Relationship Between Chemokine Receptor CXCR3 MRNA Expression Of PBMC And Acute Cardiac Allograft Rejections In Rats

Orthotopic heart transplantation remains the only viable option for many patients suffering from end-stage cardiomyopathies. The survival time of patients post-transplant has been prolonged and the life quality has been improved pretty much since arrival of many kinds of new immunosuppressive drugs, improvement of surgical techniques and myocardial protection and so on. However, acute and chronic cardiac rejections and transplant coronary artery diseases are still the major reason to cause patients post-transplant death and the mechanism is still unclear. Many experimental and clinical studies have shown that chemokine recptors and their ligands are targeting the progress of antigen presenting, activation of the first and second pathway of T lymphocytes and effective infiltrating of activated T lymphocytes. Gene arrays is a kind of new techniques that can be used to search among those gene profiles related with acute and chronic allograft rejection and TACD. Many studies have shown that CXCR3 and CCR5 are major targets among the chemokine receptors, while others such as CCR1 and CCR2 are not very significant. But there is little known about the relationship between CXCR3 mRNA of peripheral blood mononuclear cells(PBMC) and acute cardiac allograft rejections.In this study, using Wistar rats as donors and SD rats as recipients the abodominal heterotopic heart transplantation models were established to investigate the dynamic changes of chemokine receptor CXCR3 mRNA expression of PBMC, the ratio of CD4+/CD8+ T lymphocytes in peripheral blood and several cytokines such as IL-10, IL-2, IFN-γ and TNF-α in peripheral blood serum, in order to explore CXCR3 in the possible application of post-transplant monitoring in heart transplant recipients as a non-invasive monitoring target. There were three parts in the experiment as following.Part I Establishment and comparison of non-working and working abodominal heterotopic heart transplantation models in ratsObjective To establish the non-woking and working model of abodominal heterotopic heart transplantations in rats and compare with each other. Methods Healthy male Wistar rats were used as donors and healthy male Sprague-Dawley rats was used as recipients. The donor hearts were harvested in the same way, while the ways to anastomose with the recipients were different. In the non-working heart transplant model, the ascending aorta of the donor was attached to the abodominal artery of the recipient first, and the pulmonary artery of the donor was then anastomosed to the inferior vena cava of the recipient. While in the working heart transplant model, the pulmonary artery of the donor was attached with the left atrial appendage of the recipient first, and then the ascending aorta of the donor was then anastomosed to the abodominal artery of the recipient just as the former. The time spent in each step was counted and compared with each other. After transplant, general conditions of the recipients, survival time of cardiac allografts, pathological evaluation and ultrasound echo examinations of cardiac allografts were observed. Results The successful rate of operations in non-working and working models were 100% and 90% separately. All the hearts of donors recovered to beat within 30 seconds. Conclusions The procedure of the heterotopic heart transplantation is viable and convenient. The technique to establish non-working model is mature and all the procedures could be done alone.It is an ideal model to observe acute cardiac rejection. While the working model need two persons to co-operate, the operation is more difficult than non-working model. However, the left and right ventricles have blood flow so that the heart can work similarly as a normal heart. It will be a good model to study chronic cardiac rejections and transplant coronary artery diseases.Part II Relationship between chemokine receptor CXCR3 mRNA expression of PBMC and acute cardiac allograft rejections in ratsObjective To explore the relationship between dynamic changes of CXCR3 mRNA expression of PBMC and acute cardiac allograft rejections and evaluate its role for prediction of early acute cardiac rejections in rats. Methods Three Groups of rats underwent non-working heterotopic cardiac transplantations: the donor and receptor of group A were both SD rats, untreated after transplant and served as control group. Group B and C underwent Wistar to SD transplantation, group B was untreated and group C was treated with Cyclosporine A(5mg/Kg/d i. p. ×14d). The rats of three