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Ann Thorac Surg 2004;78:1932-1939
© 2004 The Society of Thoracic Surgeons
a Division of Cardiothoracic Surgery, Department of Surgery, Washington University School of Medicine, St. Louis, Missouri, USA
b First Department of Surgery, Nagasaki University School of Medicine, Nagasaki, Japan
Accepted for publication June 7, 2004.
* Address reprint requests to Dr Patterson, Division of Cardiothoracic Surgery, Washington University School of Medicine, One Barnes-Jewish Hospital Plaza, 3108 Queeny Tower, St. Louis, MO 63110-1013 (E-mail: pattersona{at}msnotes.wustl.edu).
Presented at the Fortieth Annual Meeting of The Society of Thoracic Surgeons, San Antonio, TX, Jan 26–28, 2004.
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METHODS: All experiments utilized an orthotopic left lung isograft transplant model. Donors were divided into three groups (n = 6 each) for endobronchial transfection: group I received 2 x 107 plaque-forming units of adenovirus encoding soluble type I interleukin-1 receptor IgG; group II received 2 x 107 plaque-forming units of nonfunctional control adenovirus encoding ß-galactosidase; and group III received 0.1 mL of saline. Left lungs were harvested 24 hours after transfection and stored for 18 hours before transplantation. Graft function was assessed 24 hours after reperfusion using three measurements: isolated graft oxygenation, wet-to-dry lung weight ratio, and tissue myeloperoxidase activity. Transgene expression of soluble type I interleukin-1 receptor IgG was also evaluated using enzyme-linked immunosorbent assay and immunohistochemistry.
RESULTS: Isolated graft arterial oxygenation was significantly improved in group I compared with groups II and III (281.8 ± 134.8 versus 115.7 ± 121.5 and 88.0 ± 58.9 mm Hg, p = 0.0197 and p = 0.0081, respectively). Myeloperoxidase activity was also significantly reduced in group I compared with groups II and III (0.083 ± 0.044 versus 0.155 ± 0.043 and 0.212 ± 0.079 optical density units per minute per milligram protein, p = 0.0485 and p = 0.0016, respectively). Expression of soluble type I interleukin-1 receptor IgG was detected only in lungs from group I.
CONCLUSIONS: Endobronchial gene transfer of soluble type I interleukin-1 receptor IgG to donor lung grafts subjected to prolonged cold ischemia ameliorates ischemia-reperfusion injury by improving graft oxygenation and reducing lung edema and neutrophil sequestration.
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Gene therapy is emerging as a new strategy to combat disease in many different areas of thoracic surgery [3]. Some of the arenas in which gene therapy has found fruitful experimental application include malignant diseases of the chest [4], cystic fibrosis [5], and lung transplantation [6]. Our laboratory and others have demonstrated the feasibility of gene therapy in ameliorating ischemia-reperfusion injury after lung transplantation using rodent orthotopic lung transplantation model [6]. Exploiting gene therapy in lung transplantation requires the consideration of many different factors. These include, but are not limited to, the type of transgene vector (eg, naked plasmid, liposome, or adenovirus), gene transfer strategy (ex vivo gene transfer to grafts versus in vivo gene transfer to either donors or recipients), and delivery route (systemic delivery by intramuscular or intravascular routes versus local delivery using endobronchial or proximal pulmonary artery segment transfection) [6]. As the number of patients awaiting lung transplantation increases and the donor pool of organs remains limited, there has been increasing pressure to find ways to maximize the use of donor organs. Using gene therapy to repair and improve the quality of donor lungs to withstand ischemia-reperfusion injury is a promising strategy that could have a tremendous impact on the number of transplants performed and on outcomes after transplantation. Unlike other therapies for ischemia-reperfusion injury, gene therapy also has the potential to significantly prolong acceptable ischemic times in transfected organs.
