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Impact of Human Interleukin-10 on Vector-Induced Inflammation and Early Graft Function in Rat Lung Transplantation

Thoracic Surgery Research Laboratory, Toronto General Hospital Research Institute, University Health Network, University of Toronto, Ontario, Canada; and The Gene Transfer Vector Core, Department of Medicine, University of Iowa, Iowa City, Iowa

Address correspondence to: S. Keshavjee, M.D., Director, Thoracic Surgery Research Laboratory, Toronto General Hospital, 200 Elizabeth Street, EN 10-224, Toronto, Ontario, Canada M5G 2C4. E-mail: shaf.keshavjee{at}uhn.on.ca

	Abstract

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This study was undertaken to examine the time course of human interleukin (hIL)-10 gene expression after transtracheal administration of adenoviral (Ad)hIL-10 and its effect on the early adenoviral proinflammatory cytokine response and on post-transplant lung function. Using a rat lung transplant model, we observed that lungs retrieved 12 h after the administration of AdhIL-10 were associated with significant improvement in post-transplant lung function. Shorter periods of transfection were associated with significantly elevated levels of tumor necrosis factor- and macrophage inflammatory protein-2 in lung tissue, leading to an increased degree of injury. The release of proinflammatory cytokines secondary to the adenoviral vector was reduced by high-dose methylprednisolone (30 mg/kg) administered 3 h before transfection. Reduction in the early adenoviral inflammatory response was associated with significant improvement in post-transplant lung function when lungs were retrieved 6 or 12 h after transtracheal administration of AdhIL-10. Transtracheal administration of adenoviral-mediated hIL-10 to donor lungs is associated with a significant early inflammatory response that may enhance ischemia-reperfusion injury if insufficient hIL-10 is expressed in lung tissue before retrieval. The period between delivery of AdhIL-10 and lung retrieval can be reduced if the early inflammatory response is suppressed with methylprednisolone.

Abbreviations: cold ischemic time, CIT • human IL-10, hIL-10 • interferon, IFN • interleukin, IL • ischemia-reperfusion, IR • low-potassium dextran, LPD • main bronchus, MB • macrophage inflammatory protein, MIP • pulmonary artery, PA • partial pressure of oxygen, Pa_O2 • peak airway pressure, PawP • positive end-expiratory pressure, PEEP • pulmonary vein, PV • Rous sarcoma virus, RSV • tumor necrosis factor, TNF • wet-to-dry, W/D

	Introduction

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Over the last two decades, lung transplantation has evolved from an experimental endeavor to the mainstay of therapy for many end-stage lung diseases. Almost 15,000 lung transplants have been performed worldwide, and over 1,500 transplants are performed annually (1). Although the number of patients on the waiting list is increasing, even in the most aggressive programs, only 20–30% of donor lungs are deemed suitable for transplantation (2). To increase the pool of available donor lungs, we and others have recently extended our donor selection criteria (3–5). However, this strategy at some point may contribute to an increased risk of immediate graft dysfunction and may lead to more severe acute rejection and impaired long-term graft function (3, 6, 7). Hence, the development of new strategies to repair and improve the quality of donor lungs could have a tremendous impact on the number of transplants performed and on the outcome after transplantation.

Genetic modification of donor organs using gene therapeutic approaches, such as viral vector-mediated gene delivery to donor lungs, is a novel and promising strategy. The use of gene therapy in the transplantation setting is facilitated by the fact that the immunosuppressive therapy used to prevent graft rejection allows more effective and prolonged transfection without developing immunization (8, 9). This strategy, however, is hindered by the poor transfection rate obtainable during the period of cold temperature required to preserve the organ and by the potential to transfect other organs if the gene is delivered systemically instead of locally. To overcome these problems, we have delivered the gene to the donor lung through the transtracheal route before retrieving and cooling the lungs to 4°C (10). This technique provides specific delivery to the lungs and avoids unnecessary transfection of other organs, such as the heart, liver, or kidneys (10).

