- Email: [email protected]
Gene transfer of interleukin-4 delays acute rejection of splenic allografts in rats
Gene Transfer of Interleukin-4 Delays Acute Rejection of Splenic Allografts in Rats H. Jiang, C. Liu, J. Xu, B. Sun, S. Pan, H. Qiao, L. Luo, and X. Sun ABSTRACT Spleen transplantation is the treatment of choice for some diseases, such as hemophilia A. However, the risk and intensity of rejection after spleen transplantation is greater and more difficult to control than other types of transplant. In the present study, we demonstrated that perfusion of IL-4 expression plasmids into donor spleens pretransplantation led to overexpression of IL-4 and downregulation of IFN-␥ in situ, associated with delayed acute rejection of the allograft. Gene transfer of IL-4 may represent a potential therapeutic approach to induce tolerance to splenic allografts.
S
PLEEN transplantation is becoming the treatment of choice for some diseases, such as hemophilia A, ever since the first report by Hathaway in 1969.1 However, delayed dysfunction of the grafted spleens was observed in a follow-up study.2 The level of factor VIII in serum declined and the symptoms reappeared associated with splenic, atrophy which have been related to the use of immunosuppressive drugs.3,4 For example, cyclosporine damages T cells and induces B-cell apoptosis, which contribute to the splenocyte injury.5 Furthermore, immunosuppressants may produce complications of infection, and malignancies,6 and retard allograft tolerance.7,8 Cytokines are key mediators that regulate the induction, amplification and effector phases of immune responses implicated in rejection versus tolerance to grafts.9 Th1 cytokine (IL-2 and IFN-␥) downregulation and Th2 cytokine (IL-10 and IL-4) upregulation have been shown to promote tolerance.10,11 IL-4, a major inducer of Th2 differentiation,12 inhibits Th1 cell function by downregulating IFN-␥ and IL-12. Upregulated IL-4 has been associated with allograft tolerance in several animal models,13 including cardiac grafts in conjunction with blockade of the CD40 pathway,14 or CD2 markers, or CD3 with monoclonal antibodies (mAbs).15 Anti-IL-4 mAb retarded the establishment of tolerance.16 Gene transfer of IL-4 adenoviral vectors significantly prolonged the cardiac graft survival by reducing Th1 cytokines (IL-2/IFN-␥),17 or of renal grafts by inhibiting activation of p21(ras) pathways.18 Therapeutic expression of IL-4 protected non-obese diabetic islet cells from insulitis by deviating the local T-cell response toward a nondestructive Th2 phenotype.19,20 It therefore represents a potential therapy for improving allograft survival without the adverse effects of immunosuppression. In the
present study, we sought to investigate whether IL-4 gene transfer into donor spleens achieved allograft tolerance, and inhibited rejection of splenic allografts. MATERIALS AND METHODS Rats Male Wistar donor rats of 180 to 240 g, and male SD (recipient), rats 200 to 260g were purchased from our animal research center. The animals maintained under standard conditions were fed rodent chow and water. All surgical procedures and care were in accordance with institutional guidelines.
Plasmid Construction Total RNA was extracted from Wistar rat, splenocytes with TRIZOL (Takara Bio Inc, Japan) according to the manufacturer’s instruction, and reversely transcribed to generate cDNA. The cDNA was used as the template to amplify the open reading frame or rIL-4 by PCR using two oligonucleotide primers 5⬘-ACGTC GACAGAGCTATTGATGGGTCTCAGCC-3⬘ and 5⬘-CAGCAT GCGTTAGACATGGAAGTGCAGGACTG-3⬘. The PCR product was subcloned into PMD18-T (Takara Bio Inc), and further subcloned into the eukaryotic expression vector pSI (Promega, USA) at the site of XbaI and AccI. This expression vector together with a SV40 enhancer was driven by a cytomegalovirus (CMV) From the Hepatosplenic Surgery Center of Heilongjiang Province, and the Department of General Surgery, The First Clinical Medical School, Harbin Medical University, Harbin, China. Supported by the National Natural Science Foundation of China, and The First Clinical College of Harbin Medical University, China. Address reprint requests to X. Sun, the Hepatosplenic Surgery Center of Heilongjiang Province, the First Clinical Medical School, Harbin Medical University, Harbin 15001, China.