Interleukin-1 (IL-1) is a proinflammatory cytokine mainly produced by macrophages [7]. Its role in a variety of inflammatory injuries has been studied extensively, and it has also been shown to be an important factor in ischemia-reperfusion injury after lung transplantation [8]. Soluble type I IL-1 receptor (sIL-1RI) functions as an extracellular binding protein of IL-1. It competitively inhibits the binding of IL-1 to receptors attached to the cell surface and therefore reduces the proinflammatory actions of IL-1 [9]. Hence, gene transfer of sIL-1RI has the potential to inhibit the inflammatory cascade mediated by IL-1 and reduce acute lung graft reperfusion injury. The aim of this study was to determine whether in vivo endobronchial gene transfection of an sIL-1RI-IgG fusion protein (sIL-1RI-Ig) to donor lungs could ameliorate lung graft ischemia-reperfusion injury after prolonged cold storage.
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Adenoviral Vectors
Adenovirus encoding sIL-1RI-Ig (Ad.sIL-1RI-Ig) is a replication-deficient recombinant type 5 adenoviral vector lacking the E1 and E3 loci [10]. The sIL-1RI-Ig cDNAs were inserted in place of the E1 region, and expression was driven by the cytomegalovirus promotor. Adenovirus encoding sIL-1RI-Ig encodes the extracellular portion of the human type I IL-1 receptor fused to the mouse IgG1 heavy chain [9]. The adenovirus used in this study was provided as a gift from Dr Paul D. Robbins, Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA [9].
First-generation replication-deficient adenovirus serotype 5 carrying the Escherichia coli LacZ gene encoding ß-galactosidase (ß-gal) was purchased from the Gene Therapy Center at the University of North Carolina, Chapel Hill, NC; ß-gal is a nonfunctional reporter gene used as an adenovirus control.
Experimental Design
In this study, two experiments were performed. Experiment 1 determined the transgene expression of sIL-1RI-Ig immediately before transplantation. Experiment 2 evaluated the effect of sIL-1RI-Ig gene transfection on lung graft ischemia-reperfusion injury. Both experiments had the same study groups. Fischer 344 rats were divided into three groups based on the transfecting vector: group I donors received 2 x 107 plaque-forming units (pfu) of Ad.sIL-1RI-Ig, group II donors received 2 x 107 pfu of adenovirus encoding ß-gal, and group III donors received 0.1 ml of normal saline. The transfection titer of 2 x 107 pfu of adenovirus was chosen based on our previous reports [11, 12].
In Vivo Endobronchial Gene Transfection of the Donor
Donor animals were anesthetized with a subcutaneous injection of ketamine chloride (25 mg/kg) and atropine sulfate (0.25 mg/kg). After endotracheal intubation with a 14-gauge catheter, animals were mechanically ventilated with a small-animal Harvard ventilator (tidal volume: 2.5 mL, respiratory rate: 60 breaths/min) with inhalational halothane used for anesthesia. Donors underwent a right thoracotomy, and the carina was dissected. A catheter was introduced through the endotracheal tube selectively into the left main bronchus and vector diluted in 0.1 mL of sterile normal saline solution was instilled. After 5 minutes of bilateral ventilation after instillation, the left main bronchus was clamped for 30 minutes at the end-inspiratory phase of mechanical ventilation. After the left main bronchus was unclamped, the right thoracotomy was closed and animals were recovered.
Donor Lung Procurement
Donor lungs were harvested 24 hours after gene transfection. The procurement technique was identical to previously published reports from our laboratory [11, 12].
Experiment 1: Expression of sIL-1RI-Ig Before Transplantation
In the three donor groups (n = 3 in each group), donor lung grafts were harvested 24 hours after transfection and sIL-1RI-Ig transgene expression was measured using sIL-1RI enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry of sIL-1RI was performed to document epithelial cell transgene expression. In addition, for the Ad.sIL-1RI-Ig group (group I), we measured transgene expression in donor lung grafts after 18 hours of cold preservation.