groups were killed at day 1,3,5 and 7 after transplant, peripheral blood and cardiac allografts were harvested. Each group 8 rats were left to observe survival time of cardiac allografts. CXCR3 mRNA was detected by real-time PCR. Cardiac allograft specimens were assessed in immunohistochemically and pathologically. Results Survival time in group A was longer than 100d and average survival time in group B and C was (7.40±1.14)d and(24.00 ± 2.35) d. Compared with group B, average survival time in group C was obviously prolonged (P<0.01) . The average survival time in group A was much longer than group B and C (P<0.01) . There was no rejection in group A and CXCR3 mRNA of both cardiac allografts and PBMC were weakly expressed. There was a positive correlation between the dynamic changes of CXCR3 mRNA of both cardiac allografts and PBMC and the process of acute rejections in group B. The peak of CXCR3 mRNA expression of PBMC (4.30 ±0.82) in group B appeared in day 5 after transplant, while the peak expression of PBMC was (1.50±0.76) in group C which appeared in day 7 after transplant, significantly lower than group B (P<0.01) . The peak of CXCR3 mRNA expression of cardiac allografts (2. 30±0. 72) in group B appeared in day 5 after transplant, while the peak expression of cardiac allografts was (1.21±0.77) in group C which appeared in day 7 after transplant, also significantly lower than group B (p<0.01) . CXCR3 waspositive on the surface of infiltrating T lymphocytes in the cardiac allografts and its ligand CXCL10/IP-10 was positive in the epithelial cells and myocardial interstitial tissues. Conclusions CXCR3 mRNA expression of cardiac allografts and PBMC were correlated with process of acute rejections, it could be a new target in the diagnosis or therapy of acute cardiac rejections.Part III Mechanism of chemokine receptor CXCR3 involving in acute cardiac allograft rejections in ratsObjective To evaluate the dynamic changes of cytokines IL-10,IL-2, IFN-γ,and TNF-α and ratio of CD4+/CD8+ T lymphocytes in peripheral blood and explore chemokine receptor CXCR3 involving in acute cardiac allograft rejections in rats. Methods Three Groups of rats underwent heterotopic cardiac transplantations: the donor and receptor of group A were both SD rats, untreated after transplant and served as control group. Group B and C underwent Wistar to SD transplantation, group B was untreated and group C was treated with Cyclosporine A(5mg/Kg/d i. p. ×14d). The rats of three groups were killed at day 1, 3, 5 and7 after transplant, peripheral blood and cardiac allografts were harvested. Each group 8 rats were left to observe survival time of cardiac allografts. Cytokines IL-10, IL-2, IFN-γ and TNF-α in peripheral blood serum were measured by streptavidin biotin complex ELISA. CD3+, CD4+ and CD8+ T lymphocytes were counted by flow cytometry. Results The CD3+ lymphocytes and the ratio of CD4+/CD8+ lymphocytes were not enhanced in group A compared with normal healthy SD rats. While they were increased with the progress of acute rejections in group B and the peak level 70.17 %±4.04% and 2.38±0.35 shown at day 5 after operation. After given CsA treatment, the level of CD3+ T lymphocytes and the ratio of CD4+/CD8+ lymphocytes were both decreased compared with group B and the level ofCD3+ T lymphocytes and CD4+/CD8+ ratio was 54. 67%±3.95% and 1.46 ±0.19 (P<0.01). The level of cytokines such as IL-10, II-2, IFN-γ and TNF-α in group A was same as normal healthy SD rats. The level of IL-10 was temporarily increased in group B and peaked at (61.41±2.29) pg/mL in day 5 and then decreased in day 7. But in group C the level of IL-10 was enhanced slowly and seemed as continuously. The other three cytokines were also related with the progress of cardiac allograft rejection in group B and the level were all decreased in group C. Conclusions The chemokines and cytokines were both involved in acute cardiac allograft rejections. In cardiac allograft rejections cytokines IL-2.IFN-γ and TNF-α in peripheral blood serum were increased and chemokine receptor CXCR3 expression was increased so that ThO cells were differentiated to be Th1 cells. Thl/Th2 balance turned to Th1 side and the ratio of CD4+/CD8+ was increased. Given cyclosporine A therapy, cytokines IL-2, IFN-γ and TNF-α in peripheral blood serum were decreased and IL-10 was increased, the balance turned to Th2 side and the ratio of CD4+/CD8+ was decreased, helpful to prolong cardiac allograft survival. Combined with CXCR3 mRNA of PBMC, those factors were helpful to evaluate the immunological status of the recipients and for prediction of early acute cardiac rejections in rats.