0041-1345/04/$–see front matter doi:10.1016/j.transproceed.2004.05.041
© 2004 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
1600
Transplantation Proceedings, 36, 1600 –1603 (2004)
GENE TRANSFER OF IL-4
1601
immediate-early promoter. DNA sequence analysis confirmed that the cDNA sequences (GeneBank accession: AY496861) were inserted in the proper reading frame, and no mutations had been incorporated.
Preparation of DNA/Liposome Complex The preparation of DNA/Liposome has been described previously.21 Briefly, equal volumes of purified plasmids and Lipofect AMINE Regent (Invitrogen) were mixed, and incubated at room temperature for 30 min. Then the mixture was dissolved in cold WMO-1 reserving solution (4°C) at a final concentration of 200 g/mL.
Operative Procedures All rats were anesthetized by intraperitoneal injection of ketamine. The donor spleen vascularized was via a portion of the portal vein with the splenic vein, and a portion of abdominal aorta with the splenic artery which had been excised after in situ perfusion with cold WMO-1 (4°C) solution. Donor spleens were randomly divided into three groups, each receiving 1 mL of DNA/liposome solution either containing rIL-4/ pSI (n ⫽ 24), or empty pSI vector (n ⫽ 24), or 1 mL WMO-1 solution (n ⫽ 24), via the splenic artery, and incubated inside the spleens for 2 hours. Then the donor spleens were transplanted to the recipients using side-side anastomoses of donor abdominal aorta to recipient abdominal aorta and donor portal vein to recipient infrarenal inferior vena cava (IVC).
Histologic Analysis The spleens were fixed in 10% formalin. Transverse 10-m sections were mounted on the poly-L-lysine-coated slides and stained with hematoxylin and eosin. The slides were observed under microscopy.
Quantification of IL-4 and IFN-␥ in Spleens With ELISA The spleens were homogenized in 1% heparin solution, and centrifuged for 5 minutes at 1000 RPM to collect the supernates. The concentration of IL-4 or IFN-␥ was detected with an IL-4 EIA kit (Biosource, USA) or an IFN-␥ EIA kit (Biosource).
Statistical Methods SPSS10.0 statistical software was used to analyze all experimental data. The results were expressed as mean values ⫾ standard deviation (SD), and a LSD test was used to evaluate statistical significance. P ⬍ .05 was considered to be statistically significant.
RESULTS Perfusion with IL-4/pSI Led to Overexpression of IL-4 and Decreased Levels of IFN-␥ in Spleens.
Donor spleens were reserved inside the organs for 2 hours before transplantation. Recipients were sacrificed 3 days after transplantation of spleens that had received the DNA/liposome complex. The concentration of IL-4 was significantly increased (up to 10-fold) in spleens treated with IL-4/pSI plasmids compared with empty vector (P ⬍ .001), or WMO-1 solution (P ⬍ .001) (Fig 1). There was no significant difference between the spleens perfused with empty vector or WMO-1 solution. On the other hand, IL-4
Fig 1. IL-4 and IFN-␥ concentration in homogenates from donor spleens perfused with WMO-1 solution (control), empty vector, or IL-4/pSI, three days after transplantation. *Significant difference in IL-4 concentration obtained with IL-4/pSI versus control or empty vector (indicated by a single asterisk). **Significant difference in IFN-␥ concentration obtained with IL-4/pSI versus control or empty vector.
