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Ann Thorac Surg 2002;73:1092-1097
© 2002 The Society of Thoracic Surgeons

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Original article: general thoracic

Transplant immunosuppression enhances efficiency of adenoviral-mediated gene retransfection: inhibition of interferon- and immunoglobin G

^a Thoracic Surgery Research Laboratory, University Health Network, Toronto General Hospital Research Institute, Department of Surgery, University of Toronto, Toronto, Ontario, Canada

Accepted for publication November 19, 2001.

* Address reprint requests to Dr Liu, Department of Surgery, Thoracic Surgery Research Laboratory, Toronto General Hospital, Room CCRW 1-816, 200 Elizabeth St, Toronto, Ontario, M5G 2C4, Canada
e-mail: mingyao.liu{at}utoronto.ca

	Abstract

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Background. Transplant immunosuppression regimen facilitates successful adenovirus-mediated gene transfection and retransfection in the rat lung. Herein, we investigated the effect of this strategy on circulating cytokines and antiadenoviral immunoglobin G antibody.

Methods. Male Lewis rats were transfected with 1 x 10⁹ pfu/mL of E1-deleted Ad5CMVLacZ vector transtracheally. Rats were randomly assigned to receive daily intraperitoneal triple immunosuppression regimen consisting of cyclosporine (15 mg/kg per day), azathioprine (6 mg/kg per day), and methylprednisolone (2.5 mg/kg per day), or normal saline solution. Retransfection was performed 35 days later to all nonimmunosuppressed animals, whereas immunosuppressed rats were further randomized to receive retransfection or phosphate-buffered saline. Animals were sacrificed on days 1, 2, 7, 35, 42, and 49 after the initial transfection. ß-Galactosidase activity was measured on lung homogenates. Interferon-, tumor necrosis factor-, and antiadenoviral immunoglobin G were measured from the serum.

Results. Enhanced and prolonged transgene expression was observed in immunosuppressed animals, especially after retransfection. Concentrations of serum tumor necrosis factor- in both groups were less than 12 pg/mL throughout the study. A significant increase in serum interferon- levels was observed in nonimmunosuppressed animals after retransfection; this was not seen in the immunosuppressed animals. Serum antiadenoviral immunoglobin G titers in both groups were sharply elevated on day 1, and declined to basal levels by day 7, reflecting a preexisting level of humoral immunity to adenovirus. The titer in nonimmunosuppressed rats was significantly increased after retransfection, but remained at very low level in immunosuppressed animals.

Conclusions. Inhibition of interferon- and antiadenoviral immunoglobin G production by triple immunosuppressants may be part of the mechanisms that lead to enhanced and prolonged transgene expression after retransfection.

	Introduction

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Adenoviral-mediated gene transfer has been used as an experimental tool to study underlying mechanisms, and as a potential therapeutic strategy for bronchiolitis obliterans, a chronic complication of lung transplantation. Adenoviral-mediated human interleukin 10 gene transfer in a rat tracheal transplant model [1] effectively inhibited obliterative airway lesions [2]. Delivering soluble transforming growth factor-ß type III receptor, an antagonist of transforming growth factor-ß, with an adenovirus vector demonstrated significant protective effects on allograft tracheal transplant-induced obliterative lesions [3]. Early graft ischemia-reperfusion injury is another major obstacle in lung transplantation. The early inflammatory injury of lung grafts may also contribute to the development of chronic graft dysfunction and rejection—seen as bronchiolitis obliterans. Using a rat single-lung transplant model, we have optimized adenoviral gene transfer to donor lungs [4, 5]. Adenoviral-mediated human interleukin 10 gene transfer ameliorated ischemia-reperfusion-induced injury of lung transplants in this model [5]. However, the successful use of adenoviral-mediated gene transfer as a therapy is limited by the nonspecific and specific immune responses of the host to the adenoviral vector [6–8], which not only limits the amount and duration of transgene expression but also obviates successful retransfection [6, 7, 9].