SIL-1RI ENZYME-LINKED IMMUNOSORBENT ASSAY
Lungs were homogenized and total protein was extracted using methods identical to previously published reports from our laboratory [11, 12]. The human sIL-1RI enzyme-linked immunosorbent assay (ELISA) was performed using monoclonal antihuman IL-1RI antibody, biotinylated antihuman IL-1RI antibody and recombinant human sIL-1RI (purchased from R&D Systems, Minneapolis, MN) according to manufacturer protocol. There is no cross reactivity between the human and rat sIL-1RI proteins. Optical density was determined using an Ultra Microplate Reader (EL808; Bio-Tek Instruments, Winooski, VT) set to 450 nm with appropriate wavelength correction. The extract was subsequently assayed for total soluble protein (pg/mg total protein) using the method of Pierce Laboratories (Rockford, IL) [13].
IMMUNOHISTOCHEMISTRY OF SIL-1RI
Tyramide Signal Amplification (TSA) Biotin System kits (NEN Life Science Products, Boston, MA) were used for immunohistochemistry. Briefly, at sacrifice, lungs were perfused with 20 mL of normal saline and 20 mL of HistoChoice (Amresco, Solon, OH). Specimens were fixed, cut, mounted, deparaffinized, and then treated with Dako Target Retrieval Solution (Dako, Carpinteria, CA), followed by 3% hydrogen peroxide in methanol. The sections were then incubated with an Fc
II Receptor blocker (Purified antirat CD32, BD PharMingen, San Diego, CA), and with Super Block Blocking Buffer (Pierce Chemical, Rockford, IL) including 1% bovine serum albumin and 1% normal goat serum, followed by TNB Blocking Buffer in TSA kits. The sections were then incubated overnight with a biotinylated goat antihuman IL-1RI antibody (R&D Systems, Minneapolis, MN) at 1:20 dilution in TNB Blocking Buffer at 4°C. They were then incubated with Streptavidin-Horseradish Peroxidase, followed by Biotinyl Tyramide solution, and then Streptavidin-Alkaline Phosphatase (NEN Life Science Products, Boston, MA). Specific binding was detected using the BCIP/NBT substrate working solution (Vector Laboratories, Burlingame, CA) containing 5 mmol/L levamisole (Vector Laboratories). The slides were then counterstained with nuclear fast red, dehydrated and mounted, and cover slips were placed.
Experiment 2: Effect of sIL-1RI-Ig Gene Transfection on Lung Graft Ischemia-Reperfusion Injury
In all three groups (n = 6 in each group), donor lungs were harvested 24 hours after endobronchial transfection and transplanted into recipient animals after 18 hours of cold preservation. Briefly, recipient animals were anesthetized, intubated, and had a left thoracotomy performed. The pulmonary vessels and bronchus were anastomosed using a modification of the previously described "cuff technique" [14]. Graft assessment was performed 24 hours after reperfusion. Recipient animals were reanesthetized with pentobarbital. After tracheostomy, animals were mechanically ventilated with 100% oxygen. The right main bronchus and pulmonary artery were clamped to isolate the left lung graft. Animals were ventilated for 5 minutes using a tidal volume of 1.5 mL, respiratory rate of 100 breaths per minute, and positive end-expiratory pressure of 1.0 cm H2O. At the end of 5 minutes of isolated graft ventilation, arterial blood gas analysis was performed using blood samples obtained from the abdominal aorta. Then, the lungs were immediately flushed with 20 mL of cold (4°C) saline solution. Each left lung graft was isolated from the heart-lung block and divided into three sections. The upper third was used for ELISA of sIL-1RI and endogenous rat tumor necrosis factor-
(TNF) and IL-1ß. The middle third of the lung graft was used for myeloperoxidase activity measurements. The lower third of the graft was weighed, dried at 80°C for 48 hours, and then reweighed for calculation of the wet-to-dry lung weight ratio.
MYELOPEROXIDASE ACTIVITY
Quantitative myeloperoxidase (MPO) activity was determined as previously described [15]. Optical density was measured at 460 nm with a spectrophotometer (Model PMQ II; Carl Zeiss, Oberkochen/Wuett, Germany). Color development was linear from 5 minutes to 20 minutes. One unit of enzyme activity was defined as 1.0 optical density units per minute per milligram of tissue protein at room temperature.