gene transfer led to a statistically significant (P ⬍ .001) reduction (71% and 69%, respectively) in IFN-␥ levels, compared with donor spleens treated with empty vector or WMO-1 solution (Fig 1). Gene Transfer of IL-4/pSI into Donor Spleens Delays Acute Rejection of Allografts
To investigate whether the exogenous IL-4 produced by gene transfer could inhibit rejection to the allografted spleens, groups of 4 recipient rats were sacrificed on days 1, 3, 5, 7, 10, or 14 after transplantation, in excised spleens. The typical signs of acute rejection include mononuclear cell infiltration, increased splenocytes in the white pulp, dilated or destroyed periarterial lymphatic sheath (PALS), and mural thrombosis in splenic arteries. Gene perfusion with IL-4 greatly reduced these signs of acute rejection. Representative sections of allografted spleens at 7 days are shown in Fig 2. Figure 3 shows that acute rejection was detected at 2 to 3 days after operation in empty vector or WMO-1 treated groups. In contrast, acute rejection was delayed 7 days in IL-4/pSI transfected donor spleens (Fig 3). There was a significant difference between the IL-4/pSI treated groups and the empty vector or the WMO-1 solution treated groups (both P ⬍ .001). DISCUSSION
The spleen is the largest peripheral lymphoid organ, and the risk of rejection of splenic grafts is higher than other types of transplants. The transplanted spleen may also produce an immune reaction against the host (GVHR), posing difficulties in posttransplantation management.22 Meanwhile, immunosuppressive agents to combat acute rejection may also destroy lymphocytes in the spleen, contributing to delayed dysfunction. The rejection of allotransplanted organs is mediated predominantly by host T cells that are specifically activated
1602
JIANG, LIU, XU ET AL
Fig 2. Histologic observation in donor spleens perfused with WMO-1 solution (control), empty vector, or IL-4/pSI, 7 days after transplantation.
by donor alloantigen.23 Acute rejection seems to require the presence of Th-1 cytokines and tolerance may result from an up-regulated Th-2 activity.24 –27 As a main Th-2 cytokine, IL-4 plays a key role in immune mediated infectious and autoimmune diseases by inhibiting the production of several cytokines, such as IL-1, TNF-␣, IL-8 and in PGE2.28 Despite its potential activity in inducing tolerance,13–15 the clinical application of IL-4 has been hindered by its short half-life (several minutes) in the circulation, potential cytotoxicity, and autoimmune reaction. Gene transfer represents an alternative method to deliver IL-4. Local perfusion with IL-4 genes is a more practical and safer approach. Gene modification to grafts during cold preservation avoids abnormal systemic expression in recipients. Although the most efficient way to introduce transgenes into grafts is via recombinant viral vectors, their immunologic response, safety issues, and toxicity preclude their use in humans in the near future.29,30 In principle plasmid DNA of low immunogenicity does not pose the health risks of viral infection, is easy to propagate on a large scale at high
quality, and carries relatively large DNA sequences. The advantages of localized compared to the systemic delivery of IL-4 genes are obvious: it can induce a high level of transgenic proteins in situ and reduce side effects. Furthermore, the unique anatomic features of the spleen facilitate, regional gene transfer. The transgenes have been expressed in endothelial vascular cells and in cardiac muscles after gene delivery of a liposome/DNA system.31 The method used in this study was modified from the report of Dalesandro et al,22 where a complex of CAT reporter gene/ liposome was perfused in to donor heart grafts, and preserved for 41 ⫾ 6 minutes. The transgenes were observed to be expressed at 24 hours after transplantation. In the present study, we demonstrated for the first time that gene perfusion of IL-4 delayed the occurrence of acute rejection in splenic allografts, although it could not be considered long-term survival. Overexpression of IL-4 inhibited the production of IFN-␥ and Th1 cell activation, the ratio of IL-4:IFN-␥ was used as a indicator to determine whether unresponsiveness had been achieved.32 The present study suggests that the pathway mediating IL-4 action in splenic rejection is more complex than expected. Further studies of its mechanism, seeking therapeutic approaches, possibly in combination with agents blocking costimulatory pathways, are required. We also assume that the plasmid/liposome mediated gene transfer only resulted in transient expression of exogenous IL-4. Generation of a proper expression vector and improvement in the transfection strategies, may lead to long-term expression, enhancing the therapeutic efficacy of IL-4 gene transfer to induce tolerance to splenic allografts. For instance, recently Xu et al33 reported that intraportal transfusion of a novel transfection system led to persistent expression of transgenes in livers for 6 months.
Fig 3. The time after transplantation when acute rejection was detected. *Significant difference in days after transplantation obtained from treatment groups with IL-4/pSI versus WMO-1 solution (control) or empty vector.
