J Thorac Cardiovasc Surg 2005;129:926-931
© 2005 The American Association for Thoracic Surgery
Cardiothoracic Transplantation |
Recipient intramuscular cotransfection of transforming growth factor ß1 and interleukin 10 ameliorates acute lung graft rejection
Takashi Suda, MDa,
Niccoló Daddi, MDa,
Tsutomu Tagawa, MDa,
Samer A. Kanaan, MDa,
Benjamin D. Kozower, MDa,
Jon H. Ritter, MDb,
G. Alexander Patterson, MD, FRCS(C)a,*
a Division of Cardiothoracic Surgery, Washington University School of Medicine, St Louis, Mo
b Department of Pathology, Washington University School of Medicine, St Louis, Mo
Received for publication March 18, 2004; revisions received July 8, 2004; accepted for publication July 13, 2004.
* Address for reprints: G. Alexander Patterson, MD, FRCS(C), Division of Cardiothoracic Surgery, Washington University School of Medicine, One Barnes-Jewish Hospital Plaza, 108 Queeny Tower, St Louis, MO 63110 (E-mail: pattersona{at}msnotes.wustl.edu).
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Abstract
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OBJECTIVE: Multiple gene transfer might permit modulation of concurrent biochemical pathways involved in acute lung graft rejection. We investigated whether gene cotransfection into the recipient reduces acute lung graft rejection.
METHODS: Brown Norway rats were used as donors, and F344 rats were used as recipients. Recipient animals were injected with saline (groups I/VI) or 1 x 1010 pfu of adenovirus encoding ß-galactosidase (groups II/VII), transforming growth factor ß1 (groups III/VIII), interleukin 10 (groups IV/IX), or both transforming growth factor ß1 and interleukin 10 (groups V/X) into both leg muscles 2 days before transplantation (groups I-V) or at the time of harvest (groups VI-X). The Kruskal-Wallis test for rejection score and 1-way analysis of variance were used to compare groups.
RESULTS: Oxygenation was significantly improved in the cotransfected groups treated 2 days before transplantation and at the time of harvest. Rejection scores were also reduced in the cotransfected groups. In group V cotransfection suppressed endogenous interleukin 2 but not interferon 
and tumor necrosis factor 
.
CONCLUSION: Recipient intramuscular cotransfection of transforming growth factor ß1 and interleukin 10 suppressed interleukin 2 expression and provided a synergistic effect that reduced acute lung graft rejection. This approach might be applied to the clinical setting because transplant recipients could be treated at the time of implantation.
Although lung transplantation has proved to be a therapeutic option for a variety of end-stage pulmonary diseases, acute and chronic rejection continue to affect lung transplantation. Donor lung gene transfer has tremendous potential for the treatment of pulmonary disease by providing local immunosupression.1,2 Gene transfer into the recipient is another potential therapeutic option. Recipient intramuscular gene transfection of major histocompatibility complex class I antigen and transforming growth factor ß1 (TGF-ß1) decreases experimental graft rejection.3,4 These studies suggest that the transgene products enter into the recipient’s circulation, resulting in beneficial effects on the transplanted organs.
TGF-ß1 is one of a number of closely related and multifunctional molecules that play a central role in embryonic development, tumorigenesis, wound healing, fibrosis, and immunoregulation.4,5 The immunomodulator function occurs by suppressing the proliferation of B and T cells; by antagonizing inflammatory cytokines, such as interleukin 2 (IL-2), tumor necrosis factor (TNF-), and interferon (IFN-); and by inhibiting natural killer cells.5–7 In experimental models expression of TGF-ß1 has been clearly implicated in the establishment of tolerance.8 IL-10 is produced by TH2 cells and macrophages and plays an immunosuppressive role by decreasing both the expression of proinflammatory cytokines and the expression of class II major histocompatibility complex antigens and antigen-presenting cell function.9 Multiple gene transfer of these cytokines might provide synergistic benefits and might permit modulation of concurrent biochemical pathways involved in acute lung graft rejection.