ELISA OF ENDOGENOUS RAT TNF AND IL-1ß
The upper third of reperfused lung grafts from all groups were used for ELISA of endogenous rat TNF and IL-1ß by a procedure similar to that described earlier for sIL-1RI. The rat TNF and IL-1ß ELISA kits were purchased from R&D Systems (Minneapolis, MN).
Statistical Analysis
All values are described as mean ± SD. Data not normally distributed were analyzed after logarithmic correction. One-way analysis of variance with pairwise comparison by Fisher's least significant difference method was used to compare overall differences among multiple groups.
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ELISA OF ENDOGENOUS RAT TNF and IL-1ß IN REPERFUSED LUNG GRAFTS
Endogenous rat TNF and IL-1ß levels in reperfused lung grafts were measured to determine the effect of sIL-1RI-Ig transfection on the production of these proinflammatory cytokines. There was a noticeable trend toward lower levels of both cytokines in the group transfected with sIL-1RI-Ig compared with each of the control groups. The levels of TNF for group I compared with groups II and III were 2.86 ± 1.70 versus 3.20 ± 1.22 and 5.34 ± 3.24 pg/mg total protein, respectively (p = 0.8074 and p = 0.0804, respectively). Levels of IL-1ß for group I compared with groups II and III were 197.2 ± 100.3 versus 224.1 ± 53.7 and 319.6 ± 73.4 pg/mg total protein, respectively (p = 0.5693 and p = 0.0191, respectively). The difference in levels of endogenous IL-1ß between groups I and III reached statistical significance.
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and IL-1ß have been described. The type I IL-1 receptor is an 80 kDa transmembrane protein that is expressed predominantly by T cells, fibroblasts, and endothelial cells [7, 23]. Recombinant sIL-1RI is a potent antagonist of IL-1 action [9]. The purpose of this study was to evaluate whether adenovirus-mediated gene transfer of sIL-1RI-Ig could inhibit IL-1 activity and reduce acute lung injury induced by prolonged cold ischemia.
In the present study, the overexpression of sIL-1RI-Ig as demonstrated by ELISA resulted in physiologic improvement of transplanted lung grafts with corresponding reduction in ischemia-reperfusion injury. The mechanism for this improvement is that gene transfer of sIL-1RI-Ig produces sIL-1RI protein expression, which competitively inhibits the action of IL-1 and attenuates the proinflammatory cascade of ischemia-reperfusion injury. In assessing the function of the isolated lung graft after transplantation, gas exchange is perhaps the most important factor. Isolated isograft oxygenation was more than twofold higher in lungs transfected with sIL-1RI-Ig compared with controls. In addition, lungs transfected with sIL-1RI-Ig demonstrated significantly reduced pulmonary edema and neutrophil sequestration as measured by wet-to-dry lung weight ratio and MPO activity, respectively. Also, production of the endogenous proinflammatory cytokines TNF and IL-1ß was lower in lungs transfected with sIL-1RI-Ig than in controls, providing further evidence that IL-1 inhibition reduced the proinflammatory environment characteristic of ischemia-reperfusion injury.
We have demonstrated that in vivo donor intravenous transfection or recipient intramuscular transfection of functional genes such as heat shock protein 70 [24], endothelial nitric oxide synthase [25], and human interleukin-10 [26, 27] attenuates ischemia-reperfusion injury after experimental lung transplantation. Our laboratory has also shown that in vivo donor endobronchial gene transfer of human interleukin-10 [11] and soluble type I tumor necrosis factor receptor [12] ameliorates ischemia-reperfusion injury. In vivo systemic gene delivery to the donor or recipient requires large amounts of vector (5 x 109 pfu or 1 x 1010 pfu, respectively) and the systemic side effects of such delivery routes are not clear. In contrast, gene delivery through an endobronchial route, as used in the present study, requires a much lower titer of adenovirus vector (2 x 107 pfu) to ameliorate ischemia-reperfusion injury. Such data shows that localized endobronchial gene transfer can be as effective as systemic intravenous or intramuscular delivery in ameliorating ischemia-reperfusion injury while requiring more than 250-fold less adenovirus vector. Endobronchial gene delivery is a graft-targeted transfection strategy unique to lung transplantation. The advantages of endobronchial transfection are that it is relatively simple to perform, results in minimal host inflammatory response to adenoviral transfection, and provides transfection of the lung graft alone. It transfects respiratory epithelium without significant systemic expression in heart, liver, native right lung, or plasma [12, 28]. Therefore, endobronchial transfection of donor lung grafts provides an organ-selective and effective transfection strategy for use in lung transplantation.