REFERENCES 1. Hathaway WE, Mull MM, Githens JH, et al: Attempted spleen transplantation in classical hemophilia. Transplantation 7:73, 1969
GENE TRANSFER OF IL-4 2. Hongchi J, Anlong Z, Jun X, et al: Living related-donor spleen transplantation to treat 5 patients with hemophilia A. Chinese Journal of Hepatobiliary Surgery 7:475, 2001 (Chinese) 3. Suisheng X: Promoting development of splenic surgery. Chinese Journal of Practical Surgery 19:707, 1999 (Chinese) 4. Zhishui C, Suisheng X, Changsong L: The effects of different recipes of immunosuppressants on rejection and survival in splenic transplantation. Chinese Journal of Hepatobiliary Surgery 6:358, 2000 (Chinese) 5. Nemlander A, Soots A, Willebrand E, et al: Effect of cyclosporine A on the in situ inflammatory response of rat renal allograft rejection. Scand J Immunol 16:91, 1982 6. Koshiba T, Kitade´ H, Van Damme B, et al: Regulatory cell-mediated tolerance does not protect against chronic rejection. Transplantation 76:588, 2003 7. Jenkins MK, Schwartz RH, Pardoll DM: Effects of cyclosporine A on T cell development and clonal deletion. Science 241:1655, 1988 8. Sirak JH, Orosz CG, Roopenian DC, et al: Cardiac allograft tolerance: failure to develop in interleukin-4-deficient mice correlates with unusual allosensitization patterns. Transplantation 65: 1352, 1998 9. Nickerson P, Steiger J, Zheng XX, et al: Manipulation of cytokine networks in transplantation. False hope or realitice opportunity for tolerance. Transplantation 63:489, 1997 10. Babcock GF, Alexander W: The effect of blood transfusion on cytokine production by Th1 and Th2 lymphocytes in the mouse. Transplantation 61:465, 1996 11. Shirwan H, Barwari L, Khan NS: Predominant expression of T helper 2 cytokines and altered expression of T helper 1 cytokines in long-term allograft survival induced by intrathymic immune modulation with donor class I major histocompatibility complex peptides. Transplantation 66:1802, 1998 12. Chomarat P, Banchereau J: An update of interleukin-4 and its receptor. Eur Cytokine Netw 8:333, 1997 13. Saggi BH, Fisher RA, Bu D, et al: Intragraft cytokine expression and tolerance induction in rat renal allografts. Transplantation 67:206, 1999 14. Larsen CP, Pearson TP: The CD40 pathway in allograft rejection, acceptance, and tolerance. Curr Opin Immunol 9:641, 1997 15. Punch JD, Tono T, Qin L, et al: Tolerance induction by anti-CD2 plus anti-CD3 monoclonal antibodies. evidence for an IL-4 requirement. J Immunol 161:1156, 1998 16. Field EH: Balancing the immune system for tolerance: a case for regulatory CD4 cells. Transplantation 64:1, 1997 17. Takeuchi T, Ueki T, Sunaga S, et al: Murine interleukin 4 transgenic heart allograft survival prolonged with down-regulation of the Th1 cytokine mRNA in graft. Transplantation 64:152, 1997
1603 18. Kato H, Ritter T, Ke B, et al: Adenovirus-mediated gene transfer of IL-4 prolongs rat renal allograft survival and inhibits the p21(ras)-activation pathway. Transplant Proc 32:245, 2000 19. Mueller R, Krahl T, Sarvetnick N: Pancreatic expression of interleukin-4 abrogates insulitis and autoimmune diabetes in nonobese diabetic (NOD) mice. J Exp Med 184:1093, 1996 20. Rabinovitch A, Suarez-Pinzon WL, Sorensen O, et al: Combination therapy with cyclosporine and interleukin-4 or interleukin-10 prolongs survival of synergeneic pancreatic islet grafts in nonobese diabetic mice: islet graft survival does not correlate with mRNA levels of type 1 or type 2 cytokines, or transforming growth factor-beta in the islet grafts. Transplantation 64:1525, 1997 21. Dalesandro J, Akimoto H, Gorman CM, et al: Gene Therapy for donor hearts: ex vivo liposome-mediated transfection. J Thorac Cardiovase Surg 111:416, 1996 22. Xiang WZ, Jie ZW, Sheng XS: Clinical observation on hemophilia A treatment by cadaveric spleen transplantation. Transplant Proc 34:1929, 2002 23. Versluis DJ, Ten Kate FJW, Wenting GJ, et al: Mononuclear cells infiltrating