Cytokines have been shown to play a critical role in modulating inflammatory processes and in enhancing cellular infiltration in transplanted organs. In human lung transplantation, we have observed that pro- and anti-inflammatory cytokines, such as tumor necrosis factor (TNF)-, interferon (IFN)-, interleukin (IL)-10, IL-12, and IL-18, are elevated in lung tissue during the cold ischemic time (CIT) and decrease over time after reperfusion. The chemokine IL-8, however, significantly increased after reperfusion, and its levels correlated negatively with lung function and outcome (11). In addition, we and others have observed that IL-8 was significantly higher in lungs from donors that subsequently developed severe graft dysfunction immediately after reperfusion (11, 12). Hence, we hypothesized that upregulation of a potent anti-inflammatory cytokine, such as interleukin-10, in the donor lung before graft retrieval could limit ongoing lung inflammation and prevent further injury related to ischemia and reperfusion.

We have recently shown that the transtracheal administration of adenoviral-mediated human IL-10 (hIL-10) gene to donor rat lungs 24 h before lung retrieval reduces ischemia-reperfusion (IR) injury and improves post-transplant graft function in an isogeneic rat single lung transplant model (13). In clinical practice, however, the time between brain death declaration of a potential donor and organ retrieval is kept as short as possible to reduce the risk of potential complications related to brain stem death and usually ranges from 6–12 h. Hence, a period of < 24 h between gene delivery and lung retrieval is a desirable and important practical prerequisite to deliver genes to the donor in the setting of clinical lung transplantation.

A short period between gene delivery and lung retrieval raises two important issues. The first is related to the minimum time necessary to obtain sufficient gene expression before lung retrieval, and the second relates to the effect of the nonspecific inflammation associated with the administration of an adenoviral vector on IR injury and post-transplant lung function. The present study was performed to determine the minimal time required after transtracheal administration of adenoviral (Ad)hIL-10 to the donor that provides improved function of the lung after transplantation.

	Material and Methods

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Animals
Experiments were performed using male inbred (250–350 g) Lewis rats (Charles River, Montreal, QC, Canada). All animals received care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research, the Guide for the Care and Use of Laboratory Animals (NIH publication no.85–23, Revised 1996, US Government Printing Office, Washington, DC), and the Guide to the Care and Use of Experimental Animals formulated by the Canadian Council on Animal Care. The experimental protocol was approved by the Animal Care Committee of the Toronto General Hospital Research Institute.

Recombinant Adenovirus Expressing hIL-10
Second-generation adenoviral vectors (serotype 5) containing the hIL-10 gene with a Rous sarcoma virus (RSV) promotor (Ad5RSVhIL-10) and "empty" vectors (Ad5BGL2) were constructed at the Gene Transfer Vector Core of the University of Iowa College of Medicine. The hIL-10 adenoviral construct is referred to as "AdhIL-10," and the Ad5BGL2 is referred to as "empty vector."

hIL-10 cDNA was obtained by polymerase chain reaction with 5' and 3' flanking primers (5'-hIL-10BamHI: 5'-CGCGGATCCCATGCACAGCT-CAGCACTG-3'; 3'-hIL-10BamHI: 5'-CGCGGATCCGCCACCCTGATGTCTCAGT-3') using the clone pSRhIL-10 as template (kindly provided by E. Field, University of Iowa). The polymerase chain reaction product was cloned, using the BamHI restriction-site tails added to the oligonucleotide sequences, in a shuttle plasmid (pAdRSV4). This shuttle plasmid contains the RSV promotor, the SV40-polyA signal, and the genomic adenoviral sequences from 0 to 1 and 9 to 16 map units of human adenovirus type 5. Recombinant adenovirus expressing IL-10 was generated by homologous recombination between pAdRSVhIl-10 and human adenovirus serotype 5 derivative d1309 using standard methods (14). AdBGL2 has the same viral backbone as AdRSVhIL-10.

In Situ Transfection Procedure
Donor rats were anesthetized in a halothane chamber, intratracheally intubated with a 14-gauge intravenous canula, and connected to a volume-controlled ventilator (Model 683; Harvard Rodent Ventilator, South Natick, MA). All animals were ventilated with a fraction of inspired oxygen (FI_O2) of 1.0 and a tidal volume of 10 ml/kg at 80 breaths/min. A 1-ml syringe containing 0.5 ml of the transfection solution was connected to the lateral outlet of a three-way stopcock placed in the circuit at the endotracheal catheter.

For intratracheal injection, the ventilator outlet of the three-way stopcock was closed, and the solution was injected. Ventilation was continued until the animal resumed spontaneous breathing ( 60 s after intratracheal injection). We have previously demonstrated that this strategy provides effective and uniform delivery of vector to the lungs (13). All transfected animals were kept in microisolators until the lung retrieval procedure was started. Food and water was supplied ad libitum.

