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*Substance via MeSH
Medline Plus Health Information
*Heart Transplantation
The Journal of Immunology, 2005, 174: 6983-6992.
Copyright © 2005 by The American Association of Immunologists

Liposome-Mediated Combinatorial Cytokine Gene Therapy Induces Localized Synergistic Immunosuppression and Promotes Long-Term Survival of Cardiac Allografts1

Hiroshi Furukawa*, Kiyohiro Oshima*, Thomas Tung*, Guanggen Cui*,{dagger}, Hillel Laks* and Luyi Sen2,*,{dagger}

* Division of Cardiothoracic Surgery, Department of Surgery, and {dagger} Division of Cardiology, Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA 90095


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Localized gene transfer has the potential to introduce immunosuppressive molecules only into the transplanted allograft, which would limit systemic side effects, and prolong allograft survival. However, an applicable gene transfer strategy is not available, and the feasible therapeutic gene(s) has not yet been determined. We developed an ex vivo liposome-mediated gene therapy strategy that is able to intracoronary deliver the combination of IL-4 and IL-10 cDNA expression vectors to the allograft simultaneously. We examined the efficiency, efficacy, and cardiac adverse effects of this combinatorial gene therapy protocol using a rabbit functional cervical heterotopic heart transplant model. Although the efficiency was moderate, the expression of both transgenes was long lasting and localized only in the target organ. The mean survival of cardiac allograft was prolonged from 7 to >100 days. Synergism of overexpressed IL-4 and IL-10 in the inhibition of T lymphocyte infiltration and cytoxicity, and modulation of Th1/Th2 cytokine production promote long-term survival of cardiac allografts.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Cardiac transplantation has been a reliable surgical strategy for end-stage heart failure. However, there are major obstacles about acute and chronic rejection. Systemically administering pharmacological agents can easily induce immunosuppression; however, it usually results in multiple, deleterious side effects requiring major dosage adjustments, and true tolerance is rarely achieved (1, 2). Recently, the evolution of localized gene therapy for preventing cardiac allograft rejection has attracted attention. Various genes and vectors were tested in experimental models, but only a few successful results were reported (3, 4). The feasibility of gene therapy in allograft rejection remains unproven mainly due to the lack of a candidate gene, and an applicable and safe gene transfer technique.

Immunosuppressive cytokines are the key mediators for alloimmune responsiveness (5). Among all of three immunosuppressive cytokines, IL-4, IL-10, and IL-13, IL-10 is the most potent and has a wide immunosuppressive spectrum (6). IL-10 produced by Th2 cells, suppresses the induction of Th1 cells, inhibits Th1 cell cytokine production (7, 8), and promotes the development of Th2 cells (9). IL-10 is also a macrophage-deactivating factor and suppresses NO synthesis (10). As a major immunosuppressive cytokine and an anti-inflammatory agent, IL-10 holds the potential in the treatment of allograft rejection (11). However, systemic administration of IL-10 after transplantation did not show any benefit in humans, which is mainly due to the significant pleiotropic effect (12). Localized IL-10 gene transfer mediated by adenovirus or liposome has been the most effective gene therapy, other than CTLA4Ig, for prolonging allograft survival in various animal models (4, 13, 14); however, true tolerance was never achieved (15, 16). IL-4 shares many of its activities with IL-10, and some activities with IL-13; however, unexpectedly, the observations of its role as a mediator of allograft rejection are inconsistent (17, 18).

Recently, the synergy of IL-4 and IL-10 was observed in both in vitro and in vivo studies. Schmidt-Weber et al. (19) reported that IL-4 enhanced both IL-10 gene expression and protein expression in Th2 cells. Another in vitro study also found that IL-10 synergized with IL-4 to inhibit NO production and macrophage cytotoxic activity (20). Powrie et al. (21) found IL-4 and IL-10 synergize to inhibit delayed-type hypersensitivity responses to Leishmania major in mice immune to Leishmania. Most recent experimental animal studies suggested that both synergistic and antagonistic immunoregulatory capacities of IL-4 and IL-10 may led to a superior suppression of inflammatory arthritis (22, 23). Additionally, three studies showed that the systemic coinjection of IL-4 and IL-10 or plasmids encoding IL-4 and IL-10 significantly prevented the nonfunction of transplanted islets in NOD mice (24, 25, 26).

Here, we developed an ex vivo liposome-mediated gene therapy strategy that is able to deliver intracoronary the combination of IL-4 and IL-10 cDNA expression vectors to the cardiac allografts. We examined the efficiency, efficacy, and cardiac adverse effects of this localized combinatorial gene therapy protocol using a rabbit functional cervical heterotopic heart transplant model. Although the efficiency was moderate compared with viral-mediated gene transfer, the expression of both transgenes was stable, long lasting, and localized only in the target-organ (4, 27). Localized overexpression of IL-4 and IL-10 synergistically suppressed the alloimmune responses by significantly reducing T lymphocyte infiltration and cytoxicity, and promoted the long-term survival of cardiac allografts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Liposome-gene complex preparation

The plasmid pSVhIL-4 and pSVhIL-10 containing human recombinant IL-4 (hIL-4)3 and IL-10 (hIL-10) cDNA coupled with SV40 early promoter were used (4, 16, 27, 28). The new generation of the cationic liposome GAP:DLRIE in the 2,3-dioxypropaniminium class of cationic lipid basic skeleton that also includes (+)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propaniminium bromide (DLRIE), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl ammonium, 1,2-bis(oleoyloxy)-3-(trimethyl-ammonio)propane, and 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanammonium was provided by Invitrogen Life Technologies (4). Optimized concentration of recombinant human IL-4 cDNA (50 µg), recombinant human IL-10 cDNA (50 µg) (4, 16), or both were complexed with liposome (50 or 100 µg for two genes) by a 20-min gentle vortex.