The results of immunohistochemical staining in the present study showed heterogeneous distribution of transgene expression with staining only in respiratory epithelium and not in vascular endothelium, smooth muscle or interstitial tissue cells. It is interesting to note that endobronchial gene transfection affords protection against ischemia-reperfusion injury despite the fact that vascular endothelial cells, important in the genesis of reperfusion injury, are not transfected. That may be because endobronchial gene transfer works in a paracrine manner, although the data from the present study are not sufficient to confirm this hypothesis.
An important complication of airway gene delivery is the local inflammation that occurs as a response to adenovirus transfection [29]. Local inflammation may be due to the heterogeneous volume of vector solution, direct toxicity of the adenovirus vector [30], or the host immune response to adenovirus transfection, with the result being dose-dependent local inflammation [29]. Lungs transfected with adenoviral vectors may display this inflammatory response in different forms, such as pneumonia, atelectasis or edema. Low dose endobronchial transfection significantly reduces the local inflammatory response to adenovirus transfection, as demonstrated by the immunohistochemistry findings in this study (no neutrophils in the alveoli of lungs transfected with low-dose endobronchial adenovirus). The advantages of using less vector include decreased costs, reduced local inflammation, and reduced viral antigenicity in cellular and humoral immune responses [31].
In order to apply gene therapy to clinical lung transplantation, experimental studies need to demonstrate functional improvement in transplanted lung grafts with minimal pathophysiologic side effects rather than merely document transgene expression alone. The protein expression of transfected genes is dose dependent [32], and finding the optimal titer for gene transfection to produce optimal levels of the desired protein is crucial for eventual therapeutic applications. The demonstration of adequate transgene expression without improvement in graft function can mean that either the dose of transgene administered was lower than that required for functional improvement or so large that the inflammatory response to gene transfection overwhelmed the desired positive effects of transgene expression. Therefore, the detection of transgene expression combined with proof of physiologic improvement and minimal side effects are essential in the study of functional gene therapy. Further efforts in this area should be directed toward searching for the best strategy of gene transfection, using minimal amounts of vector necessary to produce functional transgene expression with physiologic improvement, to apply this strategy to clinical settings.
It has been recently demonstrated that IL-10 gene therapy, delivered in an adenoviral vector endoscopically, is effective in reducing ischemia-reperfusion injury in a large-animal model of lung transplantation [33]. This indicates that human lung protection from ischemia-reperfusion and immunologic injury by gene therapy is feasible and may soon be possible. One of the main challenges to the implementation of gene therapy for ischemia-reperfusion injury in clinical lung transplantation is finding approaches to reduce the vector-associated inflammatory response to allow for minimum time between transfection and transplantation. Clearly, the 24-hour incubation period between gene transfection and donor lung harvest used in this study is impractical in the clinical setting. Recent work by de Perrot and colleagues [34] shows that the minimal period between transfection and harvest that allows for sufficient transgene expression to produce functional graft improvement may be reduced to as little as 6 hours if steroids are used to blunt the inflammatory response to transfection itself. Low-titer endobronchial gene transfection as developed by our laboratory represents another method by which the inflammatory response to transfection itself may be reduced. Recent advances in gene therapy vector technology provide the promise of great improvements in safety and efficiency of gene transfer and the hope that clinical trials for gene therapy in lung transplantation may soon be possible.