In the setting of transplantation, patients receive life-long systemic immunosuppression regimens to prevent graft rejection. We have demonstrated that transplant immunosuppression regimen enhances and prolongs adenovirus transgene expression [10]. A unique feature of the lung is that repeated local transgene delivery can be easily achieved through the airway. We have further demonstrated that immunosuppression regimen permits successful adenoviral gene retransfection [11]. These studies demonstrate the feasibility of gene therapy not only for ischemia-reperfusion-related acute lung injury, but also for the treatment of bronchiolitis obliterans, by repeated gene delivery.

T-cell-dependent antigen-specific immunity appears to be responsible for the initial immune-mediated inflammation. Both CD8⁺ and CD4⁺ T cells can act together to limit duration of adenoviral-mediated transgene expression [6, 7]. We have shown that transplant immunosuppression regimen reduces CD8⁺ and CD4⁺ T cell infiltration, as well as the perivascular, peribronchial, and alveolar inflammation, induced by the initial transfection of adenoviral vectors in the lung [10]. To gain further understanding of the mechanisms responsible for successful retransfection of adenoviral vectors with systemic immunosuppression regimen, we examined the effects of immunosuppression regimen on adenoviral vector-induced cytokines and antiviral immunoglobin G (IgG) responses in the circulation. Proinflammatory cytokines such as interferon- (IFN-) and tumor necrosis factor- (TNF-) may have both early antiviral activity and late cytotoxic immunoreaction against the virus or transfected cells [12, 13]. Transfection of adenoviral vector in rat lungs resulted in an increase in circulating antiadenoviral IgG antibodies [14]. Repeated adenovirus administration induced an increase in serum IgG and IgM in rats [9]. In the present study, we hypothesized that standard transplant triple immunosuppression regimen may suppress the production of proinflammatory cytokines or antiadenoviral IgG, which might be a mechanism responsible for the successful retransfection demonstrated in our previous studies.

	Material and methods

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Animals
Male Lewis rats weighing 300 to 350 g were purchased from Harlan Sprague-Dawley Inc (Indianapolis, IN). Animal care was provided according to National Institutes of Health guidelines (Guide for the Care and Use of Laboratory Animals, NIH publication 86-23, revised 1985), the "Guide to the Care and Use of Experimental Animals" formulated by the Canadian Council on Animal Care, and the policies stated by the Toronto General Hospital Research Institute Animal Care Committee. Gene manipulation protocol in the study was reviewed and approved by the Toronto General Hospital Biohazard Committee.

Gene transfer and immunosuppression regimen
Adenovirus-mediated gene transfection and retransfection was performed as previously described [4, 10, 11]. Briefly, after intraperitoneal administration of ketamine (Rogar; London, Ontario, Canada) and acepromazine (Ayerst; Montreal, Quebec, Canada), rats were intubated with a 16-gauge polytetrafluoroethylene (Teflon) angiocatheter (Becton-Dickinson, Sandy, UT) and then transfected intratracheally with 1 x 10⁹ pfu/mL of an E1-deleted Ad5CMVLacZ vector (University of Iowa Gene Transfer Vector Core, Iowa City, IA) in phosphate-buffered saline (PBS). Starting on the day of initial transfection, rats were randomly assigned to two groups. Animals in the first group (n = 32) received 15 mg/kg per day of cyclosporine A, 6 mg/kg per day of azathioprine, and 2.5 mg/kg per day of methylprednisolone intraperitoneally (immunosuppression regimen group, or group I). Animals in the other group (n = 24) received normal saline (nonimmunosuppression regimen group, or group NI). In addition, 4 rats were used as normal control animals to measure the basal cytokine and antiviral antibody levels in the serum. At days 1, 2, 7, and 35 after transfection, 4 animals from each group were sacrificed. Thirty-five days later, all animals remaining in the NI group (n = 8) and half the number of animals remaining in group I (8 of 16), randomly selected, were retransfected with the same adenoviral vector in the same fashion (this subgroup is referred to as I/RT). Other immunosuppressed rats (n = 8) received 1 mL of PBS solution (I/PBS subgroup). Four animals from each group were sacrificed on days 42 and 49. For all animals, their lungs were dissected for ß-galactosidase (ß-gal) activity analysis, and blood serum was collected for cytokine and antiadenoviral IgG assays.