Heterotopic functional cervical heart transplantation model

New Zealand White donor rabbits weighing 3.5 kg (Charles River Laboratories) and recipient rabbits weighing 4 kg (Mytrle’s Rabbitry) were purchased from geographically unrelated vendors. The pathologic characteristic of this mismatch acute rejection model has been described previously (4, 15). Briefly, under general anesthesia, median sternotomy was performed on donor rabbits, both superior and inferior venae cavae were ligated, then cardiac arrest was induced by infusion of University of Wisconsin solution (4°C, 20 ml/kg, 120 ml/h) through an aortic cannula (4). Pulmonary artery and left and right atrium appendage were cut and kept open. After pulmonary vein was ligated, donor heart was excised and placed in University of Wisconsin solution (4°C). Liposome-gene complex in 10 ml of normal saline was administrated by ex vivo intracoronary infusion at 20 ml/h. Donor aorta and pulmonary artery were anastomosed to the recipient’s proximal right carotid artery and common jugular vein, respectively, and the left and right atrium were anastomosed to the recipient’s distal right carotid artery and common pulmonary artery, respectively. All animal experimental protocols received approval from Institutional Board of Animal Research Committee. Functioning allografts were retrieved from recipient rabbits after euthanasia on postoperative days (POD) 1–2, 4–5, 7–8, 27–28, and >60 in the IL-4 and IL-10 combined gene therapy group, days 1–2, 4–5, 7–8, and 27–28 in the IL-10 gene therapy group and days 1–2, 4–5, and 7–8 in the IL-4 gene therapy group and control group.

Quantitative comparative RT-PCR (qcRT-PCR) and Northern blot analysis for detecting the gene expression

qcRT-PCR was performed to detect the transgene expression of hIL-4 and hIL-10 in cardiac allograft, and the expression level of intrinsic Th1 and Th2 genes (TNF-{alpha}, IL-1{beta}, IL-2, IFN-{gamma}, rIL-4, rIL-10) using the primers and methods described previously (4, 16, 29, 30). Briefly, one pair of primers (sense, 5'-ATGGAGCGAAGGTTAGTGGTCA-3'; antisense, 5'-CTCGCTTTAATTGTCATGTATGCT-3') were used to amplify a 461-bp region of the hIL-10 cDNA and another pair of primers (sense, 5'-AAAGCAAACCACAAGGCGGA-3'; antisense, 5'-ATCGAGGTCAGGAGCCT-3') were used for amplify the rabbit IL-10 cDNA. Two different sets of primers were also used for amplify the hIL-4 and rIL-4. There was no species cross-reactivity detected while using these primers. Competitive templates (CT) were constructed for each gene and a housekeeping gene, GAPDH. The amplification product of each CT differs in size from the original cDNA product of 70–179 bp. To control for the efficiency of individual reverse transcriptase (RT) reactions from which sample cDNA templates were drawn, the same amplification technique was used first to measure the expression of the housekeeping gene GAPDH. Samples of testing gene cDNA equivalent to 50 ng of total RNA from each individual RT reaction products were diluted appropriately to contain equal concentrations of CT cDNA, normalized to the expression of GAPDH in the sample. For each particular gene, 5 µl of the normalized RT product were coamplified with the constant amount of the gene-specific CT DNA. The relative amounts of testing gene cDNA in the various samples were determined by comparing their respective sample ratios of testing gene cDNA/CT DNA multiplied by the constant amount of CT DNA used for the particular gene in the CT RT-PCR. In addition, all samples were normalized against the respective GAPDH cDNA to CT DNA ratio. These normalization controls for the quantity of cDNA loaded in all samples. PCR samples were run on 2% agarose gel. The intensity of ethidium bromide luminescence was measured using Eagle Sight 3.0 Software (Stratagene) to obtain digital-image acquisition, processing, and analysis system. This software provides an analysis of the relative densities of gel images, which represent two-dimensional arrays of pixels. Gel images were further analyzed and quantified using NIH Image 1.54 program.

In situ hybridization for evaluating the gene transfer efficiency

To determine the gene transfer efficiency, antisense and sense digoxygenin-labeled riboprobes (Boehringer Mannheim) of hIL-4 and hIL-10 mRNA were synthesized and used for in situ hybridization on paraffin section as described previously (31, 32). The gene transfer efficiency was determined as the percentage of blue-stained positive cells in total cardiac myocytes counted in 10 high power microscopic field (magnification, x400) per section. Transgenes were not only expressed in the cardiac myocytes, but in endothelial cells and vascular smooth muscle cells as well. Only those observation fields without vessel were used for analysis. The subsequent slide of a serial section stained with H&E was used to distinguish the cardiac myocytes from other cells.