Lung Transplantation Procedure
Retrieval and storage. Donor rats were anesthetized by an intraperitoneal injection of 1 ml of sodium pentobarbital (Somnotol; MTC Pharmaceuticals, Cambridge, ON, Canada) and intubated through a tracheostomy with a 14-gauge intravenous catheter. The tracheostomy tube was then connected to a volume-controlled ventilator (Harvard Rodent Ventilator), and the animals were ventilated at a rate of 80 breaths/min, a tidal volume of 10 ml/kg, a FI_O2 of 1.0, and a positive end-expiratory pressure (PEEP) of 2 cm H₂O. After this, a median laparo-sternotomy was performed, and 300 USP units of heparin (Hepalean; Organon Teknika, Toronto, ON, Canada) were injected into the inferior vena cava. After 5 min, 0.5 ml of arterial blood was taken from the abdominal aorta for blood gas analysis. For the retrieval of the heart-lung block, the inferior vena cava was incised, the left atrial appendage was excised, and a 14-gauge intravenous catheter was placed into the main pulmonary artery (PA) through an anterior incision in the right ventricular outflow tract. The lungs were flushed through this catheter with 20 ml of low-potassium dextran (LPD) preservation solution (Perfadex; Vitrolife, Gotenberg, Sweden) from a height of 30 cm. Immediately after flushing the lungs, the tracheostomy tube was clamped after inspiration to preserve the lungs in the inflated state. The heart-lung block was removed and placed in LPD at 4°C. The left lung was prepared for transplantation with the placement of three 14-gauge cuffs into the left PA, left main bronchus (MB), and the left pulmonary vein (PV), respectively. The left lung was then placed into 40 ml of LPD at 4°C.

Transplantation. Recipient animals were anesthetized in a halothane chamber, and a tracheostomy was performed as described for the donor animals. The recipient animals were ventilated with 100% O₂ mixed with 1.5% halothane at a rate of 80 breaths/min, a tidal volume of 10 ml/kg, and a PEEP of 2 cm H₂O. A left thoracotomy was performed through the fifth intercostal space. The left lung was mobilized by dividing the pulmonary ligament. The hilar structures were dissected free. The left PA, PV, and MB were identified and clamped with microsurgical aneurysm clamps. A ventral incision was made in each of these structures. The cuffs on the donor lung structures were placed into the corresponding recipient structures. The anastomoses were secured with 6.0 polypropylene ties. The implantation time (warm ischemic time) was standardized at 20 min. After the 20-min warm ischemic time, the transplanted lung was reinflated after removal of the MB clamp, and blood was reintroduced by unclamping the PV followed by the PA. The animals were ventilated with a FI_O2 of 1.0 at a rate of 80 breaths/min, a tidal volume of 10 ml/kg, and a PEEP of 2 cm H₂O during the 2-h reperfusion period. One minute after the beginning of the reperfusion period, each animal received 1 ml of 0.9% normal saline intraperitoneally for volume replacement. Heparin was not given to the recipient animals because it is not routinely administered to lung transplant recipients before graft reperfusion at our institution. Each animal was covered during the reperfusion period to prevent hypothermia.

Study Phases
The study was divided into three phases. Five animals were included in each group.

Phase I: Timing of gene delivery. The goal of the first phase was to determine the optimal timing of transtracheal administration of AdhIL-10 (5 x 10⁹ pfu) to donor rats before retrieval. The optimal timing was defined by the minimum time required from transtracheal administration of AdhIL-10 to lung retrieval to obtain significant improvement in lung graft function after transplantation. We have previously demonstrated that the transtracheal administration of AdhIL-10 24 h before lung retrieval was sufficient to improve graft function (13). Hence, we examined the effect of lung retrieval 24, 12, 6, and 3 h after donor transtracheal administration of AdhIL-10 on post-transplant lung function and compared the results to a control group that was not transfected. Lungs were preserved at 4°C for 18 h, transplanted, and reperfused for 2 h in each group. An 18-h storage period was chosen based on previous studies demonstrating that this period provided a sufficiently injured lung to be able to demonstrate a difference related to a therapeutic intervention.