Chemiluminescent assay for ß-galactosidase
ß-Galactosidase activity in lung homogenates was measured using Galacton-light-plus cellular chemiluminescent assay (Tropix; Bedford, MA) [4, 10, 11]. Briefly, lung tissue sections were homogenized and sonicated in 5 mL of PBS and 200 µL of lysis solution. After centrifugation, a fraction of the supernatant was incubated with reaction buffer for 1 hour, followed by addition of the accelerator solution. Finally, color strength of the sample was measured in relative light units using a chemiluminometer (Berthold; Bad Wildbad, Germany) and standardized by protein content.

Measurement of serum concentration of cytokines and antiadenoviral immunoglobin G
Tumor necrosis factor- and IFN- levels in the rat serum were measured using a rat interferon-gamma enzyme-linked immunosorbent assay kit and a rat tumor necrosis factor- enzyme-linked immunosorbent assay kit (Biosource International; Camarillo, CA). Enzyme-linked immunosorbent assay for antiadenoviral IgG was performed as described previously [14]. Briefly, 96 microwell plates (Nunc Maxisorb microplates; Nalge Nunc International; Rochester, NY) were precoated with 50 µL/well of diluted lysate made from adenovirus-infected HeLa cells (100 ng/mL) at 4°C overnight. Each well was incubated with 50 µL of Tris-Tween reagent diluent (0.24% Tris-HCl, 0.876% NaCl, 0.037% KCl, 0.05% Tween 20, 0.05% bovine serum albumin, 0.02% NaN3, and 0.01% bromocresol purple, pH 7.4) at 37°C for 30 minutes, followed by diluted serum samples (50 µL/well) at 37°C for 60 minutes, 50 µL/well of 1:1000 diluted bionylated antirat IgG antibody at 37°C for 30 minutes, 50 µL/well of 1:2000 diluted extravidin-peroxidase conjugate at 37°C for 15 minutes, and finally substrate TMB for 20 minutes at room temperature. Plates were washed three times between each of these steps. The color development was stopped by adding 12.5 µL of 2 mol/L H2SO4. The optical density was measured at 450 nm. The amount of antiadenovirus IgG was expressed as titer determined by following formula:

The diluted sample optical density that immediately reached two times higher value than that of average background was adopted for calculation.

Statistical analysis
Data are expressed as mean values ± standard error of the mean. Two-way analysis of variance was used for analysis of differences among groups. Multiple pairwise comparisons were performed using Student-Newman-Keuls test. Data are considered statistically significant if p values are less than 0.05. All analyses were performed using SigmaStat version 1.0 statistical software (Jandel Scientific, San Rafael, CA).

	Results

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Transgene expression in transfected lungs
ß-Galactosidase activity is a marker for LacZ transgene expression. After initial transfection, the activity of ß-gal was elevated in group NI, with a peak expression on day 2, and then dropped to the basal level by day 35. In group I, the activity of ß-gal increased continuously until day 7, and maintained at the higher level, which remained significantly higher than that of the NI group on day 35 (Fig 1A). Retransfection of adenovirus vector in the NI group did not induce a further increase of the ß-gal level. The ß-gal activities were 317 ± 60 relative light units/µg protein on day 42, and 746 ± 87 RLU/µg protein on day 49, respectively. Both cannot be shown on the figure at the scale used (Fig 1B). Retransfection of adenoviral vector to immunosuppressed animals (I/RT group), however, induced a marked increase in ß-gal activity on day 42, which decreased on day 49 (Fig 1B). In the absence of retransfection (I/PBS group), the initial transfection-induced elevated ß-gal level was maintained up to 42 days (Fig 1B). The dynamics of ß-gal expression levels was consistent with our previous observations [10, 11].