ELISA and Western blot for evaluating the level of protein expression

The standard BD Pharmingen protocol for sandwich ELISA was used to quantify the amount of hIL-10 and hIL-4 in myocardium (3, 4, 27). Standard 96-well flat-bottom plates were coated overnight with 50 µl of monoclonal mouse anti-human-IL-4 or IL-10 Ab (BD Pharmingen), then samples were added. Avidine-HRP conjugate mixed with biotinylated anti-human IL-4, or IL-10 Ab (100 µl) was added. Then, 0.1% hydrogen peroxide in ABTS substrate was used to visualize detection. Quantification was performed on an automated ELISA plate reader (Dynatech Laboratories). To confirm the data from ELISA, IL-4 and IL-10 protein level in myocardium was also assessed by Western blot. Protein (100 µg) were denatured and subjected to a SDS-PAGE, then transferred to a Hybond-ECL nitrocellulose membrane. The blot was incubated with a 1/3000 diluted mouse-anti-human IL-4 or IL-10 mAb, then with peroxide-conjugated anti-mouse IgG secondary Ab (Jackson ImmunoResearch Laboratories).

Rejection score of cardiac allografts

Serial sections of left ventricle were cut from cardiac allografts and embedded in ornithine carbamoyltransferase, and made standard H&E stain for histological evaluation, respectively (4, 27). Rejection scores of cardiac allograft were assigned in a blind study by two medical doctors and determined based on the standardization of nomenclature in the diagnosis of heart rejection established by the International Society for Heart and Lung Transplantation (33).

Histology analysis and immunohistochemical staining for examination of infiltrating cells

The serial sections for detecting the infiltrating cell Ag markers were fixed in 100% acetone, rehydrated in PBS with 0.3% hydrogen peroxide, then incubated in PBS with 1% BSA as a blocking buffer to avoid nonspecific reaction. They were incubated with biotinylated primary Abs against rabbit CD3, CD4, CD8, CD20, and CD68 (Spring Valley Laboratories) for 1 h. Ab-biotin conjugate was detected with AutoProbe III kit (Biomed). Slides were counterstained with hematoxylin (3, 4, 27).

Assessment of lymphocytic infiltrate cytotoxicity

Splenocytes from donor rabbits as the target cells were isolated at the time of transplant and cultured in 10 ml of tissue culture medium consisting of RPMI 1640 supplemented with 10% FCS, 24 mM HEPES buffer, 4 mM L-glutamine, 400 U penicillin G, and 400 µg/ml streptomycin. Target cells were treated with mitomycin C at a concentration of 50 µg/ml for 60 min at 37°C before use, then labeled with 51Cr (16). Graft infiltrating lymphocytes isolated from transplanted hearts were used as effector cells. A total of 1 x 104 labeled target cells (in a volume of 100 µl) were plated in 96-well plates and coincubated with a serial dilution of effector cell suspension (100 µl), resulting in a range of effector cell to target cell ratios. The plates were incubated overnight at 37°C, then supernatants were collected and counted in a Beckman Coulter {gamma}-400. All samples were run in triplicate. Specific lysis was calculated according to the formula: specific lysis = 100x (experimental release – spontaneous release/maximum release – spontaneous release) (16).

Statistical analysis

All data are expressed as means ± SD. Paired or unpaired Student’s t test was performed to compare the difference between two groups. A value of p < 0.05 was regarded as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Efficiency of localized combinatorial cytokine gene transfer in cardiac allografts

Cationic liposome was able to transfer two therapeutic gene vectors, hIL-4 and hIL-10, to the target allografts simultaneously. The significant increase in hIL-10 transgene expression assessed by comparative RT-PCR and Northern blot analysis could be observed in the donor hearts as early as POD 2, reached a peak at POD 7–8 and followed by a slow decline (Fig. 1a). The amount and the time-course of transgene expression in the cardiac allografts remained the same in hIL-4 and hIL-10 combinatorial gene-transfer as that in the hIL-10 only gene transfer. The time course of hIL-4 transgene expression in the allografts was similar as hIL-10. However, the peak mRNA level of hIL-4 was only half of hIL-10 in the cardiac allografts. Both IL-4 and IL-10 transgene expression was dose dependent, and the optimized cDNA concentration was 50 µg (Fig. 1b). Both transfected genes were only expressed in the cardiac allografts, not in recipient’s heart, brain, lung, liver, spleen, kidney, and skeletal muscle (Fig. 1c). The efficiency of liposome-mediated ex vivo hIL-10 gene transfer in cardiac allograft evaluated by in situ hybridization was moderate (15.7%) and slightly higher than hIL-4 gene transfer (13.2%) (Fig. 1, d and e). In combinatorial gene transfer, the efficiency for hIL-10 gene transfer remained the same and hIL-4 gene transfer was slightly reduced (12.1%) compared with that transferred alone; that resulted in a significant difference of gene transfer efficiency between two genes. The discrepancy in the gene transfer efficiency between two genes remained the same, regardless of whether the two plasmid vectors were complexed with liposome, then delivered, or each plasmid vector was complexed with liposome individually. The hIL-4 transgene mRNA level was significantly lower than hIL-10 regardless of transferring alone or combined with hIL-10, suggesting also that a transcription discrepancy is present between two genes. Although transfected cells are mostly cardiac myocytes, endothelia cells and smooth muscle cells were also transfected with slightly higher transfection efficiency (18%, data not shown).



