| The immunosuppression research has been developed greatly and improved in clinical application nowadays, but acute rejection is still a major cause for renal allograft loss. Therefore, early diagnosis, treatment and immune tolerance inducing are significant to the restoration of allograft function after renal transplantation. With the development of molecular biology, the role of cytokine in rejection is now paid much more attention. Now it is hot to study the cytokine molecule gene expression and intervene to them during acute rejection.It is fully explained that IL-2/IL-2R plays an important role during acute renal allograft rejection. In both human and animal experiments, anti-IL-2R antibody remarkably reduces the acute rejection rate, but there is still a portion of subjects that developed acute rejection. The facts implied there is other pathways which independent of IL-2/IL-2R system participated in acute rejection. However, it is rare research about other cytokine activated pathways. IL-18 is a cytokine that secreted by macrophage and dendritic cell (DC) et al. As an IFN-gamma inducing factor, IL-18 has significant role in immunity regulation and been mainly involved in Th1. Recently, researches have been found that IL-18 has been involved in Th2 inducing at different immunological microenvironment. As we known that Th1 cells are involved in immunological rejection and Th2 cells are involved in immunological tolerance. As a regulatory factor in both in Th1 and Th2 cells, IL-18 is significant in organ transplantation. Recently, researches show high expression of IL-18 has been found in early acute rejection of liver, renal, heart and borrow transplantation. These results indicate that IL-18 is involved in acute rejection proceed. Although perforin and IL-18 detection have been reported in early acute rejection of renal transplantation, it is rare reported about the role of IL-18 in pathogenesis of acute rejection in kidney transplantation, the interaction role with other cytokines and damage mechanism of perforin and inducible nitric oxide synthetase (iNOS) in grafts, especially about anti-IL-18 antibody to induce immunity tolerance has no reported, so it is still to research about this work.New ideas in our research as follows: (1) IL-18 may potentially drive the process of allograft rejection via INF-γelevation and induce largely IFN-γsecretion by synergy with IL-12 in acute rejection. Through suppression of IL-10, IL-18 acts as an immunomodulator on cytokine production level influencing the balance between Th1 and Th2 cytokines toward Th1 in acute rejection; (2) Some reports that IL-18 maybe induce iNOS to perform the destructive effect to kidney graft tissue abroad. However, it is no report that IL-18 and perforin are involved in the destructive effect to graft tissue and cells during acute rejection in rat kidney transplantation. (3) It is no report that Anti-IL-18 antibody may induce immunotolerance by intervening IL-18 in rat kidney transplant.In this study, we investigated the role of IL-18 in renal allograft rejection and the expression of IL-18, IFN-γ, IL-12, IL-10, perforin and iNOS and the intervening role of anti-IL-18 antibody during acute rejection basing on model of rat kidney transplantation. The thesis is divided into four parts:Part I Establishment of acute rejection model of rat kidney transplantationObjective: To establish experimental acute rejection model of rat kidney transplantation and provide basic investigation of AR in rat kidney transplantation. Methods: In allograft group, male Wistar rats and male SD rats were separately used as donors and recipients. In syngraft group, SD rats served as both donors and recipients. We performed with end to end anastomosis of donor and recipients'renal artery, renal vein respectively. The donor ureter was sutured with ureter bladder-flap to the recipient bladder under microscope. The graft and heart blood specimens were harvested at 3, 5 and 7 days postoperatively. BCr and BUN were examined in serum. The grafts were examined histologically after staining with hematoxylin. According to the Banff 97 classification, the rejection score was classified semi-quantitatively into grades by Banff classification.Results: The post-operation complication rates were lower in both groups. At day 3, 5, 7 postoperatively, BCr and BUN were significantly increased in allograft groups when compared to syngraft group. Semi-quantitative scores at day 3, 5 and 7 after operation were (1. 