We hypothesized that intramuscular TGF-ß1 and IL-10 gene cotransfection into the recipient would inhibit the immune response and suppress acute allograft rejection. The aim of the present study was to investigate the feasibility of gene cotransfection with TGF-ß1 and IL-10 into the recipient by using an adenoviral vector to study its synergistic effects on acute rejection in a lung transplant model.
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Materials and methods
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The aim of the first study (nontransplant setting, experiment 1) was to demonstrate the feasibility of in vivo gene delivery in muscle. The second study (experiment 2) was conducted to evaluate the effect of TGF-ß1 and IL-10 gene cotransfection into the recipient in a rat lung transplant model of acute rejection and to measure endogenous cytokine expression in the grafts.
Animals
F344 rats and Brown Norway rats (Harlan Sprague Dawley Inc, Indianapolis, Ind) weighing 250 to 270 g were used in all experiments. All animal procedures were approved by the Animal Studies Committee at Washington University. Animals received humane care in compliance with "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (National Institutes of Health publication no. 85–23, revised 1996).
Adenoviral vectors
Adenovirus encoding rat TGF-ß1 (AdCMVTGFß1; provided by Dr Debra A. Hullett, Department of Surgery, University of Wisconsin, Madison, Wis) is a replication-deficient adenoviral vector encoding the mutated bioactive TGFB1 gene driven by the cytomegalovirus immediate early promoter. This contains a mutation of cysteine to serine at positions 223 and 225, rendering the expressed TGF-ß1 biologically active.4
Adenovirus encoding human IL-10 (AdRSIL-10; purchased from Gene Therapy Vector Core at the University of Iowa School of Medicine, Iowa) is a replication-deficient adenoviral vector encoding the human IL-10 gene and driven by the rous sarcoma virus (RSV) promoter.
First-generation replication-deficient adenovirus serotype 5 carrying the Escherichia coli LacZ gene encoding for ß-galactosidase (ßgal) and driven by the constitutive cytomegalovirus promoter (AdCMVßgal) served as a control adenoviral vector. It was provided as a gift from the Gene Therapy Center at the University of North Carolina, Chapel Hill.
Adenoviral amplification was achieved by means of propagation in 293 cells to obtain high-titer stocks, as determined by using the plaque assay (courtesy of Dr R. Jude Samulski and Dr Douglas McCarty, Gene Therapy Center Vector Core Facility, University of North Carolina, Chapel Hill, NC). Purified viral aliquots were stored at –80°C in 10% glycerol buffered with 10 mmol/L Tris, 140 mmol/L NaCl, and 1 mmol/L MgCl2. Immediately before use, these stocks were thawed and diluted in 1 mL of sterile normal saline.
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Experimental groups
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The expression of TGF-ß1 and IL-10 in muscle has been demonstrated in our previous studies.4,10
Effects of active TGF-ß1 and IL-10 gene transfection (groups I-X)
Animals were randomly divided into 10 groups (n = 5 per group). Brown Norway rats (RT1n) served as donors, and F344 rats (RT11v1) served as recipients. This strain combination was chosen because of the strong major and minor histocompatibility locus mismatch that results in well-documented complete lung graft rejection within the fifth postoperative day in control animals without immunosuppression. Recipient animals received 1 x 1010 pfu of adenovirus encoding active TGF-ß1 (groups I and VI), IL-10 (groups II and VII), both TGF-ß1 and IL-10 (groups III and VIII), ßgal as an adenoviral control (groups IV and IX), or normal saline without adenovirus (groups V and X) 2 days before transplantation (groups I-V) or at the time of harvest (groups VI-X). Donor lungs were harvested as described below. Briefly, after achievement of general anesthesia, mechanical ventilation, and systemic heparinization, donor rat lungs were flushed through the main pulmonary artery with 20 mL of cold (4°C) low-potassium dextran-1% glucose solution at 20 cm H2O pressure. The heart-lung bloc was removed with the lungs inflated at end-tidal volume. The left lung was stored at 4°C in low-potassium dextran-1% glucose until implantation. Recipient animals were anesthetized and intubated and underwent a left thoracotomy. The pulmonary vessels were anastomosed by a modification of the previously described cuff technique.11 The bronchial anastomosis was performed by a running 8–0 Prolene suture (Ethicon, Inc, Somerville, NJ). Ventilation and perfusion were restored, and a temporary chest tube was placed, which was removed after recovery from anesthesia. In all groups no immunosuppressive drugs were used, and recipients were put to death on the fifth postoperative day.4 Recipient animals were reanesthetized with the donor technique described above and mechanically ventilated with 100% oxygen. Median laparotomy-sternotomy was performed, and the contralateral right hilum was clamped. Animals’lungs were then ventilated for 5 minutes with a tidal volume of 1.5 mL, a respiratory rate of 100 breaths/min, and a peak end-expiratory pressure of 1.0 cm H2O to assess the function of the isolated left lung graft by means of arterial blood gas analysis with blood samples obtained from the ascending aorta. After the animals were put to death, the lung graft was flushed with cold saline solution, and the grafts were subjected to enzyme-linked immunosorbent assay (ELISA) testing to investigate the endogenous cytokine expression in the transplanted lungs.