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  		FIGURE 1. Efficiency of liposome-mediated localized ex vivo hIL-4 and hIL-10 combined gene transfer in cardiac allografts. a, Time-course of increase in hIL-4 and hIL-10 transgene overexpression in cardiac allografts transfected pSVIL-4 and IL-10 compared with that in allografts treated with empty liposome (n = 15). Transgene expression level in the left ventricular tissue of cardiac allografts was assessed by qcRT-PCR at POD 1–2, 4–5, 7–8, 27–28, and >60 (n = 8–15 in each data points. RT-cDNA values for hIL-4 and IL-10 are plotted as a ratio of GAPDH cDNA. b, Dose-dependence of ex vivo intracoronary delivered liposome-pSVIL-4 and IL-10 cDNA induced of localized hIL-4 and hIL-10 transgene overexpression in cardiac allografts (n = 2–15, *, p < 0.01 compared with that in cDNA at 0 µg). c, Superimposed illustration shows hIL-4 and hIL-10 transgene expression was detected only in donors’ hearts (lanes 5 and 6) by quantitative competitive RT-PCR analysis, not in the recipient’s brain, lung, skeletal muscle, and heart (lanes 1–4, respectively). d, In situ hybridization analysis using a riboprobe specific for hIL-10. Transgene was expressed in a mosaic pattern. Blue stained transfected cardiac myocytes are diffusely and uniformly distributed in the left ventricular myocardium and whole heart. There is no transfected cell in cardiac allografts transfected with empty liposome. e, The efficiency of gene transfer in cardiac allografts transfected with liposome-hIL-4 (Lip-IL4, n = 15), liposome-hIL-10 (Lip-IL10, n = 15), or liposome-hIL-4 and hIL-10 (Lip-IL4&IL10, n = 15). The transfection efficiency was determined as blue stained positive cells (percentage) in total cells counted in 10 high power microscopic fields (magnification, x400) per section. —, comparison made between two groups; *, p < 0.05; NS, p > 0.05.

 
The significant increase in protein expression of IL-4 revealed at POD 2–3 and reached the peak at POD 7–8, then slowly decreased in a long period of time (Fig. 2a). The time course of IL-10 expression in the allografts was the same as IL-4, but the amount of the IL-10 was 2-fold of IL-4. The increment of IL-4 and IL-10 expression in combinatorial gene therapy was similar as that in IL-4 (8.3 ± 1.2- vs 8.0 ± 1.8-fold; p > 0.05) or IL-10 only gene therapy (14.3 ± 2.5- vs 14.1 ± 1.7-fold, p > 0.05). However, unlike mRNA expression, the decline of IL-4 and IL-10 protein expression was slower in combined gene therapy group than that in single gene therapy groups. As shown in Fig. 2, b and c, the expression of IL-10 as well as IL-4 was higher in combined gene transfer group that transfected alone at POD 8. At POD 27–28, IL-4 and IL-10 were only decreased 27 ± 3 and 18 ± 3% from the peak level in combined gene therapy group (Fig. 2a), but 43 ± 4 and 68 ± 5% in IL-4 or IL-10 single gene therapy groups (p < 0.01, data not shown).



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  		FIGURE 2. IL-4 and IL-10 protein overexpression in cardiac allografts induced by liposome-mediated ex vivo intracoronary gene delivery. a, Time-course of IL-4 and IL-10 protein overexpression in the left ventricle of cardiac allografts transfected with SVhIL-4 and hIL-10 assessed by ELISA at POD 1–2, 4–5, 7–8, 27–28, and >60 (n = 8–15 in each data points). b, Superimposed illustration of Western blot analysis shows IL-4 and IL-10 protein overexpression in the left ventricular tissue of cardiac allografts induced by ex vivo intracoronary lipsome-hIL-4 (Lip-IL4), liposome-SVhIL-10 (Lip-IL10) or liposome-SVhIL4 and hIL10 (Lip-IL4&IL10) gene transfer compared with that transfected with empty liposome (Lip). c, Histogram summarizes the statistics analysis of IL-4 and IL-10 protein overexpression in the left ventricular tissue of cardiac allografts induced by ex vivo intracoronary Lip-IL-4, Lip-IL-10 or Lip-IL-4 and IL-10 gene transfer compared with that transfected with Lip. Left ventricular tissue was collected at POD 7. *, p < 0.01 verses IL-4 in Lip; **, p < 0.05 verses IL-10 in Lip. d, The histogram shows the IL-4 and IL-10 protein level assessed by ELISA in left ventricle (LV), interventricular septum (IVS), right ventricle (RV), left atrium (LA) and right atrium (RA) from allografts at POD 7–8 (n = 15 in each data point). Overexpressed IL-4 and IL-10 protein was homogeneously distributed in the whole heart.

 
The distribution of IL-4 and IL-10 was similar in left ventricle, interventricular septum, right ventricle, left atrium, and right atrium (Fig. 2d). There was no significant increase in IL-4 and IL-10 concentration in the recipients’ serum, brain, lung, spleen, liver, kidney, and skeletal muscle in all time phases examined by ELISA, compared with those recipient rabbits treated with "empty" liposome (data not shown).

