The Study On Immunological Tolerance Of Rat Islets Transplantation Induced By Transfusion Of Donor Apoptotic Cells

BackgroundIslets transplantation to re-establish the secretion system of insulin has beenconsidered as a most promising treatment to cure diabetes mellitus. Especially thesuccess of Edmonton Protocol in 2000, has demonstrated the great progress whichhas been made in the territory of islets transplantation, and accordingly exploited anextensive application perspective of islets transplantation to cope with type 1 diabetes.However, transplantation rejection and the potential risk coming with the applicationof immunosuppressive agents are still the obstacles unable to overcome completely.Now it is generally thought that the key to solve the rejection of allografts is to inducethe recipient's immune tolerance to the grafts from the donor. Though there are somany means to induce the tolerance, none of them can achieve the completepermanent specific tolerance. Apoptosis is a kind of physiological or pathologicalphenomenon generally existing in the biosystem, and plays an important role inmaintaining the balance and stabilization of the host's immune state. Apoptotic cellscan secrete lipidic haemostatic factors, and present "eat-me" signals on the surface to attracted the phagocyte to remove themselves; at the same time, antigent-presentingcells (monocytes and macrophages, etc ) can secrete many inhibitive immune factorssuch as TGF-β,PGE2,IL-10 ere, after the phagocytosis. All of these creat amicroenvironment for antigen recognition and induce the related lymphocytes'tolerance to apoptotic antigen rather than the inflammatory response. On thesegrounds, Sun et al proposed to make use of the transfusion of apoptotic cells toinduce the donor's specific tolerance. Our workgroup has demonstrated that apoptoticcells can make an inhibitive effect on the proliferation of T lymphocytes in vitro, andresearchers have reported that transfusion of apoptotic cells can notably prolong thesurvival time of rat allografts after the heart transplantation or liver transplantation.So we plan to induce the allogenic islets transplantation tolerance in diabetic SD ratby pre-transfusion of apoptotic cells, accordingly we observe the survival time ofislets grafts and investigate the kind of T lymphocytes, cytokine and molecularmechanism involving the regulative immune response of apoptotic cells.ObjectiveTo investigate the methods and mechanism of islets transplantation toleranceinduced by transfusion of the apoptotic cells. To provide experimental evidences forestablishing new protocols which can induce islets transplantation tolerance.Methods1. To investigate the effect of X-irradiation from electron linear accelerator onthe rats spleen cell in vitro: the Wistar rat spleen cells obtained by the method ofgrinding were divided into four groups: control (A) and B, C, D group were irradiatedby the absorbed dose of 1.5Gy, 2.0Gy, 3.0Gy respectively. And then incubated in37℃, 5ï¼…CO₂. The early apoptotic cells at 4h, 8h, and 12h were measured by flowcytometry (FCM) with Annexin V-FITC/PI.2. Isolation and purification of rat islets: The pancreatic islets were digested and isolated by perfusing collagenase P via pancreatic duct and purified by Ficoll-400discontinuous density gradients after 1-2d incubation.3. To establish animal model of diabetes mellitus: SD rats were rendered diabeticvia single intraperitoneal injection of streptozotocin at 56mg/kg·weight (continuoustwice plasma glucose＞16.7mmol/L).4. To study the effect of transfusion of donor apoptotic spleen cells on thesurvival of islet grafts: Wistar and SD rats were taken respectively as donor andrecipient. Diabetic SD rats were randomly divided into 4 groups, and recived infusionof Hank's, normal donor cells, apoptotic donor cells and necrotic donor cells(respectively as A, B, C, D group) via dorsal vein of penis, then, 7 days later, all ratsreceived islets (1000IEQ per STZ SD rat) under their left renal capsule respectivelyand the survival time of islet grafts were compared. The insulin lever in serum weremeasured by RIA at 0d, 7d, 14d, after transplantation and at the time of rejection.5. Grafts from recipients were examined for