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Adenoviral vectors containing the CTLA4IG-gene inhibit chronic rejection in heterotopically transplanted rat tracheas
Adenoviral Vectors Containing the CTLA4IG-Gene Inhibit Chronic Rejection in Heterotopically Transplanted Rat Tracheas Y. Kita, X.- K. Li, M. Ohba, N. Funeshima, S. Enosawa, H. Nogimura, S. Ohi, Y. Kageyama, K. Matsushita, Y. Ito, T. Takahashi, K. Suzuki, S. Suzuki, and T. Kazui
O
BLITERATIVE airway disease (OAD) is one of the most common complications after lung transplantation. The pathologic hallmark of OAD is fibrosis of small cartilaginous airways along with a variable peribronchiolar inflammatory infiltrate. The chronic inflammation may lead to fibroblast recruitment, extracellular matrix formation, and fibrosis of the bronchioles. This lesion differs from that of acute rejection, which is defined by perivascular mononuclear cell infiltration. A model for OAD after heterotopic tracheal allografting in rats undergoes tracheal obliteration with the features resembling that of human obliterative bronchiolitis.1 Mononuclear cell infiltration, denudation of epithelium, fibroproliferation, and obliteration of the airway was found subsequently. These phenomena suggest that a process of graft rejection leads to OAD. In this model, T cells play an important role in the development of disease.2 CTLA4Ig, a soluble recombinant fusion protein that contains the extracellular domain of the CTLA4 and Fc portion of IgG1, strongly adheres to the B7 molecule to block CD28-mediated costimulatory signals and inhibits in vitro and in vivo immune responses. In vivo gene transfer using adenovirus vector 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 (adCTLA4Ig) to the OAD model. AdCTLA4Ig, administered at the time of transplantation, markedly inhibited the obliteration of airway lumen by fibrous tissue.
Table 1. Pathologic Scoring Criteria 0. 1. 2. 3. 4.
Normal Mononuclear cell infiltrate within graft tissue. Epithelial squamous metaplasia and/or subepithelial thickening. Severe epitherial abnormalities (sever metaplasia or denudation). Lumenal fibrosis.
genome. The recombinant viruses were subsequently propagated with 293 cells. The prepared vector solutions were stored at ⫺80°C.
Experimental Groups Adult male DA (RT-1a) and LEW (RT-1l) rats, weighing 200 to 250 g, were used as donors and recipients, respectively. The animals were maintained under standard conditions and fed rodent chow and water. Under ether anesthesia, the isolated donor tracheas were transplanted into the subcutaneous pocket in the back of recipients, essentially as described previously.1 The recipients were divided into the following groups: group 1: (n ⫽ 6), DA-to-LEW rats injected with 1 ⫻ 109 plaque-forming units (p.f.u.) of control vector, adLacZ; group 2: (n ⫽ 6), DA-to-LEW rats administered 1 ⫻ 109 p.f.u. of adCTLA4Ig; group 3: (n ⫽ 6), syngeneic grafts (LEW-to-LEW) without any treatment. The vectors were administered via 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
MATERIALS AND METHODS Adenoviral Vector
The grafts were removed on 7, 14, 21, 28, and 35 days after transplantation for light microscopic study and estimated by the pathologic scoring criteria, as described previously (Table 1).2
The recombinant adenoviruses, 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 beta-galactosidase gene (LacZ) was constructed by homologous recombination between the expression cosmid cassette (pAdex/CAhCTLA4Ig) and the parental virus
From the First Department of Surgery (Y.K., H.N., S.O., Y.K., K.M., Y.I., T.T., K.S., S.S., T.K.), Hamamatsu University School of Medicine, Hamamatsu and the Department of Experimental Surgery and Bioengineering (X-K.L., M.O., N.F., S.E.), National Children’s Medical Research Center, Tokyo, Japan. Address reprint requests to Yusuki Kita, MD, First Department of Surgery, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu, 431-3192 Japan.