kidney allografts in the absence of rejection. Transplant Int 1:205, 1988 24. Carlquist J, Shelby J, Hammond EH, et al: Recovery and phenotype identification of in vivo activated lymphocytes from mouse cardiac allografts. Transplantation 50:349, 1990 25. Bishop DK, Shelby J, Eichwald EJ: Mobilization of Tlymphocytes following cardiac transplantation. Transplantation 53: 849, 1992 26. Lowry RP, Takeuchi T: The Th-1, Th-2 paradigm and transplantation tolerance: cellular and molecular mechanisms: In: Alexander JW, Good RA (eds): Transplantation Tolerance Induction. Austin, Tex; R.G. Landes; 1996, 91 27. Lowry RP: The relationship of IL-4, IL-10 and other cytokines to transplantation tolerance. Transplant Sci 3:104, 1993 28. Brown MA, Hural J: Functions of IL-4 and control of its expression. Crit Rev Immunol 17:1, 1997 29. Zabner J, Ramsey BW, Meeker DP: Repeat administration of an adenovirus vector encoding cystic fibrosis transmembrane conductance regulator to the nasal epithelium of patients with cystic fibrosis. J Clin Invest 97:1504, 1996 30. Fisher KJ, Choi H, Burda J: Recombinant adenovirus deleted of all viral genes for gene therapy of cystic fibrosis. Virology 217:11, 1996 31. Sawa Y, Kadoba K, Suzuki K, et al: Efficient transfer of oligonucleotide and plasmid DNA into whole heart through coronary artery. J Thorac Cardiovase Surg 112:1409, 1997 32. Chen N, Field EH: Enhanced type 2 diminished type 1 cytokines in neonatal tolerance. Transplantation 59:933, 1995 33. Xu R, Sun X, Chan D, et al: Long-term expression of angiostatin suppresses liver metastatic cancer in mice. Hepatology 47:1451, 2003
S
PLEEN transplantation is becoming the treatment of choice for some diseases, such as hemophilia A, ever since the first report by Hathaway in 1969.1 However, delayed dysfunction of the grafted spleens was observed in a follow-up study.2 The level of factor VIII in serum declined and the symptoms reappeared associated with splenic, atrophy which have been related to the use of immunosuppressive drugs.3,4 For example, cyclosporine damages T cells and induces B-cell apoptosis, which contribute to the splenocyte injury.5 Furthermore, immunosuppressants may produce complications of infection, and malignancies,6 and retard allograft tolerance.7,8 Cytokines are key mediators that regulate the induction, amplification and effector phases of immune responses implicated in rejection versus tolerance to grafts.9 Th1 cytokine (IL-2 and IFN-␥) downregulation and Th2 cytokine (IL-10 and IL-4) upregulation have been shown to promote tolerance.10,11 IL-4, a major inducer of Th2 differentiation,12 inhibits Th1 cell function by downregulating IFN-␥ and IL-12. Upregulated IL-4 has been associated with allograft tolerance in several animal models,13 including cardiac grafts in conjunction with blockade of the CD40 pathway,14 or CD2 markers, or CD3 with monoclonal antibodies (mAbs).15 Anti-IL-4 mAb retarded the establishment of tolerance.16 Gene transfer of IL-4 adenoviral vectors significantly prolonged the cardiac graft survival by reducing Th1 cytokines (IL-2/IFN-␥),17 or of renal grafts by inhibiting activation of p21(ras) pathways.18 Therapeutic expression of IL-4 protected non-obese diabetic islet cells from insulitis by deviating the local T-cell response toward a nondestructive Th2 phenotype.19,20 It therefore represents a potential therapy for improving allograft survival without the adverse effects of immunosuppression. In the
present study, we sought to investigate whether IL-4 gene transfer into donor spleens achieved allograft tolerance, and inhibited rejection of splenic allografts. MATERIALS AND METHODS Rats Male Wistar donor rats of 180 to 240 g, and male SD (recipient), rats 200 to 260g were purchased from our animal research center. The animals maintained under standard conditions were fed rodent chow and water. All surgical procedures and care were in accordance with institutional guidelines.