Synergistic prevention of allograft rejection and promoting long survive of cardiac allograft

The mean survival of cardiac allografts was significantly prolonged from 9 ± 2 days in control group (allografts treated with empty liposome) to 135 ± 25 days in IL-4 and IL-10 gene combined group (p < 0.01). The mean survival of IL-4 gene-treated allografts was only slightly prolonged (14 ± 3 days), and it was substantially prolonged in IL-10 gene-treated allografts (28 ± 7 days, p < 0.01) (Fig. 3a). However, the mean survival of cardiac allograft in liposome-mediated antisense IL-10 gene-transfected groups (9 ± 2 days) was not prolonged. Rejection score of cardiac allograft in control group was grade 3.4 ± 0.4 at POD 4–5 and 3.6 ± 0.4 in POD 7 (Fig. 3, b and c). IL-4 alone only reduced rejection score in POD 1–2. In IL-10 gene-treated allografts, the rejection process was delayed, but it was still exaggerated at POD 27–28. To determine the synergistic effect of IL-4 and IL-10 combined gene therapy, we compared the rejection score in IL-4 or IL-10 gene alone-treated groups, and combined gene therapy group to that in control group at POD 1–2, 4–5, and 7–8. As shown in Fig. 3d, the effect of combinatorial IL-4 and IL-10 gene therapy on the rejection score was significantly greater than the sum of IL-4 and IL-10 (Fig. 3c, p < 0.05). In comparison to the allograft treated with IL-10 gene alone, the rejection score was also remarkably lower in IL-4 and IL-10 combined gene therapy group at POD 28 (p < 0.01), further declined in the long period of time.



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  		FIGURE 3. Efficacy of liposome-mediated localized ex vivo IL-4 and IL-10 combined gene therapy in cardiac allografts. a, Kaplan-Meier survival curves for allografts treated with empty liposome (n = 15), liposome-SVhIL-4 group (n = 15, p < 0.05 compared with that in the empty liposome group), liposome-SVhIL-10 (n = 15, p < 0.01 compared with that in the empty liposome group and liposome-SVhIL-4 group) and liposome-SVhIL-4 and SVhIL-10 (n = 15, p < 0.001 compared with that in the empty liposome group, liposome-SVhIL-4 group or liposome-SVhIL-10 group, and the sum of liposome-SVhIL-4 group and liposome-SVhIL-10 group). b, Photomicrograph of H&E staining of sections from allografts treated with empty liposome or liposome-SVhIL-4 at POD 7, and liposome-SVhIL-10 or liposome-SVhIL-4 and SVIL-10 at POD 7 and POD 28. c, Histologic rejection grades for allografts treated with empty liposome, liposome-SVhIL-4, liposome-SVhIL-10 and liposome-SVhIL-4 and SVhIL-10. *, p < 0.05 vs that in the allografts treated with empty liposome at the same time period. **, p < 0.05 vs that in the allografts treated with liposome-SVhIL-10 at the same time period.

 
Synergistic suppression of T lymphocyte infiltration in cardiac allografts induced by localized overexpression of IL-4 and IL-10

Combinatorial cytokine gene therapy markedly reduced the total number of graft infiltrating cells compared with that in controls (Fig. 4, a and b). The synergistic inhibition of IL-4 and IL-10 on T lymphocytes was most remarkable (Fig. 4b). In IL-4 gene alone-treated allografts, a slight decrease of CD3+ cell was revealed in the early stage and maintained in the later stage. However, there was also a significant increase in other cell compartment that was mostly consisted with B cells; therefore, the total amount of infiltrates were not significantly decreased. In IL-10 gene-treated allografts, a significant decrease of CD3+ cells was initiated in the early stage and more profound decrease was observed at POD 7–8. However, the rebound was occurred at POD 27–28, whereas transgene expression was reduced. In the combined gene therapy group, localized overexpression of IL-4 and IL-10 synergistically inhibits T lymphocyte infiltration, this effect was significantly greater than that in IL-4 or IL-10 gene treated alone (p < 0.01). CD3+ cell infiltration was almost eliminated at the early stage and markedly reduced at POD 7–8. Unlike in the IL-10 gene therapy, the number of CD3+ cell infiltration was profoundly reduced in the combined gene therapy group at POD 27–28, and in the long term (Fig. 4b). B cell and monocytes/macrophage were initially decreased, but slightly increased in the long term in the combined gene therapy group compared with that in the control group. The reduction of CD3+ cells in the allografts was significantly correlated with the IL-4 and IL-10 overexpression in the combined gene therapy group (Fig. 5, a and b).



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  		FIGURE 4. Effects of liposome-mediated IL-4 and IL-10 combined gene therapy in alloreactive infiltrates. a, Photomicrographs of immunohistochemical staining of CD3+ lymphocytes, B lymphocytes, and monocytes and macrophages in sections from the allograft treated with empty liposome (Control) and liposome-SVhIL-4 and SVhIL-10 (IL-4&10) at POD 7. b, Histograms summarizing the percentages of various infiltrating cell types in the allografts treated with empty liposome (c), liposome-SVhIL-4 (IL4), liposome-SVhIL-10 (IL10) and liposome-SVhIL-4 and SVhIL-10 (IL4&IL10) at POD 4–5, 7–8, 27–28, and >60. CD3+ cells; monocytes and macrophages (CD68+); B cells (CD22+). *, p < 0.05 vs that in c; **, p < 0.05 vs that in IL-10.

 


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  		FIGURE 5. Correlation between localized IL-4 and IL-10 overexpression and the reduction of infiltrative CD3+ T lymphocytes in cardiac allografts. a, The correlation between IL-4 expression and the reduction of infiltrating CD3+ T cells in cardiac allografts treated with liposome-SVhIL-4 and SVhIL-10 (n = 24). b, The correlation between the protein expression of IL-10 and the reduction of subpopulation of CD3+ T cells was revealed as more significant data (n = 24).

