Liposome-Mediated Combinatorial Cytokine Gene Therapy Induces Localized Synergistic Immunosuppression and Promotes Long-Term Survival of Car
http://www.100md.com
免疫学杂志 2005年第11期
Abstract
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
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.
Results
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).
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-, 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- 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- 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).
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- 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).
Discussion
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- and IL-1 production could be through this mechanism. However, significantly less effectiveness in the inhibition of IFN- 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.
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.
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
3 Abbreviations used in this paper: h, human; POD, postoperative day; qcRT-PCR, quantitative comparative RT-PCR; CT, competitive template; RT, reverse transcriptase.
Received for publication July 15, 2004. Accepted for publication March 22, 2005.
References
Cotts, W. G., M. R. Johnson. 2001. The challenge of rejection and cardiac allograft vasculopathy. Heart Fail. Rev. 6: 227-240.
Zuckermann, A., H. Reichenspurner, T. Birsan, H. Treede, E. Deviatko, B. Reichart, W. Klepetko. 2003. Cyclosporine A versus tacrolimus in combination with mycophenolate mofetil and steroids as primary immunosuppression after lung transplantation: one-year results of a 2-center prospective randomized trial. J. Thorac. Cardiovasc. Surg. 125: 891-900.
Kita, Y., X. K. Li, M. Ohba, N. Funeshima, S. Enosawa, A. Tamura, K. Suzuki, H. Amemiya, S. Hayashi, T. Kazui, S. Suzuki. 1999. Prolonged cardiac allograft survival in rats systemically injected adenoviral vectors containing CTLA4Ig-gene. Transplantation 68: 758-766.
Sen, L., Y. S. Hong, H. Luo, G. Cui, H. Laks. 2001. Efficiency, efficacy, and adverse effects of adenovirus- vs. liposome-mediated gene therapy in cardiac allografts. Am. J. Physiol. 281: H1433-H1441.
Moore, K. W., A. O’Garra, R. de Waal Malefyt, P. Vieira, T. R. Mosmann. 1993. Interleukin-10. Annu. Rev. Immunol. 11: 165-190.[
Morita, Y., M. Yamamoto, M. Kawashima, T. Aita, S. Harada, H. Okamoto, H. Inoue, H. Makino. 2001. Differential in vitro effects of IL-4, IL-10 and IL-13 on proinflammatory cytokine production and fibroblast proliferation in rheumatoid synobium. Rheumatol. Int. 20: 49-54.
de Waal Malefyt, R., J. Abrams, B. Bennett, C. G. Figdor, J. E. de Vries. 1991. Interleukin 10 (IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes. J. Exp. Med. 174: 1209-1220.(Hiroshi Furukawa*, Kiyohi)
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
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.
Results
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).
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-, 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- 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- 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).
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- 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).
Discussion
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- and IL-1 production could be through this mechanism. However, significantly less effectiveness in the inhibition of IFN- 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.
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.
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
3 Abbreviations used in this paper: h, human; POD, postoperative day; qcRT-PCR, quantitative comparative RT-PCR; CT, competitive template; RT, reverse transcriptase.
Received for publication July 15, 2004. Accepted for publication March 22, 2005.
References
Cotts, W. G., M. R. Johnson. 2001. The challenge of rejection and cardiac allograft vasculopathy. Heart Fail. Rev. 6: 227-240.
Zuckermann, A., H. Reichenspurner, T. Birsan, H. Treede, E. Deviatko, B. Reichart, W. Klepetko. 2003. Cyclosporine A versus tacrolimus in combination with mycophenolate mofetil and steroids as primary immunosuppression after lung transplantation: one-year results of a 2-center prospective randomized trial. J. Thorac. Cardiovasc. Surg. 125: 891-900.
Kita, Y., X. K. Li, M. Ohba, N. Funeshima, S. Enosawa, A. Tamura, K. Suzuki, H. Amemiya, S. Hayashi, T. Kazui, S. Suzuki. 1999. Prolonged cardiac allograft survival in rats systemically injected adenoviral vectors containing CTLA4Ig-gene. Transplantation 68: 758-766.
Sen, L., Y. S. Hong, H. Luo, G. Cui, H. Laks. 2001. Efficiency, efficacy, and adverse effects of adenovirus- vs. liposome-mediated gene therapy in cardiac allografts. Am. J. Physiol. 281: H1433-H1441.
Moore, K. W., A. O’Garra, R. de Waal Malefyt, P. Vieira, T. R. Mosmann. 1993. Interleukin-10. Annu. Rev. Immunol. 11: 165-190.[
Morita, Y., M. Yamamoto, M. Kawashima, T. Aita, S. Harada, H. Okamoto, H. Inoue, H. Makino. 2001. Differential in vitro effects of IL-4, IL-10 and IL-13 on proinflammatory cytokine production and fibroblast proliferation in rheumatoid synobium. Rheumatol. Int. 20: 49-54.
de Waal Malefyt, R., J. Abrams, B. Bennett, C. G. Figdor, J. E. de Vries. 1991. Interleukin 10 (IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes. J. Exp. Med. 174: 1209-1220.(Hiroshi Furukawa*, Kiyohi)