Phase II: Early inflammatory response. In the second phase of the study, we examined the early inflammatory response that occurs in the lung 3 and 6 h after transtracheal administration of AdhIL-10, empty vector, and diluent. We also examined if a high dose of steroids (30 mg/kg methylprednisolone) administered intraperitoneally 3 and 6 h before the transtracheal administration of AdhIL-10 could reduce the degree of inflammation secondary to the adenoviral vector and improve gene expression.

Phase III: Lung transplantation. In the third phase, we applied the optimized strategy from information obtained during the first and second phases of the study. We administered high-dose steroids (30 mg/kg of methylprednisolone) intraperitoneally 3 h before transtracheal administration of AdhIL-10 and retrieved the lungs 3, 6, and 12 h later. Two additional groups were used as controls: one was not transfected, and the other was transfected for 12 h but did not receive steroids before the transtracheal administration of AdhIL-10. The lungs were preserved at 4°C for 18 h, transplanted, and reperfused for 2 h.

Assessment of Lung Function
Lung function in the donor was determined by measuring the partial pressure of oxygen (Pa_O2) in the blood taken from the abdominal aorta at the time of retrieval. Post-transplant graft function was assessed by measuring Pa_O2 levels in blood taken from the graft pulmonary vein at the completion of the 2-h reperfusion period under direct vision using a heparinized needle. Peak airway pressures (PawP) were obtained after recipient thoracotomy (baseline) and throughout the reperfusion period. Wet-to-dry (W/D) lung tissue weight ratios were calculated at the end of the reperfusion period as a measurement of lung edema. To measure the PawP, a three-way stopcock was inserted between the endotracheal tube and the ventilator circuit and connected to a pressure transducer. For W/D weight ratios, lung tissue was weighed and placed in an oven at 96°C for 24 h. After this drying procedure, the portion was re-weighed, and the ratio of the lung weight before and after drying was calculated.

Lung Tissue Samples
Lung tissue samples were obtained at the time of retrieval ("retrieval"), at the end of the CIT, and at the end of the reperfusion period ("reperfusion"). The retrieval and CIT samples were obtained from the right lung so that the left lung was preserved intact for transplantation. At the end of the reperfusion period, the transplanted lung was divided into three parts. The superior third was used for W/D weight ratio, the middle third was fixed in 10% buffered formalin and embedded in paraffin, and the inferior third was used for cytokine analyses.

Cytokine Measurements
Samples were immediately snap frozen in liquid nitrogen and stored at -70°C. Tissue specimens were homogenized and incubated at 4°C in cell lysis buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM ethylenediaminetetraacetic acid, 0.1 mM ethyleneglycol-bis-(ß-aminoethyl ether)-N,N'-tetraacetic acid, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 0.6% octylphenoxy-polyethoxy-ethanol (Nonidet P-40). Homogenates were sonicated and centrifuged at 12,000 rpm for 10 min at 4°C. Supernatants and plasma samples were assayed in duplicate using specific enzyme-linked immunosorbent assay kits for rat TNF-, rat IFN-, rat IL-10, rat IL-12, rat macrophage inflammatory protein (MIP)-2, and hIL-10 according to the manufacturer's instructions. Specific Cytoscreen Immunoassay Kits (BioSource International, Camarillo, CA) were used for all cytokines. The optical density of each well was read at 450 nm with an NM-600 microplate reader (Dynatech Laboratories, Chantilly, VA). The final concentration was calculated by converting the optical density readings on a standard curve. The protein content of each sample was determined by the Bradford method (15). Because there was no significant difference in protein levels between the groups, all cytokines are expressed in pg/mg of protein. The hIL-10 kit did not cross-react with endogenous rat IL-10 in rat lungs that were not transfected with AdhIL-10. The absence of cross-reactivity was confirmed by assaying the respective standards provided with each kit (rat and human) with the opposite kit (human and rat).

Statistical Analysis
All data are expressed as mean ± standard deviation. A one-way analysis of variance was used to determine statistical significance. For differences in peak airway pressures in transplanted lungs over the 2-h reperfusion period, the Friedman repeated measures analysis of variance on ranks was performed. P < 0.05 was considered statistically significant. When statistical significance was reached, it was followed by a post hoc analysis using the Bonferroni method. The Graphpad software package (Graphpad Software, San Diego, CA) was used for all statistical analyses.