52±0. 46), (3. 67±1. 14), (5.61±0. 35) in allograft groups and (0. 61±0. 49), (0.65±0. 52), (0. 71±0. 38) in syngraft group, respectively. At day 3, 5, 7 postoperatively, the scores of allograft groups were significantly higher than syngraft groups. In allograft groups, the scores at day 5 and 7 postoperatively were significantly higher than those on day 3 (P < 0.05), and the scores at day 7 postoperatively were significantly higher than those at 5 days (P < 0.05). In syngraft groups, the scores at day 3, 5 and 7 postoperatively were not significantly different (P>0.05).Conclusions: Using this method can reduce the effect of recipient circulation system and avoid the stricture of ureter. Because of their good tissue compatibility, SD rats can serve as both donors and recipients, the rejection is mild. The rejection of Wistar-SD rat kidney transplantation is rapid and severe, which can serve as good animal model of acute rejection. The model could be repeated stably and be suitable for investigation of the immunity mechanism of organ transplantation.Part II IL-18 induce IFN-γto the result of acute rejection in rat kidney transplantationObjective: To compare serum levels of IL-18, IL-12, IFN-γand IL-10 and their mRNA expression on allografts after rat kidney transplantation and to understand the expression of IL-18 in macrophage, and then to investigate the effect of IL-18 to the Th1/Th2 balance and synergistic effect of IL-18 and IL-12 in acute rejection.Methods: The model of rat kidney transplantation was established. The rats were divided to three groups: Syngraft group (Syn) (SD-SD) (n=24); Acute rejection group (AR)(Wistar-SD)(n=24); CsA group (Wistar-SD)(n=24). Six recipients per group were sacrifice in POD 1, 3, 5 and 7, recpients'blood, kidney grafts and spleen were collected. Serum levels of IL-18, IL-12, IFN-γand IL-10 were detected by ELISA and the expression of IL-18, IL-12, IFN-γand IL-10 were detected by Real time PCR. The expression of IL-18 in macrophage in POD 7 was detected by flow cytometer. Results: Compared to Syn group and CsA group, the serum levels of IL-18, IL-12, IFN-γwere significantly elevated (P<0.01) in POD 1, 3, 5 and 7, the expression of IL-18, IL-12, IFN-γmRNA were also significantly increased (P<0.01) after transplantation. In AR group, the serum levels of IL-18, IL-12 and IFN-γin day 5 and 7 were significantly higher than in day 3 (P<0.01). The expression of IL-18, IL-12 and IFN-γmRNA in POD 5 and 7 were significantly higher than in day 3 (P<0.01). Contrary, when compared to Syn group, the serum levels of IL-10 and the expression of IL-10 mRNA in kidney grafts of AR group were rapidly decreased in POD 3, 5 and 7 (P<0.01). Compared to Syn group, the serum levels of IL-10 and the expression of IL-10 mRNA in kidney grafts of CsA group were significantly increased in POD 3, 5 and 7 (P<0.01). The positive rate of expression of IL-18 in macrophage was max in AR group.Conclusion: Macrophage maybe the main source of IL-18 in acute rejection of rat kidney transplantation. IL-18 may potentially drive the process of allograft rejection via INF-γelevation and induce largely IFN-γsecretion by synergy with IL-12 in acute rejection. Through suppression of IL-10, IL-18 acts as an immunomodulator on cytokine production level influencing the balance between Th1 and Th2 cytokines toward Th1 in acute rejection.Part III IL-18 induce the lethal effect of iNOS and perforin in acute rejection of rat kidney transplantationObjective: To compare serum levels of IL-18, IFN-γ, iNOS, perforin and their mRNA expression on allografts after rat kidney transplantation, expression of perforin