Effects Of Donor Spleen Leukomonocytes Transfusion And FK506 On Cardiac Allo-transplantation In Mice During Acute Rejection

【Background】Transplantation of solid organs has been established as the only therapy for end-stage disease of these organs, but graft rejective reaction is one of the most important factors for function of transplantated organs and recipient's longer survival after operation. Since the introduction of effective immunosuppressive agents, such as cyclosporine(CsA) and Tacrolimus(FK506), transplantatioin of solid organs has been greatly promoted. However, the level of immunosuppression needed to block rejection with current immunosuppressive agents is associated, in some degree, with decreased ability to mount necessary immune responses against infection and neoplasia.Therefore,each drug presenting toxic side effects specific to the agent limited its long-term use. Combination immunosuppression therapy has been used in solid organ transplantation to reduce drug toxicity. Donor blood transfusions(DST)prior to heart transplantation can decrease the dose of drug and improve organ allograft survival.FK506 (tacrolimus) is a powerful and selective macrolide immunosup- pressant that was discovered by scientists at Fujisawa Pharmaceuticals ( Ibaraki, Japan ) in 1984. The experimental results show,in vitro, FK506 inhibited the response of graft rejection 10 to 100 times higher than that of CsA. FK506 is also helpful to decrease the dose of steroid hormones used in experiments and clinical cases. Since 1993, FK506 has been used to treat graft rejection after organ transplantation, such as liver transplantation,kidney transplantation and heart transplantation in clinical trails in more than 70 countries. FK506 was permitted to be used for heart transplantation by FDA on March 30, 2006. FK506 mainly induces tremors, headache, sleep disturbances, hypertension, decreasing of renal function and sugar tolerance, and other side effects.【Objective】To investigate the effect of donor-specific transfusion (DST) and different doses of FK506 on the survival of homologous cardiac transplantation in mice. We wanted to find the suitable doses of immune depressant for cardiac transplantation, and supply experimental datum for further study.【Methods】1. Preparation of splenic lymphocyte cell suspension. Harvestting spleens from BALB/C mice, then we add 5ml EZ-SepTM Mouse 1X lymphocyte separating medium in sterile dish. Single cell suspensions were made in the dish by passing the cell population through a nylon mesh with 200 pores. Ten×107 splenic lymphocytes in 1ml PBS were ready to be used.2. Donor-specific transfusion (DST). Five×106 splenic lymphocytes in 0.5ml PBS were injected intravenously via the caudal vein into each C57BL/6 mouse once one day before operation.3. Transplantation Model. Male BALB/C (H-2d) mouse hearts were transplanted to male C57BL/6(H-2b) mice using Chen's model. We often used 0.3% pentobarbital sodium solution 40-50mg/kg body weight for intraperitoneal injection. Briefly, the donor aorta and pulmonary artery were anastomosed to the recipient internal carotid artery and internal jugular vein, respectively.4. Experimental design. Forty male C57BL/6 mice were randomly divided into four groups(n=10): Group 1, the control group; Group 2, the DST group; Group 3, the DST/FK506 (2mg/kg/d) group; Group 4, the DST/FK506 (0.3mg/kg/d) group. C57BL/6 mice in Group 3 and Group 4 were treated with FK506 once daily by intraperitoneal injection from the day of operation to the day when the transplanted hearts stopped or the 40th day after operation.5.Management and observation after operation. Postoperative recipient mice should be rewarmed as soon as possible, and be raised alone.①The function of the transplanted heart could be monitored daily by direct palpation and observation, and beating for more than 72 hours with the normal heart rate (200-300/min) indicated the operation was successful. If the transplanted heart beated slowly or stopped in 72 hours after operation indicated the operation was failed. If the operation was successful, the day of heart stop should be considered as the end of transplant rejection.②Pathological examination. At the 7th day after operation, transplanted hearts were harvested from the allogeneic group, fixed by hematoxylineosin (HE), then observed by optics microscope. The intensity of acute rejection reaction should be grading refered to Stanford standard.③At the 7th day after operation, serum IL-2, IL-4, IL-10 and INF-γlevels were compared as instruction of ELISA Kit.④Immune responsiveness was tested in recipient mice treated with FK506 after DST. Fifteen days after operation, the spleen cells of recipient mice in Group 3 and Group 4 were assayed for direct lymphocyte-mediated cytotoxicity (control: normal C57BL/6 mice), for the ability to respond in mixed lymphocyte culture (MLC) assays to BALB/C and C3H spleen cells.【Results】1. Survival time of cardiac allograft. Data were analyzed using SPSS software. Graft survival data were compared by Kaplan-Meier analysis and the log-rank test. A P value less than 0.05 was considered to be significant. Results were expressed as mean values±SD. DST used at the day before operation and FK506 used sequently from the beginning to the end, the cardiac allograft survival was longer in the Group 3 and the Group 4 than those in Group 1 (P<0.01). Among DST/FK506 groups, the low dose of FK506 as aid to DST was the most effective (group 4 vs group 3, P<0.05).2. Pathological changes: Seven days after transplantation, different rejections were found in recipient mice.①Group 1. Severe rejections were found in mice in Group 1 with histological grading 4. Many lymphocytes and monocytes infiltration were seen in the myocardium of Group 1;Large cadiocytes degeneration and necrosis; The myocardium have hemorrhage, edema, and microvascular injury;Small vessels with severe luminal narrowing by intimal proliferation of cells and deposition of connective tissue.②Group 2. Rejections were found in mice in Group 2 with histological grading 3. Significant lymphocytes and monocytes infiltration with myocardial small pieces degeneration and necrosis were seen in the myocardium of Group 2.③Group 3. Mild rejections were found in mice in Group 3 with histological grading 2. Moderate lymphocytes and monocytes infiltration with a focus of myocardial cell degeneration and necrosis were seen in the myocardium of Group 3. The edema of the myocardium is slight.④Group 4. Rejections were found in mice in Group 4 with histological grading 2. What we saw in this group is similar to that of Group 3.3. Serum cytokine levels: IL-2 level in Group 3 and Group 4 was higher compared with that in Group 1 and Group 2 (P<0.01). IL-4 level in Group 3 and Group 4 was lower compared with that in Group 1 and Group 2 (P<0.01). IL-10 level in Group 3 and Group 4 was lower compared with that in Group 1 and Group 2 (P<0.01). INF-γlevel in Group 3 and Group 4 was higher compared with that in Group 1 and Group 2 (P<0.01).4. Mixed lymphocyte response (MLR): When assayed 15 days after operation, the MLC response of recipient mice in Group 3 or Group 4 was specifically suppressed to the donor spleen cells after DST (P<0.01). At the same time, the MLC response of recipient mice was also suppressed to the C3H spleen cells (P<0.01).【Conclusion】1. We establish the model of the acute rejection in heart transplantation of mice successfully. Postoperative transplanted hearts came back to beat fast, and the operative chievement ratio was high.2. The preparation of spleen cells suspension has important effects on treatment of DST. The method of grinding spleen in this experiment can help to harvest enough cells to DST treatment and reduce non-lymphocyte foreign matter.3. DST and FK506 can effectively suppress acute rejection after cardiac allo-transplantation in mice, and prolong the murine cardiac allograft survival. Low dose of FK506 suppresses Th2 cytokines such as IL-4 and IL-10, less than high dose. Th2 cytokines can inhibit proliferation of antigenic-specific T cell, and improve the process of graft rejective reaction. Low dose of FK506 less suppresses regulate T cell (Treg cell) and memory T cell (Tm cell). While under certain circumstances they may contribute to immunological reaction turning to tolerance, and significantly prolong the murine cardiac allograft survival.