	Results

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Phase II.
In the first phase of the study, we determined that a period of at least 12 h was required after the transtracheal administration of AdhIL-10 before retrieving the lungs to significantly improve post-transplant lung function. The groups that underwent retrieval 12 and 24 h after the administration of AdhIL-10 had a significantly higher Pa_O2, lower PawP, and lower W/D weight ratio after reperfusion than the nontransfected group (Figure 1). This improvement in lung function was associated with significantly lower levels of IL-12 in lung tissue at the end of the CIT and significantly lower levels of TNF- and MIP-2 after reperfusion than in the nontransfected group (Figure 2).

View larger version (18K): [in this window] [in a new window]   Figure 1. Donor lungs were retrieved 3 (squares), 6 (circles), 12 (diamonds and solid lines), and 24 h (diamonds and dotted lines) after transtracheal administration of AdhIL-10. Post-transplant lung function was compared with a control group that was not transfected (triangles). Donor lungs that were retrieved after 12 and 24 h had a significantly higher PaO2 (A), lower PawP (B), and lower W/D lung weight ratio (C) than the control group. Donor lungs that were retrieved after 3 h, in contrast, had a lower PaO2 and higher PawP than the control group. *P < 0.05, **P < 0.01, and ***P < 0.001 versus no transfection. Each group included five animals.

View larger version (46K): [in this window] [in a new window]   Figure 2. Lung tissue samples were obtained at the time of retrieval (retrieval), at the end of the CIT, and at the end of the 2-h reperfusion period (reperfusion). Lung tissue cytokines were compared with the nontransfected group at each time point. The expression of hIL-10 was significant 6, 12, and 24 h after transtracheal administration of AdhIL-10. The levels of TNF- and MIP-2 were higher at the time of retrieval and at the end of the CIT in the lungs that were retrieved 3 and 6 h after the administration of AdhIL-10. The levels of IL-12 at the end of the CIT and the levels of TNF- and MIP-2 at the end of the reperfusion period were lower in the lungs that were retrieved 12 and 24 h after the administration of AdhIL-10. *P < 0.05, **P < 0.01, and ***P < 0.001 versus no transfection. Each group included five animals.

Phase II.
In the second phase of our study, we confirmed the presence of a significant release of TNF- and MIP-2 in lung tissue 3 and 6 h after the transtracheal administration of AdhIL-10 or empty vector (Figure 3). Donor lung oxygenation was also significantly decreased after the administration of AdhIL-10 or empty vector (Figure 4). In contrast, the transtracheal administration of a diluent, which contained saline with 3% sucrose without any adenoviral vector, caused almost no inflammation. This indicates that the cytokine release is primarily caused by the adenoviral vector itself (Figure 3).

View larger version (22K): [in this window] [in a new window]   Figure 3. The levels of TNF- and MIP-2 were elevated in lung tissue 6 h after the transtracheal administration of an empty vector or AdhIL-10. In contrast, the transtracheal administration of the diluent (saline with 3% sucrose) induced almost no inflammatory cytokine response. The release of TNF- was prevented by the administration of high-dose steroids (methylprednisolone 30 mg/kg) given 3 and 6 h before transtracheal administration of AdhIL-10. Expression of hIL-10 was not significantly increased by the steroid administration. *P < 0.05 and ***P < 0.001 versus diluent. Each group included five animals.

View larger version (16K): [in this window] [in a new window]   Figure 4. The administration of empty vector or AdhIL-10 was associated with a lower PaO2 at the time of lung retrieval 6 h later. The effect was prevented by the administration of a high dose of steroids (methylprednisolone 30 mg/kg) given intraperitoneally 3 and 6 h before the transtracheal administration of AdhIL-10. ***P < 0.001 versus diluent. Each group included five animals.

Phase III.
In the third phase of our study, we administered steroids 3 h before administering the AdhIL-10 transtracheally and retrieved the lungs 3, 6, and 12 h after the administration of AdhIL-10. Two additional groups included a nontransfected group and a group that underwent retrieval 12 h after the transtracheal administration of AdhIL-10 without prior steroid administration. The dose of methylprednisolone reduced the levels of TNF- at the time of retrieval 3 and 6 h after the administration of AdhIL-10 (Figure 5). In addition, there was no significant difference in donor Pa_O2 at the time of retrieval between groups (515 ± 25 mm Hg and 536 ± 53 mm Hg, 3 and 6 h after the transtracheal administration of AdhIl-10, respectively, versus 584 ± 27 mm Hg in the nontransfected group; P = 0.2). At the end of the 2-h reperfusion period, the groups that had the donor lungs retrieved 6 and 12 h after the administration of AdhIL-10 had significantly better Pa_O2 and PawP than the nontransfected group (Figure 6). There was a trend toward better lung function in the 12-h steroid-treated group as compared with the 12-h untreated group, but the difference did not reach statistical significance. However, the 6-h steroid-treated group had significantly better lung function than the 6-h untreated group (Pa_O2 of 214 ± 28 mm Hg versus 163 ± 32 mm Hg, respectively; P = 0.03).