Plasmid Construction Total RNA was extracted from Wistar rat, splenocytes with TRIZOL (Takara Bio Inc, Japan) according to the manufacturer’s instruction, and reversely transcribed to generate cDNA. The cDNA was used as the template to amplify the open reading frame or rIL-4 by PCR using two oligonucleotide primers 5⬘-ACGTC GACAGAGCTATTGATGGGTCTCAGCC-3⬘ and 5⬘-CAGCAT GCGTTAGACATGGAAGTGCAGGACTG-3⬘. The PCR product was subcloned into PMD18-T (Takara Bio Inc), and further subcloned into the eukaryotic expression vector pSI (Promega, USA) at the site of XbaI and AccI. This expression vector together with a SV40 enhancer was driven by a cytomegalovirus (CMV) From the Hepatosplenic Surgery Center of Heilongjiang Province, and the Department of General Surgery, The First Clinical Medical School, Harbin Medical University, Harbin, China. Supported by the National Natural Science Foundation of China, and The First Clinical College of Harbin Medical University, China. Address reprint requests to X. Sun, the Hepatosplenic Surgery Center of Heilongjiang Province, the First Clinical Medical School, Harbin Medical University, Harbin 15001, China.
0041-1345/04/$–see front matter doi:10.1016/j.transproceed.2004.05.041
© 2004 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
1600
Transplantation Proceedings, 36, 1600 –1603 (2004)
GENE TRANSFER OF IL-4
1601
immediate-early promoter. DNA sequence analysis confirmed that the cDNA sequences (GeneBank accession: AY496861) were inserted in the proper reading frame, and no mutations had been incorporated.
Preparation of DNA/Liposome Complex The preparation of DNA/Liposome has been described previously.21 Briefly, equal volumes of purified plasmids and Lipofect AMINE Regent (Invitrogen) were mixed, and incubated at room temperature for 30 min. Then the mixture was dissolved in cold WMO-1 reserving solution (4°C) at a final concentration of 200 g/mL.
Operative Procedures All rats were anesthetized by intraperitoneal injection of ketamine. The donor spleen vascularized was via a portion of the portal vein with the splenic vein, and a portion of abdominal aorta with the splenic artery which had been excised after in situ perfusion with cold WMO-1 (4°C) solution. Donor spleens were randomly divided into three groups, each receiving 1 mL of DNA/liposome solution either containing rIL-4/ pSI (n ⫽ 24), or empty pSI vector (n ⫽ 24), or 1 mL WMO-1 solution (n ⫽ 24), via the splenic artery, and incubated inside the spleens for 2 hours. Then the donor spleens were transplanted to the recipients using side-side anastomoses of donor abdominal aorta to recipient abdominal aorta and donor portal vein to recipient infrarenal inferior vena cava (IVC).
Histologic Analysis The spleens were fixed in 10% formalin. Transverse 10-m sections were mounted on the poly-L-lysine-coated slides and stained with hematoxylin and eosin. The slides were observed under microscopy.
Quantification of IL-4 and IFN-␥ in Spleens With ELISA The spleens were homogenized in 1% heparin solution, and centrifuged for 5 minutes at 1000 RPM to collect the supernates. The concentration of IL-4 or IFN-␥ was detected with an IL-4 EIA kit (Biosource, USA) or an IFN-␥ EIA kit (Biosource).
Statistical Methods SPSS10.0 statistical software was used to analyze all experimental data. The results were expressed as mean values ⫾ standard deviation (SD), and a LSD test was used to evaluate statistical significance. P ⬍ .05 was considered to be statistically significant.
RESULTS Perfusion with IL-4/pSI Led to Overexpression of IL-4 and Decreased Levels of IFN-␥ in Spleens.