View larger version (14K): [in this window] [in a new window]   Fig 1. Immunosuppression regimen enhances and prolongs adenovirus-mediated transgene expression in rat lungs. (A) After the initial adenoviral gene transfection, animals were randomly assigned into a nonimmunosuppressed group (solid line, triangles) and an immunosuppressed group (dashed line, squares). Immunosuppression regimen significantly enhanced and prolonged the transgene expression (p < 0.001, as analyzed with two-way analysis of variance). *p < 0.05 compared with all other data points except the value in group I on day 35; #p < 0.05 compared with the value of the nonimmunosuppressed group on day 35, as determined by Student-Newman-Keuls test. (B) Five weeks later, animals in the nonimmunosuppressed group received a second transfection (blank bars, too small to show on the scale). Animals in the immunosuppression regimen group were further randomized to receive adenoviral vector retransfection (filled bars), or phosphate-buffered saline (hatched bars) as sham-operation controls (p < 0.01 as analyzed with two-way analysis of variance). *p < 0.05 versus all other data points as determined by Student-Newman-Keuls test. (ß-gal = ß-galactosidase.)

View larger version (15K): [in this window] [in a new window]   Fig 2. Immunosuppression regimen inhibits adenoviral vector retransfection-induced elevation of serum interferon- (IFN-) concentrations. The experimental design and figure legends are the same as described in Figure 1. Each data point represents the mean ± standard error of the mean of four samples. (A) Initial adenoviral transfection did not change serum interferon- levels significantly. (B) Adenoviral retransfection significantly increased serum interferon- levels in the nonimmunosuppressed group (p < 0.001 as analyzed with two-way analysis of variance). *p < 0.05 versus all other data points as determined by Student-Newman-Keuls test.

View larger version (18K): [in this window] [in a new window]   Fig 3. Immunosuppression regimen inhibits adenoviral vector retransfection-induced elevation of serum antiadenoviral immunoglobin G (IgG) concentrations. The experimental design and figure legends are the same as described in Figure 1. Each data point represents the mean ± standard error of the mean of four samples. (A) Initial adenoviral transfection induced antiviral immunoglobin G in both groups, with a peak at day 1 (p < 0.001, as analyzed with two-way analysis of variance). *p < 0.05 compared with data in both groups on days 0, 7, and 35; #p < 0.05 compared with the value of the immunosuppressed group on day 35, as determined by Student-Newman-Keuls test. (B) Retransfection significantly increased antiviral immunoglobin G in the nonimmunosuppressed group (p < 0.01 as analyzed with two-way analysis of variance). *p < 0.05 versus all other data points as determined by Student-Newman-Keuls test.

	Comment

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Cytokines and adenoviral gene transfer
We have previously shown that transtracheal delivery of adenoviral vector induces infiltration of CD4⁺ and CD8⁺ cells in rat lungs, and that this can be inhibited by the triple immunosuppression regimen strategy, which also inhibits the inflammatory reaction in the alveolar space and in the perivascular and peribronchial areas [10]. Several studies have demonstrated that adenovirus-mediated gene delivery induces systemic cellular and humoral immune responses that inhibit the effectiveness of repeated transfection of therapeutic genes. Therefore, in the present study, we have focused on the role of cytokine production and circulating antiviral antibody. In response to adenovirus stimulation, immune cells in the host secrete various inflammatory cytokines such as TNF- and IFN-. In vitro studies have shown that IFN- [12, 13] and TNF- [13] inhibit expression of transgenes delivered by adenoviral vectors in cell cultures. Administration of neutralizing anti-IFN- monoclonal antibody leads to enhanced adenoviral transgene expression in vivo [13]. Kagami and coworkers [9] induced tolerance to adenovirus by per oral feeding of ultraviolet-inactivated virus before gene administration to rat parotid glands. Mononuclear cells from the spleens of these tolerant animals were found to produce less IFN- after virus challenge. Interestingly, Yang and colleagues [17] have shown that coadministration of IFN- (or interleukin 12, which activates TH1 cells to secrete IFN-) with the adenovirus vector into the airway of C57BL/6 mice allows for efficient readministration of adenovirus. They proposed the use of IFN- as a local short-acting immunomodulator at the time of gene delivery to overcome the problems of humoral immunity [17]. It is possible that the local and systemic IFN- may have different functions in the regulation of host immunity against adenoviral infection. Our results show that continued immunosuppression regimen prevented the adenoviral vector-induced increase in circulating levels of IFN- in response to retransfection. In contrast, without immunosuppression regimen, retransfection is associated with a significant increase in IFN- level in the blood and transgene expression is inhibited. The dynamics of IFN- in rat sera suggests that the first gene transfection may induce sensitization of animals to adenovirus vector; when animals are retransfected with the same serotype adenoviral vector, they produce relatively higher amounts of IFN- in the circulation. Therefore, our results support the notion that circulating IFN- may inhibit the efficacy of adenoviral vector retransfection.