 
Synergistic effect of IL-4 and IL-10 on T cell subpopulation

Localized overexpression of IL-4 and IL-10 induced a remarkable decrease of both CD4+ and CD8+ cells in cardiac allografts. At POD 1–2, IL-4 and IL-10 synergism was evidenced by the preventing of both CD4+ and CD8+ cell infiltration (Fig. 6, a and b). In the later stage, the synergism was manifested in two folds: 1) an extensive inhibition in the infiltration of both CD4+ and CD8+ cell was significantly more than a simple addition effect of two cytokines (p < 0.01); 2) a greater effect on the CD8+ cell resulted in an increase in the CD4+ to CD8+ ratio, which was not seen in allografts with either gene transferred alone (p < 0.05). These two effects were gradually enhanced in the late stage and revealed in all time period observed.



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  		FIGURE 6. Effect of liposome-mediated IL-4 and IL-10 combined gene therapy on the infiltrating T lymphocyte subpopulations. a, Superimposed illustrations are the representative immunohistochemical staining findings of CD4+ lymphocytes in the sections from the allograft treated with empty liposome (Control) and liposome-SVhIL-4 and SVhIL-10 (IL-4&10) at POD 7. The histogram shows the amount of infiltrating CD4+ T lymphocytes in cardiac allografts treated with empty liposome, liposome-SVhIL-4, liposome-SVhIL-10 and liposome-SVhIL-4 and SVIL-10 at POD 1–2, 4–5, 7–8, 27–28, and >60. *, p < 0.05; **, p < 0.01 vs that in the allografts treated with empty liposome at the same time period. #, p < 0.05 vs that in the allografts treated with liposome-SVhIL-10 at the same time period. b, Superimposed illustrations are the representative immunohistochemical staining findings of CD8+ lymphocytes in the sections from the allograft treated with empty liposome (Control) and liposome-SVhIL-4 and SVhIL-10 (IL-4&10) at POD 7. The histogram shows the amount of infiltrating CD8+ T lymphocytes in cardiac allografts treated with empty liposome, liposome-SVhIL-4, liposome-SVhIL-10 and liposome-SVhIL-4 and SVIL-10 at POD 1–2, 4–5, 7–8, 27–28, and >60. *, p < 0.05, **; p < 0.01 vs that in the allografts treated with empty liposome at the same time period. #, p < 0.05 vs that in the allografts treated with liposome-SVhIL-10 at the same time period.

 
Synergistic inhibition of IL-4 and IL-10 on T cell cytotoxicity

To further evaluate the synergistic effect of IL-4 and IL-10 on the cytotoxic activity of graft-infiltrating T lymphocytes, we measured the specific lysis of donor spleen cells against the graft-infiltrating lymphocytes in a cytotoxicity test. Donor spleen cells were isolated at the same time the donor heart was harvested. The recipient spleen cells were isolated at the same time infiltrating lymphocytes were collected. In the control group, the cytolytic activity was 51 ± 5%, whereas the responder cell to target cell ratio was 10:1 (Fig. 7, a and b). While recipient spleen cells were used as target cell, no cytotoxicity was observed. In combined gene therapy group, cytotoxic activity of graft infiltrating T lymphocytes was almost abolished as early as POD 2. This was also seen in the IL-10 gene-treated group, but not in the IL-4 gene-treated group. However, the cytotoxicity of T cell rebound after POD 15 in IL-10 gene-treated group. The inhibition of IL-4 and IL-10 combined gene therapy was significantly greater than the effect of IL-4 addition to IL-10 (p < 0.05). The synergism of overexpressed IL-4 and IL-10 was able to maintain the T cell cytotoxic activity in low profile without a rebound in the late stage.



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  		FIGURE 7. Effect of liposome-mediated IL-4 and IL-10 combined gene therapy on the infiltrating T lymphocyte cytotoxicity. a, Cytotoxic test of allo-Ag-specific cytotoxic T lymphocyte from allografts treated with empty liposome, liposome-SVhIL-4, liposome-SVhIL-10 and liposome-SVhIL-4 and SVhIL-10. Responder cells were isolated from the allograft heart at POD 7–8, and target cells were extracted on the each donor spleen at transplantation. Each group was included at least 15 animals. Each sample was examined by triplicate. b, Superimposed histogram shows time-dependent reduction in the cytotoxicity of cytotoxic T lymphocyte from allografts treated with empty liposome, liposome-SVhIL-4, liposome-SVhIL-10 and liposome-SVhIL-4 and SVhIL-10 at POD 4–5 and 27–28. *, p < 0.01 vs that in the allografts treated with empty liposome at the same time period. **, p < 0.01 vs that in the allografts treated with liposome-SVhIL-10 at the same time period.

