of the liver, heart, lung, skin, or spleen. No evidence of vector-mediated tissue damage was seen in any graft.
View larger version (130K):
[in this window]
[in a new window]
|
Fig 1. Photomicrographs from tracheas of group 1 (a, d, g, j), group 2 (b, e, h, k), and group 3 (c, f, i, l) on day 7 (a–c), day 14 (d–f), and day 28 (g–l). These panels were magnified x100 (a–i) or x50 (j–l). On day 7, control grafts (group 1) exhibited submucosal edema (a), whereas no subepithelial thickening in AdCTLA4Ig group (b) and syngeneic grafts (c) (hematoxylin and eosin). On day 14, epithelial damage and fibrous proliferation were prominent in group 1 (d), whereas there was no fibrous change in groups 2 (e) and 3 (f). On day 28, complete luminal obliteration was seen in the control (g, j). However, the lumen was completely patent in groups 2 (h, k) and 3 (i, l).
|
|
View larger version (17K):
[in this window]
[in a new window]
|
Fig 2. Pathologic scoring criteria (mean ± standard error). We compared the average of six grafts in each group. Grafts treated with AdCTLA4Ig (group 2 [circles]), harvested on day 5, 7, 14, 21, and 28 had significantly lower scores than the control, group 1 (p < 0.05 [#], p < 0.01 [*], unpaired Student’s t-test [diamonds]). The pathologic findings of group 2 were very similar to those of syngeneic grafts (group 3 [triangles]).
|
|
View larger version (17K):
[in this window]
[in a new window]
|
Fig 3. The infiltrating scoring criteria (mean ± standard error). We compared the average of six grafts in each group. Grafts treated with AdCTLA4Ig (group 2 [circles]), harvested on day 3, 5, and 7, had significantly lower scores than the control, group 1 (p < 0.01 [*], unpaired Student’s t-test [diamonds]). However, after day 14, the scores were not significantly different between the control and others. The scores of group 3 (triangles) were not significantly different from those of group 2 on any day investigated.
|
|
The time course of serum CTLA4Ig levels is illustrated in Figure 4. The mean level of 3 rats reached maximum at 91 µg/ml on day 5 and declined after 14 days. Detectable levels were observed more than 35 days. The rats transfected with control vector (AdLacZ) did not have any measurable CTLA4Ig at any time (data not shown).
View larger version (16K):
[in this window]
[in a new window]
|
Fig 4. Time course of serum CTLA4Ig levels in recipients after injection of AdCTLA4Ig. Values represent the mean ± standard error of three rats. The mean level reached to maximum (91 µg/ml) on day 5 and declined gradually.
|
|
|
Comment
|
|---|
OAD is a significant complication, associated with T-cell responses against graft tissue. The chronic inflammation may lead to fibroblast recruitment, extracellular matrix formation, and bronchiole fibrosis. In rat allografts, bronchial epithelium starts to express major histocompatibility complex (MHC) class II antigens during acute rejection and after insufficient cyclosporine treatment [22]. The expression of MHC class II antigen may be induced by local alloreactive T cells. These antigens on epithelial cells may stimulate the activation of the graft rejection [23].
Monocyte infiltration is a landmark feature of organ rejection. The cellular infiltrations are compromised predominantly of macrophages and T lymphocytes, which are responsible for the immunologic response to foreign bodies. For this to occur, leukocytes must undergo extravasation, interact with extracellular matrix proteins of the basement membrane, and spread in the graft tissue. AdCTLA4Ig-treatment prevents leukocyte infiltration at the early stage of graft rejection. This result suggests that peripheral leukocytes, especially T lymphocytes, were prevented from activation by the CTLA4Ig-protein and stopped the extravasation. We are studying whether this therapy is effective for ongoing rejection, such as when monocyte infiltration has been established.
In vivo gene expression by adenoviral vector was efficient. The AdCTLA4Ig enabled the persistent gene expression in vivo without repeated vector administration. The CTLA4Ig-gene was apparently expressed continuously in the recipients more than 7 weeks. Once the AdCTLA4Ig was injected systemically, intrahepatic expression of CTLA4Ig would be prominent. In our previous study, the injection of AdLacZ through mouse tail vein demonstrated high gene-expression levels in the liver by X-gal staining (> 95% of hepatocytes), although some expression was seen in the heart, lung, and spleen. No expression in the thymus, small intestine, pancreas, kidney, or brain was detected (data not shown). The CTLA4Ig protein might be produced in the gene-transfected hepatocytes and released into blood. In this study, we demonstrated the time course of serum CTLA4Ig levels after AdCTLA4Ig administration. Then immunosuppressive activity was induced in the allografts. This effect may be similar to that obtained by repeated administration of exogenous CTLA4Ig protein. However, AdCTLA4Ig induces efficient immunosuppression with its one injection.