0041-1345/00/$–see front matter PII S0041-1345(00)01547-5
© 2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
2036
Transplantation Proceedings, 32, 2036–2037 (2000)
ADENOVIRAL VECTORS CONTAINING CTLA4IG-GENE
2037
immune-privileged organs, including the retina and testis; and immunodeficient animals. In view of this limitation, the immunosuppressive product, CTLA4Ig, would be effective for prolonging gene expression by adenovirus-mediated gene transfer. Obliterative airway disease is a significant complication that is associated with a T-cell response against graft tissue. In rat allografts, the bronchial epithelium starts to express major histocompatibility complex class II antigens during acute rejection and after insufficient cyclosporine treatment.3 This expression may be induced by locally activated, alloreactive T cells. Antigens on epithelial cells may stimulate the activation of rejection and be targets of rejection.4 Adenoviral vector containing CTLA4Ig-gene markedly inhibited the obliteration of the airway lumen. ACKNOWLEDGMENT Fig 1.
Pathologic score (day 28).
RESULTS AND DISCUSSION
The grafts were removed for histologic study weekly after transplantation. The control grafts (group 1) showed a marked fibrous proliferation and luminal obstruction on day 28, whereas no fibrous change was observed in group 2 and group 3 by hematoxylin-eosin staining. DA tracheas treated with adCTLA4Ig (group 2), harvested on day 28, had significantly lower pathologic scores than the control allografts in group 1 (Fig 1). No evidence of vectormediated tissue damage was seen in any graft. In vivo gene expression using a recombinant adenovirus was highly efficient but transient except in newborn animals;
The authors gratefully acknowledge Dr H. Kimura and Dr T. Okuyama for their critical comments and useful suggestions. We also thank Dr I. Saito for providing AxCALacZ, and Dr S. Hayashi and Dr H. Hamada for providing AxCAhCTLA4Ig adenoviral vector.
REFERENCES 1. Hertz MI, Jessurun J, King MB, et al: Am J Pathol 142:1945, 1993 2. Kelly KE, Hertz MI, Mueller DL: Transplantation 66:764, 1998 3. Romaniuk A, Prop J, Petersen AH, et al: Transplantation 44:209, 1987 4. Prop J, Jansen HM, Wildevuur CRH, et al: Am Rev Respir Dis 132:168, 1985
O
BLITERATIVE airway disease (OAD) is one of the most common complications after lung transplantation. The pathologic hallmark of OAD is fibrosis of small cartilaginous airways along with a variable peribronchiolar inflammatory infiltrate. The chronic inflammation may lead to fibroblast recruitment, extracellular matrix formation, and fibrosis of the bronchioles. This lesion differs from that of acute rejection, which is defined by perivascular mononuclear cell infiltration. A model for OAD after heterotopic tracheal allografting in rats undergoes tracheal obliteration with the features resembling that of human obliterative bronchiolitis.1 Mononuclear cell infiltration, denudation of epithelium, fibroproliferation, and obliteration of the airway was found subsequently. These phenomena suggest that a process of graft rejection leads to OAD. In this model, T cells play an important role in the development of disease.2 CTLA4Ig, a soluble recombinant fusion protein that contains the extracellular domain of the CTLA4 and Fc portion of IgG1, strongly adheres to the B7 molecule to block CD28-mediated costimulatory signals and inhibits in vitro and in vivo immune responses. In vivo gene transfer using adenovirus vector 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 (adCTLA4Ig) to the OAD model. AdCTLA4Ig, administered at the time of transplantation, markedly inhibited the obliteration of airway lumen by fibrous tissue.
Table 1. Pathologic Scoring Criteria 0. 1. 2. 3. 4.
Normal Mononuclear cell infiltrate within graft tissue. Epithelial squamous metaplasia and/or subepithelial thickening. Severe epitherial abnormalities (sever metaplasia or denudation). Lumenal fibrosis.
genome. The recombinant viruses were subsequently propagated with 293 cells. The prepared vector solutions were stored at ⫺80°C.