 
Immunoregulation of exogenous IL-4 and IL-10 on endogenous Th1 and Th2 cytokine gene expression in cardiac allografts

Localized overexpression of IL-4 and IL-10 resulted in a significant decrease in the expression of endogenous Th2 cytokine genes, IL-4, IL-10 (Fig. 8a), and IL-5 (data not shown). In single cytokine gene therapy, excessive exogenous cytokine gene transfer significantly down-regulates the expression of endogenous same cytokine, IL-4 or IL-10 gene, but in a certain extent up-regulates the other Th2 cytokine gene expression, such as IL-5. In combined gene therapy, expression level of endogenous IL-4 and IL-10 genes was first decreased in the early stage, then increased at POD 18–20, and remained in a high level in a long period of time. The expression of Th1 cytokine gene, TNF-{alpha}, was significantly decreased by the synergistic inhibition of IL-4 and IL-10 because this effect was significantly greater than the combined effect of IL-4 and IL-10 used alone (p < 0.05), and maintained in the low level in the late stage (Fig. 8b). IFN-{gamma} gene expression level was only slightly decreased in the IL-4 gene therapy group (p = 0.048), but significantly decreased in the IL-10 gene therapy group. The profound decrease in IFN-{gamma} gene expression level observed in the combined gene therapy group that could be resulted from synergistic immunoregulatory effects of IL-4 and IL-10. Overall, combined gene therapy was able to maintain a slight, but significant, higher level of Th1, and the same low level of Th2 cytokine gene expression in the allograft compared with that in isografts (in that model, donor rabbit was recipients’ three-generation sibling hybrids).



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  		FIGURE 8. Effect of liposome-mediated IL-4 and IL-10 combined gene therapy on the endogenous Th1 and Th2 cytokine gene expression in cardiac allografts. a, Time-dependent changes of endogenous (rabbit) IL-4 (rIL4) and IL-10 (rIL10) mRNA levels in allograft treated with liposome-SVhIL-4 and SVhIL-10 (IL4& IL10) compared with that treated with empty liposome (control). Superimposed gel illustration shows a representative rIL-4 and rIL-10 mRNA quantitation using quantitative competitive RT-PCR assay. b, Changes of endogenous (rabbit) IL-4 (rIL4), IL-10 (rIL10), rTNF-{alpha} and rIFN-{gamma} mRNA levels in allograft treated with empty liposome, liposome-SVhIL-4, liposome-SVhIL-10 and liposome-SVhIL-4 and SVhIL-10 compared with that in isografts at POD 7–8. *, p < 0.05 vs that in the allografts treated with liposome-SVhIL-4; #, p < 0.05 vs that in the allografts treated with liposome-SVhIL-10; {dagger}, p < 0.05 vs that in the allografts treated with empty liposome; **, p < 0.05 vs that in isografts.

 
Efficacy of combinatorial cytokine gene therapy on cardiac allograft function

Using our newly developed heterotopic functional heart transplant model, we were able to evaluate the efficacy of gene therapy on the cardiac allograft function (4, 27). At POD 6, the left ventricular systolic pressure was decreased 68% in cardiac allografts compared with that in isograft (Fig. 9). Cardiac function was completely reserved in IL-10 and IL-4 and IL-10 combined gene-treated allografts, but it was only halfway improved in IL-4 gene therapy group. At POD 28, ventricular systolic pressure was significantly reduced in only IL-10 gene-treated allografts, but it remained unchanged in IL-4 and IL-10 combined gene therapy group, and for a long period of time. Overexpressed IL-4 and IL-10 significantly correlated with the reduction of total infiltrating cells in the allografts (r = 0.73, p < 0.01). Reduction of the infiltrates also significantly correlated with the left ventricular pressure (r = 0.74, p < 0.01). Both IL-4 and IL-10 expression levels were significantly correlated with the systolic pressure of the left ventricle and inversely correlated with the rejection score of cardiac allografts (p < 0.01). Additionally, reduction of TNF-{alpha} gene expression in the allografts induced by IL-4 and IL10 combined gene therapy significantly correlated with the systolic pressure of the left ventricle (r = 0.73, p < 0.01) and inversely correlated with the rejection score in the cardiac allograft (r = 0.88, p < 0.01).



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  		FIGURE 9. Synergistic effect of liposome-mediated IL-4 and IL-10 combined gene therapy induced localized immunosuppression on the function of cardiac allografts. Peak systolic pressure of left ventricle recorded from cardiac isografts (IsoG, n = 15), cardiac allografts treated with liposome-IL4 gene (IL-4, n = 15), cardiac allografts treated with liposome-IL-10 gene (IL10, n = 15) and cardiac allografts treated with liposome-IL-4 and IL-10 compared with that in cardiac allografts treated with empty liposome (Lip, n = 15) at POD 6. At POD 28, only allografts of liposome-IL-10 and liposome-IL-4 and IL-10 groups were survived for comparison and At POD 60, only allografts of liposome-IL-4 and IL-10 group were available. *, p < 0.05; **, p < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The present study demonstrates the first success of liposome-mediated ex vivo combinatorial gene transfer in cardiac allograft. Recently, cationic liposome-mediated gene transfer approach has aroused widespread interest because it has no viral-related complications (34, 35). The efficiency of liposome-mediated gene transfer is 5–15%, which is higher than all other nonviral gene transfer vehicles, although it is still 5–10 times lower than viruses (4). The lack of autoimmunogenicity allows liposome-gene complex to be administered repeatedly (36). Another feature among the advantages of liposome over viral methods is that there is no limit on the size of the vector, which offers the potential for multiple gene delivery (37). Here, we report that cationic liposome could mediate the transfer of two therapeutic genes into the target organ simultaneously. Although gene transfer efficiency is moderate compared with viral transfection, the levels of secreted cytokine proteins in allografts were increased >6-fold for IL-4 and 12-fold for IL-10. The efficiency for transferring IL-10 gene in the combined gene transfer approach was 15.7%, which is the same as that was transfected alone. The efficiency for IL-4 gene transfer was 8% lower compared with that transferred alone. The relatively lower efficiency for IL-4 remained regardless whether the two plasmid vectors were complexed with liposome, or each plasmid vector was complexed with liposome individually before transferring. Thus, this discrepancy unlikely is due to the competitive interference between two genes during liposome encapsulation of the vector. The feature of gene-specific low transfection efficiency could occur at several of the following steps: nucleic acids release from the liposome after its entry into the cell, entry of nucleic acids into the nucleus, and gene transcription (8, 37). The lower transcription of IL-4 vs IL-10 was further evidenced in the remarkable and proportional decrease at both mRNA and protein levels. Many investigators have reported the poor correlation in the gene transfer efficiency between a reporter gene and a therapeutic gene, or between two therapeutic genes (15, 38). It seems that each gene has its own rate of transcription. Even in the same animal model with same promoter-enhancer, the efficiency of gene transfer in terms of transgene expression level could differ from one gene to another (39). However, we still could not exclude the possibility of competitive interaction between two genes in the transcription step while they transferred simultaneously.