Treatment with TNF- binding protein systemically has been demonstrated to lead to prolonged expression of adenovirus-mediated LacZ gene in mice [18]. In our study, however, we were unable to demonstrate changes in TNF- levels in the serum, either with or without immunosuppression regimen. We did not measure the IFN- and TNF- levels in lung tissue. However, based on the inhibition of inflammation and reduced infiltration of CD8⁺ and CD4⁺ cells observed previously [11], we speculate that the production of these proinflammatory cytokines in the lung are likely suppressed by the immunosuppression regimen.

Circulating antibody and adenoviral gene transfer
In the present study, after the initial adenoviral transfection, the serum antiadenoviral IgG increased significantly on days 1 and 2, and returned to the basal level at day 7 in both groups. This rapid response may be related to the innate immunity of animals to adenovirus. In a phase I clinical trial, it has been reported that a single injection of adenoviral vector induced strong short-term humoral and T-cell responses in patients [19]. Preexisting immunity to adenovirus serotype 5 has been noted in humans with marked heterogeneity [20]. In this study, the animal population was strictly controlled; hence the responses were more homogenous. We used cyclosporine serum levels as an index of absorption of the immunosuppressants. At days 1 and 2, cyclosporine levels were approximately 25% and 50% of the stable levels, respectively [10], and this may not be sufficient to completely inhibit the humoral antiviral immune response during that initial period.

Interestingly, from day 7 to day 35, in the absence of immunosuppressants, the antiadenoviral IgG levels gradually increased. After retransfection, the IgG levels were further increased at day 42 and day 49. This suggests that animals developed specific IgG toward the adenovirus serotype we used. Long-term persistence of immunity to the transgene protein delivered by recombinant adenovirus has been noted clinically [21]. Vector-specific IgG antibodies against different serotypes of adenoviruses have also been reported in mice [22]. In contrast, with the treatment of triple immunosuppressants, the serum antiadenoviral IgG remained at the basal level after the initial transfection. The maneuver of retransfection slightly increased the titers of antiadenoviral IgG in both the I/RT and the I/PBS subgroups. The changes in antiadenoviral IgG levels were so similar in these two subgroups that it may indeed be attributable to a nonspecific response to the surgical procedure. The lack of antiadenoviral IgG response suggests that triple immunosuppression regimen markedly inhibited the development of antibodies against the specific serotype of adenoviral vector administered.

In summary, results of this study suggest that the inhibition of IFN- and vector-specific antiadenoviral IgG antibody production by triple immunosuppressants (cyclosporine, azathioprine, and prednisone—as used in the clinical transplantation setting) is potentially one of the mechanisms which contribute to the enhanced and prolonged transgene expression after adenovirus-mediated gene retransfection in the lung.

	Acknowledgments

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The authors thank J. Mates for his technical assistance with the animals and Anna Zganiacz for technical advice with antiadenoviral IgG measurement. This study was supported by the National Sanitarium Association of Canada, the Canadian Cystic Fibrosis Foundation, and the Canada Institutes of Health Research (MT-13270, MOP-42465, MOP-77559). Mingyao Liu is a recipient of the Canadian Institutes of Health Research New Investigator Award and the Premier’s Research Excellence Award from the Ontario Government.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Shaf H. Keshavjee Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Suga, M. Articles by Liu, M. Search for Related Content PubMed PubMed Citation Articles by Suga, M. Articles by Liu, M. Related Collections Lung - transplantation Related Article