In conclusion, in vivo endobronchial gene transfer of sIL-1RI-Ig produces sIL-1RI protein expression, inhibits the proinflammatory actions of IL-1, and ameliorates ischemia-reperfusion injury in rodent lung transplantation. Lung grafts transfected with Ad.sIL-1RI-Ig showed improved isolated graft oxygenation, less lung edema and reduced neutrophil sequestration compared with controls. Furthermore, the localized endobronchial transfection strategy employed in this study uses much less vector than other routes of adenovirus-mediated gene transfer. Endobronchial gene transfer to donor lung grafts is a potential strategy for the application of gene therapy in clinical lung transplantation and merits further investigation.
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and IL-1ß were reduced. However, in looking at that graph, I noticed that the negative control virus had about the same degree of reduction of TNF and IL-1ß. I wanted to know if you looked at any other mechanisms that may explain the reduction in injury, and also if you could explain that finding as well.
DR TAGAWA: The TNF and IL-1ß is our main proinflammatory cytokine of IR injury. So this time we used a soluble type I IL-1 receptor. This mainly inhibits the IL-1 action. The main action is an inhibitor of IL-1ß in the ischemia-reperfusion injury. But all ischemia-reperfusion injury was inhibited, so TNF- is relatively reduced, I think.
DR VAPORCIYAN: And that explains that your negative control virus caused the same reduction in TNF? The last slide that you had.
DR TAGAWA: Yes. The negative control, ß-gal, TNF are a little the same as the study group, but there is no significant difference. Especially with the saline group, both are higher than the control group, I think.
DR THOMAS K. WADDELL (Toronto, Ontario, Canada): Congratulations. Very nice studies and very well presented. It is a bit unusual model of gene delivery to a transplant donor, 30 minutes of bronchus clamping followed by 24 hours of recovery. I have three somewhat related questions for that. How did you come to that model; is it important; and would this type of therapy not work by giving it to the recipient, which could be done perhaps with a bit better time kinetics?
DR TAGAWA: This model is after the minitrial. I tried a thoracotomy on the right—your institute, Toronto—to reduce the inflammation and to make the expression I reached in this model. The second question?
DR WADDELL: This receptor, this soluble receptor strategy, that should work if it was given to the recipient and it were simply circulating in the bloodstream, should it not have, or do you think that it's necessary to have, high-level expression actually in the lung?
DR TAGAWA: I think it should work, but we are ongoing now with that work.
DR PATTERSON: Tom, it's an excellent question, and it is a question that the study section asks every time we submit this work for renewal. Of course, 24 hours ahead of time is ridiculous in the clinical donor context. What we need to do is to work on strategies to improve the speed with which gene expression occurs. We know is that the idea of utilizing IL-1 inhibition as a strategy will work. The only question is how to make it applicable, and we're working on that now.
The other question you asked, can you give it to the recipient: yes, you can. We've done quite a bit of work, actually, utilizing the strategy of intramuscular gene transfection in the recipient, which produces a pretty impressive degree of systemic expression of the cytokine. The advantage of that may not be in the reperfusion context, but in the context of rejection. One could give it repeatedly. The work that your group has done demonstrated that it is feasible to repeat the same adenovirus transfection in an immunosuppressed host. So it does work if we utilize it as a recipient strategy. The advantage of giving it endobronchially is that it is a focused strategy that can be delivered at quite low dose, using much less virus than giving it to the recipient intramuscularly.
DR WADDELL: Alec, would you like to address Ara's question about the apparent antiinflammatory effect of ß-gal transfection?
DR PATTERSON: I have no explanation for that. You wouldn't predict it. In some of the other work we have done where we have used other antiinflammatory cytokine transfection and used the adenoviral ß-gal as a viral control, we have not seen that phenomenon. It may be that if we sacrificed the animals at different time points we would not see that.
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soluble receptors to rabbit knees with experimental arthritis has local and distal anti-arthritic effects. Proc Natl Acad Sci USA. 95. 1998. pp. 4613-4618.
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  A Fisher Anti-inflammatory gene therapy in the donor improves graft function after lung transplantation Thorax, April 1, 2005; 60(4): 319 - 319. [Full Text] [PDF]
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