As the limitation of adenoviral vector, in vivo gene expression is generally transient. The immune system may play a major role in the limiting gene-transfer technology. CTLA4Ig would be effective to prolong gene expression but not permanent. We are now studying the administration timing and the dose of CTLA4Ig.
In this heterotopic tracheal implant model, all the untreated allografts develop nearly complete occlusion of the airway lumen with fibroblastic tissue and collagen scar by day 28 after transplantation, with the features resembling human obliterative bronchiolitis (OB). Mononuclear cell infiltration, epithelium denudation, fibro-proliferation, and airway obliteration are subsequently found. These phenomena suggest that graft rejection may lead to OAD. Allografts undergo the obliterative changes and structural destruction, whereas almost all the syngeneic grafts and the AdCTLA4Ig-treated grafts relatively retain their normal structures. The surgical procedure of transplantation is not so responsible for the changes observed in this model. Direct epithelial injury by infection may also trigger the development of bronchiolitis obliterans. No airway pathology other than the reimplantation response was seen in our syngeneic controls. After the grafts were denervated, we did not see evidence of mucus stasis that might have been predisposed to infection.
T cells play an important role in disease development [11]. A peak of CD2+ cell infiltration in this untreated group was recorded from 7 to 14 days. These results are in concordance with the transbronchial lung biopsy findings of human allograft rejection. Once the development toward OAD originated, the process was rapidly proceeded. It seems that OAD is a continuation of acute rejection in this model. Single dose injection of AdCTLA4Ig on day 0 resulted in significantly lower scores up to 30 days after implantation, although there were no significant differences in infiltrating scores after 14 postoperative days. For complete inhibition of OAD, we are investigating the combination therapy by using an immunosuppressant, such as FTY720. Simultaneously blocking other costimulatory pathways, such as gp39/CD40, ICAM-1/LFA-1 and CD2/CD48, also enhance the T-cell anergy.
In conclusion, the present study demonstrated that AdCTLA4Ig-gene transfer into recipient’s liver by systemic administration proved to be useful in this heterotopic tracheal allograft model. AdCTLA4Ig may be effective to inhibit OAD.
|
Acknowledgments
|
|---|
The authors gratefully acknowledge Drs Seiichi Suzuki, Xiao-Kang Li, Naoko Funeshima, and Shin Enosawa (Department of Experimental Surgery & Bioengineering, National Children’s Medical Research Center, Tokyo, Japan) for their critical comments and useful suggestions. We also thank Drs Shuji Hayashi and Izumu Saito for providing AxCALacZ; and Dr Hirofumi Hamada for providing AxCAhCTLA4Ig adenoviral vector.