Donor spleens were reserved inside the organs for 2 hours before transplantation. Recipients were sacrificed 3 days after transplantation of spleens that had received the DNA/liposome complex. The concentration of IL-4 was significantly increased (up to 10-fold) in spleens treated with IL-4/pSI plasmids compared with empty vector (P ⬍ .001), or WMO-1 solution (P ⬍ .001) (Fig 1). There was no significant difference between the spleens perfused with empty vector or WMO-1 solution. On the other hand, IL-4
Fig 1. IL-4 and IFN-␥ concentration in homogenates from donor spleens perfused with WMO-1 solution (control), empty vector, or IL-4/pSI, three days after transplantation. *Significant difference in IL-4 concentration obtained with IL-4/pSI versus control or empty vector (indicated by a single asterisk). **Significant difference in IFN-␥ concentration obtained with IL-4/pSI versus control or empty vector.
gene transfer led to a statistically significant (P ⬍ .001) reduction (71% and 69%, respectively) in IFN-␥ levels, compared with donor spleens treated with empty vector or WMO-1 solution (Fig 1). Gene Transfer of IL-4/pSI into Donor Spleens Delays Acute Rejection of Allografts
To investigate whether the exogenous IL-4 produced by gene transfer could inhibit rejection to the allografted spleens, groups of 4 recipient rats were sacrificed on days 1, 3, 5, 7, 10, or 14 after transplantation, in excised spleens. The typical signs of acute rejection include mononuclear cell infiltration, increased splenocytes in the white pulp, dilated or destroyed periarterial lymphatic sheath (PALS), and mural thrombosis in splenic arteries. Gene perfusion with IL-4 greatly reduced these signs of acute rejection. Representative sections of allografted spleens at 7 days are shown in Fig 2. Figure 3 shows that acute rejection was detected at 2 to 3 days after operation in empty vector or WMO-1 treated groups. In contrast, acute rejection was delayed 7 days in IL-4/pSI transfected donor spleens (Fig 3). There was a significant difference between the IL-4/pSI treated groups and the empty vector or the WMO-1 solution treated groups (both P ⬍ .001). DISCUSSION
The spleen is the largest peripheral lymphoid organ, and the risk of rejection of splenic grafts is higher than other types of transplants. The transplanted spleen may also produce an immune reaction against the host (GVHR), posing difficulties in posttransplantation management.22 Meanwhile, immunosuppressive agents to combat acute rejection may also destroy lymphocytes in the spleen, contributing to delayed dysfunction. The rejection of allotransplanted organs is mediated predominantly by host T cells that are specifically activated
1602
JIANG, LIU, XU ET AL
Fig 2. Histologic observation in donor spleens perfused with WMO-1 solution (control), empty vector, or IL-4/pSI, 7 days after transplantation.
by donor alloantigen.23 Acute rejection seems to require the presence of Th-1 cytokines and tolerance may result from an up-regulated Th-2 activity.24 –27 As a main Th-2 cytokine, IL-4 plays a key role in immune mediated infectious and autoimmune diseases by inhibiting the production of several cytokines, such as IL-1, TNF-␣, IL-8 and in PGE2.28 Despite its potential activity in inducing tolerance,13–15 the clinical application of IL-4 has been hindered by its short half-life (several minutes) in the circulation, potential cytotoxicity, and autoimmune reaction. Gene transfer represents an alternative method to deliver IL-4. Local perfusion with IL-4 genes is a more practical and safer approach. Gene modification to grafts during cold preservation avoids abnormal systemic expression in recipients. Although the most efficient way to introduce transgenes into grafts is via recombinant viral vectors, their immunologic response, safety issues, and toxicity preclude their use in humans in the near future.29,30 In principle plasmid DNA of low immunogenicity does not pose the health risks of viral infection, is easy to propagate on a large scale at high
quality, and carries relatively large DNA sequences. The advantages of localized compared to the systemic delivery of IL-4 genes are obvious: it can induce a high level of transgenic proteins in situ and reduce side effects. Furthermore, the unique anatomic features of the spleen facilitate, regional gene transfer. The transgenes have been expressed in endothelial vascular cells and in cardiac muscles after gene delivery of a liposome/DNA system.31 The method used in this study was modified from the report of Dalesandro et al,22 where a complex of CAT reporter gene/ liposome was perfused in to donor heart grafts, and preserved for 41 ⫾ 6 minutes. The transgenes were observed to be expressed at 24 hours after transplantation. In the present study, we demonstrated for the first time that gene perfusion of IL-4 delayed the occurrence of acute rejection in splenic allografts, although it could not be considered long-term survival. Overexpression of IL-4 inhibited the production of IFN-␥ and Th1 cell activation, the ratio of IL-4:IFN-␥ was used as a indicator to determine whether unresponsiveness had been achieved.32 The present study suggests that the pathway mediating IL-4 action in splenic rejection is more complex than expected. Further studies of its mechanism, seeking therapeutic approaches, possibly in combination with agents blocking costimulatory pathways, are required. We also assume that the plasmid/liposome mediated gene transfer only resulted in transient expression of exogenous IL-4. Generation of a proper expression vector and improvement in the transfection strategies, may lead to long-term expression, enhancing the therapeutic efficacy of IL-4 gene transfer to induce tolerance to splenic allografts. For instance, recently Xu et al33 reported that intraportal transfusion of a novel transfection system led to persistent expression of transgenes in livers for 6 months.