View larger version (47K): [in this window] [in a new window]   Figure 5. A dose of methylprednisolone (30 mg/kg) was administered 3 h before the transtracheal instillation of AdhIL-10, and the lungs were retrieved 3, 6, and 12 h later. Two additional groups included a nontransfected group and one group that underwent retrieval 12 h after the AdhIL-10 instillation without previous steroid administration. The dose of methylprednisolone prevented the released of TNF- in the group undergoing lung retrieval after a period of 6 h. *P < 0.05, **P < 0.01, and ***P < 0.001 versus no transfection. Each group included five animals.

View larger version (18K): [in this window] [in a new window]   Figure 6. The group that underwent retrieval 6 h after the transtracheal administration of AdhIL-10 (pre-treated with steroids) had a significantly higher PaO2 and lower PawP after reperfusion than the group that was not transfected. *P < 0.05, **P < 0.01, and ***P < 0.001 versus no transfection. Each group included five animals. Triangles, no transfection; squares, 3 h and steroids; circles, 6 h and steroids; diamonds with dotted lines, 12 h and steroids; diamonds with solid lines, 12 h.

	Discussion

Top Abstract Introduction Material and Methods Results Discussion References

In this study, we have demonstrated that a period of 12 h after transtracheal administration of AdhIL-10 is sufficient to improve post-transplant lung function. A 12-h donor transfection period was associated with significant expression of hIL-10 and recovery from the initial inflammatory reaction associated with the adenoviral vector transfection. A period of 6 h, in contrast, was associated with a persisting presence of significant levels of TNF- and MIP-2 that reduced the tolerance of the lungs to IR despite the presence of significantly increased expression of hIL-10.

The inflammatory reaction after in vivo administration of replication-deficient adenoviral vectors to the respiratory tract has been described previously (18–22). The degree of inflammation is dose dependent, occurs despite the absence of viral replication, and is not related to the development of any acquired immune response because it has also been observed in athymic nude rodents (23). The inflammatory reaction results from an almost immediate internalization of the adenovirus by alveolar macrophages, followed by the release of proinflammatory cytokines and the accumulation of neutrophils in lung tissue (24). TNF- and MIP-2 are released within the first few hours after transtracheal administration of the adenoviral vector and remain significantly elevated for at least 24 h (20, 24). In contrast to TNF- and MIP-2, the release of IFN- is usually delayed by several hours, but the levels peak in lung tissue within 24 h of transtracheal administration of the adenovirus (20).

We observed that transfection of an anti-inflammatory gene, such as hIL-10, with the adenovirus did not completely prevent the release of TNF- and MIP-2. However, we found that a significant expression of hIL-10 in lung tissue was associated with a significant decrease of TNF- and MIP-2 and the absence of significant release of IFN- within the initial 24 h. IL-10 prevents the release of proinflammatory cytokines such as TNF-, IFN-, and MIP-2 (25), and its expression in our model reduced the degree of early inflammation induced by the adenoviral vector. This concept is supported by Minter and colleagues (26), who have shown that intratracheal instillation of adenovirus-mediated hIL-10 gene therapy can retard the innate and humoral immune response against the adenoviral vector and suppress the bimodal release of TNF- on Days 1 and 7 after the adenoviral instillation.

The inflammation associated with the administration of adenoviral vectors has raised concern about its safety and efficacy in acute inflammatory diseases. In this study, we observed that lungs subjected to prolonged periods of ischemia soon (3–6 h) after the instillation of adenoviral vector developed a significant early inflammatory response leading to a significantly greater degree of injury after reperfusion when compared with nontransfected lungs. Macrophages have been shown to play a significant role during ischemia and in the early phase of reperfusion after lung transplantation (27). Hence, the activation of macrophages by the transtracheal administration of the adenoviral vector before ischemia could partly account for the increased IR injury that we observed when the lungs were retrieved soon after transfection. This effect, however, disappeared once sufficient hIL-10 was released into the donor lung tissue and if the early adenoviral inflammatory reaction was attenuated before the retrieval procedure.