Histologic assessment
Lungs were perfused through the pulmonary arterial trunk with 20 mL of normal saline and 20 mL of Histochoice (Amresco, Solon, Ohio). The specimens were fixed in Histochoice for 24 hours at 4°C and embedded in paraffin wax. Tissue sections 7-µm thick were cut on a microtome and mounted on slides. Lungs were stained with hematoxylin and eosin. A blinded observer (pathologist, J.H.R.) scored rejection according to the 1995 revision of the working formulation for the classification of pulmonary allograft rejection.12 Vascular and airway rejection scores ranged from 0 (no rejection) to 4 (complete destruction of the allograft).
ELISA for cytokines
Levels of the cytokines IL-10, TGF-ß1, IL-2, TNF-, and IFN- in experimental lung grafts were measured for each group by using ELISA-based techniques. Protein extraction was performed by homogenizing lung grafts in lysis solution containing 100 mmol/L potassium phosphate (pH 7.8), 0.2% triton X-100 with pepstatin A (5 µg/mL), and protease inhibitor cocktail (Complete Mini tabs, Boehringer-Mannheim). The homogenate was then centrifuged at 15,000 rpm for 15 minutes after extraction at room temperature for 15 minutes, and the supernatant was stored at –80°C until ELISA assessment. Human IL-10, human TGF-ß1, rat IL-2, rat TNF-, and rat IFN- Quantikine ELISA kits (R&D Systems, Minneapolis, Minn) were used in this study. The human TGF-ß1 and IL-10 ELISA kits are cross-reactive for human, rat, and mouse. The procedure to activate latent TGF-ß1 in the ELISA kit was not used for the detection of active TGF-ß1.
Statistical analysis
Values are reported as means ± SEM. For the pathologic rejection score, the Kruskal-Wallis rank test was used to compare groups. For other assessments, 1-way analysis of variance with pairwise comparison by the Fisher protected least significant difference method was used to compare overall differences among groups.
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Results
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Groups I-V transfected 2 days before transplantation
Left lungs from groups III, IV, and V, which had been transfected with TGF-ß1, IL-10, and both TGF-ß1 and IL-10, respectively, had superior PaO2 levels compared with control groups I or II (Figure 1, A). Mean PaCO2 levels were not significantly different in any of the groups (32.98 ± 5.44, 31.58 ± 5.7, 22.78 ± 2.4, 22.9 ± 3.2, and 25.53 ± 4.41 mm Hg for groups I, II, III, IV, and V, respectively, P > .5).
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Figure 1. Mean Pao2 after monoventilation of the allograft at the time of death on the fifth postoperative day. PaO2 values in groups III, IV, and V were superior in comparison with those in groups I and II (A). Values in group X was superior in comparison with those in groups VI, VII, VIII, and IX (B). *P < .05.
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Vascular rejection scores and airway rejection scores of the contransfection group (group V) were significantly lower compared with those of the other groups (Table 1).