Synergistic immunosuppression of IL-4 and IL-10 in cardiac allograft rejection is described the first time here. In the present study, the synergism of two cytokines was displayed in several aspects with distinct mechanisms (1). Combination of IL-4 and IL-10 exerts a synergistic effect on the suppression of cell-mediated immunity that is manifested by the synergistic inhibition of IL-4 and IL-10 on alloreactive CD3+ infiltration and synergistic inhibition of CD4+ and CD8+ subsets. This finding is consistent with previous observations that, in the delayed-type hypersensitivity reactions, both cytokines are needed to induce optimal tolerance (18, 29, 41). A significant increase in CD4+ to CD8+ ratio that was only seen in combinatorial gene therapy group, not in single gene therapy groups, suggests that the preferential inhibition of CD8+ cell may be beneficial and contributes to the prolonged allograft survival (2). IL-4 and IL-10 acts at different stages of the alloimmune-responsive process. The observations from single cytokine gene-treated allograft suggest that IL-4 acts at the induction stage and IL-10 at the both induction and effector stages (42). Exogenous administration of either IL-4 or IL-10 delays rejection times of cardiac allografts. However, the effects of IL-10 were more intense and lasting. This could be a result of the potent effect of IL-10 on the regulative T cell activation and apoptosis, and their function of cytokine production (3). In combinatorial gene therapy, overexpressed IL-4 suppresses the intrinsic IL-4 production, but up-regulates the intrinsic IL-10 gene expression (19). This was the same for IL-10. Maintaining the up-regulation of intrinsic IL-10 expression (to a certain extent, IL-4 expression) while exogenous IL-4 and IL-10 expression was declining seems to be critical for the efficacy of immunosuppression, such as suppression of CD8+ cytotoxicity in the long term (18, 43, 44) (4). IL-4 and IL-10 share many features in the regulation of Th1 and Th2 cytokine expression (44). Synergism of IL-4 and IL-10 was able to re-establish the balance of Th1 and Th2 cytokine gene expression in the allograft to the similar lever as that seen in isografts. In contrast, it has been known that IL-4 and IL-10 bind to different receptors and transduction signals by using different intracellular molecules (45). IL-4 and IL-10 synergism induced superior inhibition of Th1 cytokine, TNF-{alpha} and IL-1{beta} production could be through this mechanism. However, significantly less effectiveness in the inhibition of IFN-{gamma} production in combinatorial gene therapy compared with IL-4 or IL-10 gene therapy alone suggests a possible competitive antagonistic effect between two cytokines.

This study provides the first evidence that long survival of cardiac allograft could be induced by localized IL-4 and IL-10 combined gene therapy. The synergy between overexpressed IL-4 and IL-10 in cardiac allografts results in a remarkable localized alloimmunosupression accompanied with a great improvement of histological rejection grades. The efficacy of combinatorial gene therapy was displayed in the improvement of both cardiac mechanical function and electrophysiology.

In conclusion, liposome-mediated localized IL-4 and IL-10 combined gene therapy may promote alloreactive T lymphocytes anergy and long-term survival of cardiac allografts without systemic immunosuppression in large animals. Further studies will be necessary to determine the mechanism of synergistic immunosuppression induced by localized overexpression of IL-4 and IL-10, and to evaluate the pharmacokinetics and pharmacodynamics of combined immunosuppressive gene therapy before any clinical application.


   Acknowledgments
 
We thank Benjamin M. Sacks, Anthony Arellano-Kruse, Cornelia Gramlich, Onisuru Okotie, and Allison Linh Lam for excellent technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported in part by grants-in-aid from the American Heart Association, Western States Affiliate, the International Society of Heart and Lung Transplantation, and the Foundation of Cardiovascular Disease and Transplantation. Back

2 Address correspondence and reprint requests to Dr. Luyi Sen, Division of Cardiology, Department of Medicine, University of California Los Angeles Medical Center/David Geffen School of Medicine, University of California, 47-123 CHS, 10833 Le Conte Avenue, Los Angeles, CA 90095. E-mail address: lsen{at}mednet.ucla.edu Back

3 Abbreviations used in this paper: h, human; POD, postoperative day; qcRT-PCR, quantitative comparative RT-PCR; CT, competitive template; RT, reverse transcriptase. Back

Received for publication July 15, 2004. Accepted for publication March 22, 2005.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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