the expression of insulin byimmunohistochemical staining at the time of euglycemic or returning to ahyperglycemic state in C group. Grafts of other groups were examined for theexpression of insulin by immunohistochemical staining only after the rejection.6. To observe the effect of transfusion of donor apoptotic cells on the one-waymixed lymphocyte culture: Diabetic SD rats were randomly rendered infusion ofHank's, normal donor cells, apoptotic donor cells and necrotic donor cells, then, at 0d,7d, 14d after transplantation and at the time of rejection, all recipients' spleen wereresected and the isolated lymphocytes were co-cultured with donor spleenlymphocytes or spleen lymphocytes from the third strain F344/N rats dealed withmitomycin-C. The index of proflieration were detected by MTT method;7. To study the effect of transfusion of donor apoptotic cells on the subset of Tlymphocytes and CD4⁺CD25⁺T lymphocytes of the diabetic SD rats: Diabetic SD rats were randomly rendered infusion of Hank's, normal donor cells, apoptotic donor cellsand necrotic donor cells, then, at 0d, 7d, 14d after transplantation and at the time ofrejection, all recipients' spleen tissue were resected and made into the isolatedlymphocytes, then, the ratio of the CD8⁺,CD4⁺,CD4⁺CD25⁺/CD4⁺T cells weredetected by FCM.8. To study the effect of transfusion of donor apoptotic cells on the cytokines ofthe diabetic SD rats: Diabetic SD rats were randomly divided into groups accordingto the above-mentioned method, then, at 0d, 7d, 14d after transplantation and at thetime of rejection, all recipients' spleen tissue and peripheral blood were collected, thecontents of IL-2, IFN-γ, IL-4, IL-10 are measured by Luminex 100 and TGF-β1 byELISA respectively.Results1. The Wistar rat splenocytes were induced by X-irradiated with acceleratorlinear: the ratio of apoptotic cells in 1.5Gy group increased at 4h, and showed atime-effect relaetionship with incubation time. The 2.0Gy group has a similartendency with 1.5Gy group, and reached its apoptosis peak at 8h with a percentage of(61.17±3.70)ï¼…(P＜0.050), but began to decrease at 12h. However, the 3.0Gygroup had a high necrosis percentage compainied with a high apoptosis percentage.2. After the purification by discontinuous gradient centrifugation, about(1063.49±84.74) IEQ per rat pancreas were acquired, with an average purity of (70.51±6.20)ï¼…, and the purified islets were responded to high concentration glucosestimulation with 2.72 times increase of insulin secretion compared with lowconcentration glucose stimulation.3. Pharmacological diabetes on SD rats were induced successfully by singleintraperitoneal STZ injection (56mg/kg·weight) with an average model-performedratio of 94ï¼…. 4. The survival time of islet grafts of diabetic SD rats who were pretreated withdonor apoptotic cells were dramatically prolonged, compared with who are pretreatedwith Hank's, normal donor cells and necrotic donor cells (P＜0.050), with a MST of(31.00±6.41) days and the longest survival time of 42d. the normal donor cellsgroup also has a prolonged survival time with a MST of (12.00±2.97) days(p＜0.050). The A, D groups had no prolonged effect with a MST of (6.00±1.67),(6.00±1.10) days respectively.5. The insulin level in serum of the diabetic SD rats had a notable increase at 7d,14d, after transplantation in C group, with a content of (19.26±4.50) mIU/ml,(20.70±6.33) mIU/ml (P＜0.050), and then decreases after the rejection, the insulinlevel in serum of B group also increases notably at 7 days after transplantation(P＜0.050).The A, D groups have no significant changes.6. Grafts from groups who are pretreated with Hank's, normal donor cells andnecrotic donor cells, harvested at the time of return to the diabetic state, revealed thecomplete absence of insulin-positive cells. Compared with control grafts, the graftsfrom STZ SD rats being pretreated with donor apoptotic cells, harvested fromeuglycemic animals, showed intact islets immunohistochemically stained for insulin.In the contrast, the grafts harvested from the same group at the time return tohyperglycemia, showed no staining for islets.7. After the one-way mixed lymphocyte culture, the proliferative response oflymphocytes derived