|
References
|
|---|
- Burke C.M.J., Theodore K., Dawkins D., et al. Post-transplant obliterative bronchiolitis and other late lung sequelae in human heart-lung transplantation. Chest 1984;86:824-832.[Abstract/Free Full Text]
- Griffith B.P., Paradis I.L., Zeevi A., Rabinowich H., Yousem S.A. Immunologically mediated disease of the airways after pulmonary transplantation. Ann Surg 1988;208:371-378.[Medline]
- Milne D.S., Gascoigne A., Wilkes J., et al. The immunohistopathology of obliterative bronchiolitis following lung transplantation. Transplantation 1992;54:748-750.[Medline]
- Sharples L.D., Scott J.P., Dennis C. Risk factors for survival following combined heart-lung transplantation. Transplantation 1994;57:218-223.[Medline]
- Bando K., Paradis I.L., Similo S., et al. Obliterative bronchiolitis after heart-lung transplantation: an analysis of risk factors and management. J Thorac Cardiovasc Surg 1995;110:4-13.[Abstract/Free Full Text]
- Kroshus T.J., Kshettry V.R., Savik K., John R., Hertz M.I., Bolman R.M. Risk factors for the development of bronchiolitis obliterans syndrome after lung transplantation. J Thorac Cardiovasc Surg 1997;114:195-202.[Abstract/Free Full Text]
- Kesten S., Rajagopalan N., Maurer J. Cytolytic therapy for the treatment of bronchiolitis obliterans syndrome following lung transplantation. Transplantation 1996;61:427-430.[Medline]
- Dusmet M., Maurer J., Winton T., Kesten S. Methotrexate can halt the progression of bronchiolitis obliterans syndrome in lung transplant recipients. J Heart Lung Transplant 1996;15:948-954.[Medline]
- Hertz M.I., Jessurun J., King M.B., Savik S.K., Murray J. Reproduction of the obliterative bronchiolitis lesion after heterotopic transplantation of mouse airways. Am J Pathol 1993;142:1945-1951.[Abstract]
- al-Dossari G.A., Jessurun J., Bolman R.M., et al. Pathogenesis of obliterative bronchiolitis: possible roles of platelet-derived growth factor and basic fibroblast growth factor. Transplantation 1995;59:143-145.[Medline]
- Kelly K.E., Hertz M.I., Mueller D.L. T-cell and major histocompatibility complex requirements for obliterative airway disease in heterotopically transplanted murine tracheas. Transplantation 1998;66:764-771.[Medline]
- Linsley P.S., Brady W., Urnes M., Grosmaire L.S., Damle N.K., Ledbetter J.A. CTLA-4 is a second receptor for the B cell activation antigen B7. J Exp Med 1991;174:561-569.[Abstract/Free Full Text]
- Wu Y., Guo Y., Liu Y. A major costimulatory molecule on antigen-presenting cells, CTLA4 ligand A, is distinct from B7. J Exp Med 1993;178:1789-1793.[Abstract/Free Full Text]
- Lenschow D.J., Zeng Y., Thistlethwaite J.R., et al. Long-term survival of xenogeneic pancreatic islet grafts induced by CTLA4Ig. Science 1992;257:789-792.[Abstract/Free Full Text]
- Turka L.A., Linsley P.S., Lin H., et al. T-cell activation by the CD28 ligand B7 is required for cardiac allograft rejection in vivo. Proc Natl Acad Sci USA 1992;89:11102-11105.[Abstract/Free Full Text]
- Sayegh M.H., Zheng X.-G., Magee C., Hancock W.W., Turka L.A. Donor antigen is necessary for the prevention of chronic rejection in CTLA4Ig-treated murine cardiac allograft recipients. Transplantation 1997;64:1646-1650.[Medline]
- Pearson T.C., Alexander D.Z., Corbascio M., et al. Analysis of the B7 costimulatory pathway in allograft rejection. Transplantation 1997;63:1463-1469.[Medline]
- Glysing-Jensen T., Raisanen-Sokolowski A., Sayegh M.H., Russell M.E. Chronic blockade of CD28-B7-mediated T-cell costimulation by CTLA4Ig reduces intimal thickening in MHC class 1 and 2 incompatible mouse heart allografts. Transplantation 1997;64:1641-1645.[Medline]
- Lin H., Bolling S.F., Linsley P.S., et al. Long-term acceptance of major histocompatibility complex mismatched cardiac allografts induced by CTLA4Ig plus donor-specific transfusion. J Exp Med 1993;178:1801-1806.[Abstract/Free Full Text]
- Namii Y., Hayashi S., Yokoyama I., et al. Evidence that the adenoviral vector containing the CTLA4-Ig gene improves transgene expression and graft survival. Transplant Proc 1997;29:1738-1739.[Medline]
- Kita Y., Li X.-K., Ohba M., et al. Prolonged cardiac allograft survival in rats systemically injected adenoviral vectors containing CTLA4Ig-gene. Transplantation 1999;68:758-766.[Medline]
- Romaniuk A., Prop J., Petersen A.H., Wildevuur C.R.H., Nieuwenhuis P. Expression of class II major histocompatibility complex antigens by bronchial epithelium in rat lung allografts. Transplantation 1987;44:209-214.[Medline]
- Prop J., Jansen H.M., Wildevuur C.R.H., Nieuwenhuis P. Lung allograft rejection in the rat: V. Inhaled stimuli aggravate the rejection response. Am Rev Respir Dis 1985;132:168-172.[Medline]
Top
Abstract
Introduction