Endogenous IL-2 levels were lower in group V compared with those in groups I or II (Figure 2). Endogenous TNF- levels were not significantly different in any of the groups (10.1 ± 1.62, 11.0 ± 1.16, 10.06 ± 0.56, 8.58 ± 1.62, and 10.37 ± 1.14 pg/mg total protein for groups I, II, III, IV, and V, respectively; P > .5). Similarly, endogenous IFN- levels were not significantly different in any of the groups (241.34 ± 25.95, 214.95 ± 28.55, 180.59 ± 23.59, 185.94 ± 21.52, and 245.39 ± 35.4 pg/mg total protein for groups I, II, III, IV, and V, respectively; P > .5).
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Figure 2. Endogenous IL-2 expression in the groups transfected 2 days before transplantation. IL-2 expression was lower in group V compared with that in the control groups.
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Groups VI-X transfected at the time of harvest
Left lungs from group X, which had been cotransfected with both TGF-ß1 and IL-10, had superior PaO2 levels compared with those of the other groups (Figure 1, B). Mean PaCO2 levels were not significantly different in any of the groups (37.19 ± 5.05, 32.98 ± 3.95, 32.47 ± 4.68, 25.25 ± 2.75, and 26.47 ± 4.15 mm Hg for groups VI, VII, VIII, IX, and X, respectively; P > .5). Vascular rejection scores and airway rejection scores of group X were significantly lower compared with those of the other groups (Table 1).
Endogenous IL-2 levels were not significantly different in any of the groups (17.15 ± 1.74, 16.75 ± 2.18, 12.91 ± 3.08, 11.08 ± 6.0, and 12.3 ± 5.95 pg/mg total protein for groups VI, VII, VIII, IX, and X, respectively; P > .5). Similarly, endogenous TNF- levels were not significantly different in any of the groups (9.83 ± 0.92, 10.96 ± 1.17, 14.48 ± 3.18, 9.47 ± 0.94, and 11.16 ± 1.27 pg/mg total protein for groups VI, VII, VIII, IX, and X, respectively; P > .5). Endogenous IFN- levels were not significantly different in any of the groups (201.66 ± 21.98, 199.59 ± 30.48, 184.31 ± 18.98, 204.22 ± 49.66, and 229.78 ± 17.78 pg/mg total protein for groups VI, VII, VIII, IX, and X, respectively; P > .5).
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Discussion
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In the present study we demonstrated that recipient intramuscular cotransfection of TGF-ß1 and IL-10 suppressed IL-2 expression and provided a synergistic effect that reduced acute lung graft rejection, as shown by significantly improved lung graft oxygenation and reduced rejection scores. Gene therapy with recombinant adenoviral vectors is currently being used in many clinical gene-transfer trials for lung diseases, such as cystic fibrosis and lung cancer.13,14 In experimental lung transplantation our laboratory has demonstrated the beneficial effects of gene transfer with adenovirus vectors on ischemia-reperfusion injury and acute rejection in a rat lung graft model.4,10,15
However, most disease processes involve a combination of different gene products. This might be a significant issue for the future use of gene therapy because most current gene therapy protocols only transfect one gene. Acute rejection is a process of vascular and parenchymal injury mediated by T cells, macrophages, and antibodies. The activated T cells cause direct lysis of graft cells and produce cytokines that recruit and activate inflammatory cells, which cause necrosis. Type 1 T-regulatory cells produce high levels of IL-10 and TGF-ß, and these cytokines mediate their ability to suppress pathologic immune responses in the setting of transplantation.16
Currently, drug-induced immunosuppression is the major modality for preventing allograft rejection in the clinical transplantation setting. Clinically used immunosuppressants act in part by modulating the cytokine response of the recipient immune system to the allograft. For example, cyclosporine (INN: ciclosporin) inhibits the cytokine IL-2 and induces the synthesis of the immunosuppressive cytokine TGF-ß1; similarly, FK506 also blocks the transcription of IL-2.17,18 Thus, the prevention of allograft rejection requires, in part, effective inhibition of IL-2 and induction of immunosuppressive cytokines, such as TGF-ß1 and IL-10. Immunosuppressants, however, have serious side effects, especially when used for prolonged periods. Gene therapy for lung transplantation, however, has the potential to induce graft acceptance in the recipient without the use of immunosuppressive drugs and their side effects.