from diabetic SD rats who received donor apoptotic cells for 7days to donor lymphocytes were apparently decreased compared with who receivedHank's, normal donor cells and necrotic donor cells (P＜0.050), the suppressing effectexisted until at 14 days after transplantation and disappeares after the rejection.Compared with the response to the lymphocytes from the third F344/N rats, thedifference is also significant (P＜0.050)including the compariation between different time after transplantation.8. The ratio of CD8⁺ and CD4⁺ T lymphocytes both in peripheral blood andspleen tissue all had a significant increase in the four groups after the transplantation(P＜0.050), but the difference between the groups wasn't significant (P＞0.050).9. The ratio of CD4⁺CD25⁺ T lymphocytes both in peripheral blood and spleentissue of diabetic SD rats in C group at 7 days after the infusion of apoptotic cellswas (11.49±1.29)ï¼…,(12.44±1.63)ï¼…and it was notably different compared withthe other group (P＜0.050). This ratio kept high even at 7, 14 days aftertransplantation and began to fall after the rejection.10. The content of IL-2 both in serum and spleen tissue didn't have a significantdifference between groups before transplantation, but began to advance aftertransplantation in all groups,the content of IL-2 at 7d, 14d after transplantation wassignificantly lower than other groups (P＜0.050). But increased after rejection.11. The content of IFN-γboth in serum and spleen tissue didn't have asignificant difference between groups before transplantation, but advanced aftertransplantation in all groups, the content of IFN-γ, at 7d, 14d after transplantation wassignificantly lower than other groups (P＜0.050). But increased after rejection.12. The content of IL-4 both in serum and spleen tissue hadn't a significantdifference between groups before transplantation and there was also no differencebetween groups at 7d, 14d after transplantation (P＞0.050).13. The content of IL-10 both in serum and spleen tissue of diabetic SD rats in Cgroup at 7d after the infusion of apoptotic cells had a significant increase comparedwith other three groups (P＜0.050), and this difference existed even at 7d, 14d aftertransplantation, it decreased after rejection. There were no such changes in A, B, Dgroups.14. The content of TGF-β1 in serum of diabetic SD rats in C group at 7d after the infusion of apoptotic cells had a significant increase compared with other threegroups(P＜0.050), and this difference existed even at 7d, 14d after transplantation, itdecreased after rejection. There were no such changes in A, B, D groups.Conclusion1. Irradiation by X rays from electron linear accelerator is a convenient, safe andeffective way to induce apoptosis of Rat spleen cells.2. A large quantity of islets which still have good functions can be acquired byisolation by intraductal collagenase P and purification by Ficoll-400 discontinuousdensity gradients after culturing 1-2d.3. The transfusion of donor apoptotic cells can prolong the survival time of isletgrafts and suppress transplantation rejection; therefore, the utility of donor apoptoticcells will be a feasible pathway to induce islets transplantation tolerance.4. We have demonstrated that the transfusion of donor apoptotic cells can havean inhibitive effect on the proliferation response of the recipient's lymphocytes to thestimulation of donor's lymphocytes by MLR.5. The ratio of CD4⁺CD25⁺ T lymphocytes both in peripheral blood and spleentissue of diabetic SD rats after islets transplantation can be advanced notably by theinfusion of apoptotic cells from the donor rats.6. The contents of IL-10 and TGF-β1 of diabetic SD rats with isletstransplantation can be advanced apparently after the infusion of donor apoptotic cells.Thus, the contents of IL-2 and IFN-γshow a lower level compared with the othergroups. There is no significant changes in the content of IL-4. All these wouldindicate that the transfusion of apoptotic cells from the donor rats might produce amarked effect on accommodating the balance of Th1/Th2,Th3 and retrievingimmunological deviation. Meanwhile the participation of Tr, Tr1, especially theCD4⁺CD25⁺ T lymphocytes might be one more important regulatory mechanism.