in CD8+ T lymphocyte and to understand the effect of these factor to destructive mechanism of grafts in rat kidney transplantation.Methods: The model of rat kidney transplantation was established. The rats were divided to three groups: Syngraft group (Syn) (SD-SD) (n=24); Acute rejection group (AR)(Wistar-SD)(n=24); CsA group (Wistar-SD)(n=24). Six recipients per group were sacrifice in POD 1, 3, 5 and 7, recpients'blood, kidney grafts and spleen were collected. Serum levels of IL-18, IFN-γ, NO and perforin were detected by ELISA and the expression of IL-18, IFN-γ, iNOS and perforin were detected by Real time PCR. The expression of perforin in CD8+ T lymphocyte was detected by flow cytometer. Part allografts were taken to examine by pathology.Results: Pathological changes were significantly in AR group in day 5 and 7 post-operation. Compared to Syn group and CsA group, the serum levels of IL-18, IFN-γand perforin were significantly elevated (P<0.01) in POD 1, 3, 5 and 7, the expression of IL-18, IFN-γ, iNOS and perforin mRNA in grafts were also significantly increased (P<0.01) after transplantation. Compaered to Syn and CsA group in POD 5 and 7, serum NO levels were significantly elevated in AR group (P<0.01). The expression of iNOS mRNA in grafts was significantly increased when compared to Syn and CsA group in POD 3, 5 and 7(P<0.01). The expression of perforin in AR group was max in CD8+ T cells by flow cytometer.Conclusion: IL-18 maybe induce iNOS to perform the destructive effect to graft tissue and cells by secretion IFN-γin macrophage. IL-18 maybe up-regulate the expression of perforin to act the destructive effect to graft tissue and cells through CD8+ T lymphocyte.Part IV Anti-IL-18 antibody induces immunotolerance in rat kidney transplantationObjective: To compare serum IL-18, IL-10, IFN-γ, iNOS and perforin mRNA expression in various groups after rat kidney transplantation with anti-IL-18 antibody treatment and non- anti-IL-18 antibody treatment and the survival time of the recipients of all groups to understand the interruption of anti-IL-18 antibody in acute rejection and then induce to immunotolerance.Methods: The model of rat kidney transplantation was established. The rats were divided to five groups: Syngrafts group (Syn) (SD-SD) (n=12); Acute rejection group (AR)(Wistar-SD)(n=12); CsA group (Wistar-SD)(n=12); anti-IL-18 antibody group (Wistar-SD)(n=12); CsA+ anti-IL-18 antibody group (Wistar-SD)(n=12). The average survival time was observed in groups. The recipients'peripheral blood was collected by caudal vein in POD 5, 7, 14, 20. Serum IL-18, IL-10, IFN-γ, iNOS and perforin mRNA expression were detected by Real time PCR in POD 5 and 7.Results: The average survival time of CsA group, anti-IL-18 group, CsA + anti-IL-18 group were longer than AR group (P<0.01), and the average survival time of CsA + anti-IL-18 antibody group was longer than CsA group and anti-IL-18 antibody group (P<0.01). Serum IL-18, IFN-γ, iNOS and perforin mRNA expression in POD 5 and 7 were highest in AR group among all groups (P<0.01). Serum IL-18, IFN-γ, iNOS and perforin mRNA expression were lower in CsA group, anti-IL-18 group, CsA + anti-IL-18 group when compared to AR groups (P<0.01), and decreased significantly in CsA + anti-IL-18 group. Contrary, serum IL-10 mRNA expression in POD 5 and 7 was highest in CsA + anti-IL-18 group whereas lowest in AR group among all groups (P<0.05). IL-10 mRNA expression in POD 5 and 7 was increased in both CsA group and anti-IL-18 group, but it was no difference (P>0.05).Conclusion: Anti-IL-18 antibody may induce immunotolerance by intervening IL-18 in rat kidney transplantation. The mechanism maybe up-regulate IL-10 mRNA expression by down-regulate IL-18 mRNA expression and decrease IFN-γ, and then influence the balance of Th1/Th2 toward Th2 to induce immunotolerance. Anti-IL-18 antibody maybe reduce the destructive effect in graft during acute rejection in rat kidney transplantation by down-regulating iNOS and perforin mRNA expression.
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