In the present study cotransfection with TGF-ß1 and IL-10 two days before transplantation suppressed IL-2 expression in lung grafts. TGF-ß1 transfection alone also seemed to suppress IL-2 expression, showing that TGF-ß1, rather than IL-10, might suppress IL-2. However, significantly improved oxygenation and rejection scores resulting from the synergistic effects of cotransfection with IL-10 and TGF-ß1 suggest the existence of another unknown factor. IL-10 belongs to the TH2 cytokine family, and the balance between TH1 and TH2 cells in transplantation immunology has been studied extensively. In most experimental allotransplantation models, rejection has generally been associated with high expression of TH1 cytokines (IL-2 and IFN-), whereas TH2 cytokines (IL-10 and IL-4) have been detected only slightly or not at all.19,20 The synergistic effect of cotransfection with IL-10 and TGF-ß1 observed in this study therefore might be related to an alteration in the balance of the TH1 and TH2 cytokine responses to allograft transplantation in the recipient.
In this study the TH1 cytokines IFN- and TNF- were insufficiently not suppressed by TGF-ß1 and IL-10 overexpression. One of the potential reasons for this might be the intramuscular transfection route used in this study. Our group has recently shown that intratracheal administration of adenoviral vector encoding the desired transgene is optimal in experimental lung transplantation in that it produces the highest expression of the desired transgenic protein within the lung graft itself.21 Although one advantage of intramuscular transfection is that it avoids vector-induced inflammation within the lung graft, the disadvantage is that local concentrations of the desired transgenic protein within the lung graft are lower than with intratracheal transfection. We have also recently demonstrated that focal endobronchial transfection allows for the use of much less vector and thus much less vector-induced inflammation.15 Thus future work will examine whether endobronchial transfection can effectively inhibit acute rejection and more potently suppress the production of proinflammatory cytokines. Another reason that suppression of IFN- and TNF- could not be demonstrated might be because levels of these cytokines might peak during the early phase of the rejection process rather than at the height of acute rejection, the time point used for cytokine measurements in this study. Thus future work will also further characterize early changes in the process of graft rejection mediated by gene therapy with immunosuppressive cytokines. Finally, future work will also examine whether additional cotransfection with immunosuppressive cytokines, which specifically inhibit the action of IFN-, might create more effects on immunosuppression.
The timing of transfection is important in the clinical setting. Transfection 2 days before transplantation is not readily applicable to the clinical setting. The present study showed that cotransfection at the time of harvest reduced acute rejection. In light of the fact that only the cotransfected group showed effects against rejection, the cotransfection approach of recipient intramuscular injection of adenoviral vector can be applied in the clinical setting. However, IL-2, IFN-, and TNF- were not suppressed in the group transfected at the time of harvest. This means that TGF-ß1 and IL-10 expression might not be high enough in the early phase of acute rejection. Our laboratory has shown a synergistic benefit of cotransfection compared with transfection of either cytokine alone.22,23 The advantage of cotransfection is its synergistic effect, but its disadvantage is that the inflammatory response might be increased because of the increase in the quantity of adenovirus administered. It might augment the formation of antibodies to the adenovirus vector. Unfortunately, antiadenovirus antibodies might reduce the degree and duration of transgene expression.23 However, cotransfection might be required to regulate concurrent pathways in clinical gene therapy.
In conclusion, recipient intramuscular cotransfection of TGF-ß1 and IL-10 suppressed IL-2 expression and provided a synergistic effect that reduced acute lung graft rejection. The synergistic effects of multiple gene transfer have great potential for reducing clinical acute lung graft rejection.
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Acknowledgments
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We thank Kathleen Grapperhaus for technical assistance and Mary Ann Kelly and Dawn Schuessler for secretarial support. Statistical advice was obtained from Richard B. Schuessler, PhD.
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Footnotes
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Supported by National Institutes of Health grant 2 R01 HL041281.
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References
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- Daddi N, Suda T, D’Ovidio F, Kanaan SA, Tagawa T, Grapperhaus K, et al. Recipient intramuscular cotransfection of naked plasmid transforming growth factor ß1 and interleukin 10 ameliorates lung graft ischemia-reperfusion injury. J Thorac Cardiovasc Surg 2002;124:259-269.[Abstract/Free Full Text]
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Abstract
Materials and methods