We hypothesized that by reducing the early inflammatory response to adenoviral transfection, we could enhance the efficiency of pulmonary gene transfer and obtain an earlier and enhanced effect of hIL-10 on post-transplant lung function. Different methods have been used to reduce the early inflammatory response to adenoviral vector. These methods have included monoclonal antibodies against TNF- or MIP-2 (28, 29), neutrophil depletion (29), or the administration of dexamethasone (20, 30). In clinical practice, our protocol for lung preservation involves the administration of a high dose of methylprednisolone (30 mg/kg) immediately after the declaration of brain death to compensate for the deficit in hypophyseal hormones and to limit the inflammation related to brain stem death (31). Hence, it is practical to use this strategy to reduce the inflammation associated with the administration of the adenoviral vector in the clinical setting.

We observed that a dose of methylprednisolone (30 mg/kg) injected 3 h before administering the adenovirus prevented the release of TNF- and improved lung oxygenation. However, in this short time period, the steroids did not enhance the expression of hIL-10 despite the reduced inflammation. This finding is in contrast to previous studies and may be related to the short period of observation after the administration of AdhIL-10 in our study (i.e., 12 h or less) (20, 28, 30). Previous studies have shown that steroid administration increases gene expression only after 24–72 h or even longer (20, 30).

The expression of MIP-2 was not significantly reduced by a dose of methylprednisolone given 3 or 6 h before the administration of the adenoviral vector. This finding has also been observed by Otake and colleagues (20) and could partially be explained by the mechanical injury that occurs during the tracheal instillation. We observed that even though the level of MIP-2 was relatively low after the intratracheal administration of the diluent, it was still higher (66 ± 10 pg/mg protein) than the baseline values observed at the time of retrieval in the nontransfected group (40 ± 3 pg/mg protein, P = 0.0004). The persistence of significant expression of MIP-2 did not influence lung function in the early phase of reperfusion after transplantation in our model. However, MIP-2 (the rodent analog of human IL-8) is a potent chemokine for neutrophils, and its effect might become more prominent after prolonged periods of reperfusion. Neutrophils have been shown to play a significant role in IR injury of the lung but only after several hours of reperfusion (32–34).

Brain death has been shown to induce significant release of proinflammatory cytokines in solid organ tissue before the retrieval procedure (16, 17). However, the effect and timing of gene therapy with hIL-10 on brain-death–related inflammation remains to be elucidated. This may be important because there is always a delay between the physiologic event and the certification of brain death in clinical practice. Hence, the inflammatory reaction related to brain death may initially be worsened by the inflammatory reaction related to adenoviral vectors.

In conclusion, we have demonstrated that a period of at least 12 h is required after the transtracheal administration of AdhIL-10 to the donor lungs to improve post-transplant graft function. After 12 h, there is significant and sufficient expression of hIL-10 to decrease the early inflammatory response associated with the adenoviral vector and to prevent the release of proinflammatory cytokines in the lung during ischemia and after reperfusion. We have demonstrated that a shorter period of transfection can increase the degree of IR injury unless the inflammatory response from the adenoviral vector can be reduced. A period of transfection of 6 h may be sufficient to improve post-transplant lung function if high-dose methylprednisolone (30 mg/kg) is given before transtracheal administration of AdhIL-10. Future work in vector development should focus on decreasing the initial inflammatory response induced by the adenoviral vector to reduce the required time period between donor transfection and lung retrieval to improve the safety and efficacy of adenoviral mediated gene therapy for transplantation.

	Acknowledgments

 
The authors thank the University of Iowa Gene Transfer Vector Core (director, B. Davidson, Ph.D.), supported in part by the NIH and the Roy J. Carver Foundation, for viral vector preparations. The authors also thank Ioan Mates, D.V.M., for his technical assistance in conducting the animal procedures. Dr. Marc de Perrot is supported by a grant from the Swiss National Scientific Foundation. This work was supported by grants from the Canadian Cystic Fibrosis Foundation, the National Sanatorium Association of Canada, and the Canadian Institutes of Health Research.

Received in original form July 9, 2002

Received in final form November 26, 2002

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