Experimental Groups Adult male DA (RT-1a) and LEW (RT-1l) rats, weighing 200 to 250 g, were used as donors and recipients, respectively. The animals were maintained under standard conditions and fed rodent chow and water. Under ether anesthesia, the isolated donor tracheas were transplanted into the subcutaneous pocket in the back of recipients, essentially as described previously.1 The recipients were divided into the following groups: group 1: (n ⫽ 6), DA-to-LEW rats injected with 1 ⫻ 109 plaque-forming units (p.f.u.) of control vector, adLacZ; group 2: (n ⫽ 6), DA-to-LEW rats administered 1 ⫻ 109 p.f.u. of adCTLA4Ig; group 3: (n ⫽ 6), syngeneic grafts (LEW-to-LEW) without any treatment. The vectors were administered via 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
MATERIALS AND METHODS Adenoviral Vector
The grafts were removed on 7, 14, 21, 28, and 35 days after transplantation for light microscopic study and estimated by the pathologic scoring criteria, as described previously (Table 1).2
The recombinant adenoviruses, 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 beta-galactosidase gene (LacZ) was constructed by homologous recombination between the expression cosmid cassette (pAdex/CAhCTLA4Ig) and the parental virus
From the First Department of Surgery (Y.K., H.N., S.O., Y.K., K.M., Y.I., T.T., K.S., S.S., T.K.), Hamamatsu University School of Medicine, Hamamatsu and the Department of Experimental Surgery and Bioengineering (X-K.L., M.O., N.F., S.E.), National Children’s Medical Research Center, Tokyo, Japan. Address reprint requests to Yusuki Kita, MD, First Department of Surgery, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu, 431-3192 Japan.
0041-1345/00/$–see front matter PII S0041-1345(00)01547-5
© 2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
2036
Transplantation Proceedings, 32, 2036–2037 (2000)
ADENOVIRAL VECTORS CONTAINING CTLA4IG-GENE
2037
immune-privileged organs, including the retina and testis; and immunodeficient animals. In view of this limitation, the immunosuppressive product, CTLA4Ig, would be effective for prolonging gene expression by adenovirus-mediated gene transfer. Obliterative airway disease is a significant complication that is associated with a T-cell response against graft tissue. In rat allografts, the bronchial epithelium starts to express major histocompatibility complex class II antigens during acute rejection and after insufficient cyclosporine treatment.3 This expression may be induced by locally activated, alloreactive T cells. Antigens on epithelial cells may stimulate the activation of rejection and be targets of rejection.4 Adenoviral vector containing CTLA4Ig-gene markedly inhibited the obliteration of the airway lumen. ACKNOWLEDGMENT Fig 1.
Pathologic score (day 28).
RESULTS AND DISCUSSION
The grafts were removed for histologic study weekly after transplantation. The control grafts (group 1) showed a marked fibrous proliferation and luminal obstruction on day 28, whereas no fibrous change was observed in group 2 and group 3 by hematoxylin-eosin staining. DA tracheas treated with adCTLA4Ig (group 2), harvested on day 28, had significantly lower pathologic scores than the control allografts in group 1 (Fig 1). No evidence of vectormediated tissue damage was seen in any graft. In vivo gene expression using a recombinant adenovirus was highly efficient but transient except in newborn animals;
The authors gratefully acknowledge Dr H. Kimura and Dr T. Okuyama for their critical comments and useful suggestions. We also thank Dr I. Saito for providing AxCALacZ, and Dr S. Hayashi and Dr H. Hamada for providing AxCAhCTLA4Ig adenoviral vector.
REFERENCES 1. Hertz MI, Jessurun J, King MB, et al: Am J Pathol 142:1945, 1993 2. Kelly KE, Hertz MI, Mueller DL: Transplantation 66:764, 1998 3. Romaniuk A, Prop J, Petersen AH, et al: Transplantation 44:209, 1987 4. Prop J, Jansen HM, Wildevuur CRH, et al: Am Rev Respir Dis 132:168, 1985