Fig 3. The time after transplantation when acute rejection was detected. *Significant difference in days after transplantation obtained from treatment groups with IL-4/pSI versus WMO-1 solution (control) or empty vector.
REFERENCES 1. Hathaway WE, Mull MM, Githens JH, et al: Attempted spleen transplantation in classical hemophilia. Transplantation 7:73, 1969
GENE TRANSFER OF IL-4 2. Hongchi J, Anlong Z, Jun X, et al: Living related-donor spleen transplantation to treat 5 patients with hemophilia A. Chinese Journal of Hepatobiliary Surgery 7:475, 2001 (Chinese) 3. Suisheng X: Promoting development of splenic surgery. Chinese Journal of Practical Surgery 19:707, 1999 (Chinese) 4. Zhishui C, Suisheng X, Changsong L: The effects of different recipes of immunosuppressants on rejection and survival in splenic transplantation. Chinese Journal of Hepatobiliary Surgery 6:358, 2000 (Chinese) 5. Nemlander A, Soots A, Willebrand E, et al: Effect of cyclosporine A on the in situ inflammatory response of rat renal allograft rejection. Scand J Immunol 16:91, 1982 6. Koshiba T, Kitade´ H, Van Damme B, et al: Regulatory cell-mediated tolerance does not protect against chronic rejection. Transplantation 76:588, 2003 7. Jenkins MK, Schwartz RH, Pardoll DM: Effects of cyclosporine A on T cell development and clonal deletion. Science 241:1655, 1988 8. Sirak JH, Orosz CG, Roopenian DC, et al: Cardiac allograft tolerance: failure to develop in interleukin-4-deficient mice correlates with unusual allosensitization patterns. Transplantation 65: 1352, 1998 9. Nickerson P, Steiger J, Zheng XX, et al: Manipulation of cytokine networks in transplantation. False hope or realitice opportunity for tolerance. Transplantation 63:489, 1997 10. Babcock GF, Alexander W: The effect of blood transfusion on cytokine production by Th1 and Th2 lymphocytes in the mouse. Transplantation 61:465, 1996 11. Shirwan H, Barwari L, Khan NS: Predominant expression of T helper 2 cytokines and altered expression of T helper 1 cytokines in long-term allograft survival induced by intrathymic immune modulation with donor class I major histocompatibility complex peptides. Transplantation 66:1802, 1998 12. Chomarat P, Banchereau J: An update of interleukin-4 and its receptor. Eur Cytokine Netw 8:333, 1997 13. Saggi BH, Fisher RA, Bu D, et al: Intragraft cytokine expression and tolerance induction in rat renal allografts. Transplantation 67:206, 1999 14. Larsen CP, Pearson TP: The CD40 pathway in allograft rejection, acceptance, and tolerance. Curr Opin Immunol 9:641, 1997 15. Punch JD, Tono T, Qin L, et al: Tolerance induction by anti-CD2 plus anti-CD3 monoclonal antibodies. evidence for an IL-4 requirement. J Immunol 161:1156, 1998 16. Field EH: Balancing the immune system for tolerance: a case for regulatory CD4 cells. Transplantation 64:1, 1997 17. Takeuchi T, Ueki T, Sunaga S, et al: Murine interleukin 4 transgenic heart allograft survival prolonged with down-regulation of the Th1 cytokine mRNA in graft. Transplantation 64:152, 1997
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