Initial Study On Renal Allograft Tolerance Induced By Donor Apoptotic Cells In Rhesus Monkey

BackgroundProgress made over the last several decades in the development ofimmunosupprcssivc drugs and regimens has made organ transplantation become anacccptcd therapeutic modality for end-stage rcnal, liver, and heart disease andsignificantly reduced the incidence of acutc rejection and rcsulted in ever-increasingrates of short and long term organ allograft survival. Unfortunately, many problemsoccurred after new effective immunosupprcssivc drugs were used. First,immunosuppressant must be continued for life and nonspecifically suppresses theimmune system. Secondly, none of the numerous immunosuppressive agents that arecurrently available can abolish completely the risk of acute and especially chronicrejection. Thirdly, their usc bears the risk of malignancies and infections. Finally, alltransplant patients arc at increased risk for cardiovascular complications related tohypertension and drug-induced elevation to serum levels of cholesterol andtriglyceridcs. Cell therapy has made significant advances in recent times. Especially,intravenous infusion of apoptotic cells may subsequently induce tolerance to solidallograft. Some patients tolerate the graft indefinitely with only minimal immunosuppressant, suggesting that immune nonresponsiveness can be achieved inclinical practice.Survival duration of the heart transplant in mice has been prolonged obviouslyafter immune tolerance was induced by intravenous infusion apoptotic cells in ourresearch center. It has been repeated and proved by many laboratories. It has beenproved that the tolerance can be induced by intravenous transfusion apoptotic cells inmurine models. It suggested that the tolerance may be induced as the same method inclinical practice, and then graft survival duration will be prolonged andimmunosuppressive agents will be reduced?In order to answer this problem, we choose primate animals to study thetolerance induced by donor apoptotic cells. However, the following problems must beresolved before successful tolerance is induced by donor apoptotic cells in primateanimals: (1) To determine human-like blood group of rhesus monkey; (2) Theeffective method of inducing PBMC apoptosis must be determined;(3) To erect a newmodel which resembles clinical conditions and is of benefit of the tolerance study;Rhesus monkeys will die because of acute rejection or acute tubular necrosis7-10days after two kidney is resected in traditional surgery. Thus the upper problemsand initial study of effect of apoptotic cells on renal allograft survive and rejection innon human primate animals have been focused in this paper.ObjectiveOur study is to research the method to exanimate human-like blood group ofrhesus monkey; to create a rhesus monkey kidney transplant model; to research themethod of inducing PBMC apoptosis; to induce immune tolerance by donor apoptoticcells in kidney transplant rhesus monkey.Methods1 After 43 rhesus monkeys were injected chloramine alkone(2mg/kg,IM), venous blood 5ml was extracted respectively, using heparin to ant coagulate. Rhesus salivawas collected respectively. Human-like blood group of rhesus were tested using threemethods: monoclonal antibodies against A and B; agglutination inhibition test (testingsaliva); anti-A and anti-B agglutinins test.2 To establish the new model of rhesus kidney transplantation: 4 rhesusmonkeys were divided to two pairs random. Each rhesus contributed the left kidney,retaining the right kidney, and then simultaneously accepted:another rhesus leftkidney transplant. Donor nephrectomy: The abdomen of a rhesus was opened with amidline incision and the left kidney was exposed. Heparin(100u/kg,ⅳ) was givenbefore the vascular clips were placed. The cut kidney was flushed with 50ml HCApreservation solution at 4℃. Recipient operation: The abdomen was opened via along midline incision. The distal aorta and inferior vena were dissected free. Vascularclamps were placed after heparin (100u/kg,ⅳ) was given intravenously. The vascularanastomosis of renal artery end to aorta side and renal vein end to inferior vena sidewere done using 7-0 Prolene and microsurgical appliance. The ureter of transplantkidney is anastomozed to the bladder inserted a double pig tail as a temporary stent.Post operation recipients were given aspirin(100mg) for three days. Subjectclinical immunosuppressive protocol: Methylprednisolone+cyclophosphamide+rapamycin. Methylprednisolone 120rag is injected on the operative day and the nexttwo days. Cyclophosphamide 20rag is injected once every two days. Rapamycin isgiven 0.4mg, once a day. Antibiotic: Gentamycin Sulfate (10000u/kg/d).3 Rhesus monkey PBMC are induced apoptosis by three methods: X-rayirradiation, Co60γirradiation and ultraviolet-C(UVC) irradiation. The apoptotic rateof UVC irradiation is studied in different time, distance, FBS concentration, andheight of culture medium. The best conditions of inducing PBMC apoptosis areconfirmed. 4 The tolerance is induced by apoptotic lymphocyte injected to recipients prior torenal allograft. Six rhesus monkeys were divided into two groups, apoptotic groupand control group. Donor apoptotic cells in 20mlPBS were injected to recipient inapoptotic group. The rhesus monkeys of control group were injected 20mlPBS asempty contrast. Each rhesus contributed the left kidney, retaining the right kidney,and then simultaneously accepted another rhesus left kidney transplant.(1) A great deal of apoptotic cells preparation: PBMC were isolated fromperipheral blood 50ml extracted from donor rhesus using lymphocytes isolatedsolution. PBMC were irradiated for 60 minutes by UVC at the followingconditions:10%FBS medium 4ml in the dish of diameter 6cm, irradiating distance20cm and irradiating duration 60minutes.Apoptotic cells were transfused into arecipient monkey as soon as apoptotic cells were prepared. Apoptotic rate was testedby flow cytometry. Seven days later, allograft kidney transplantation was undergone.(2) The immunosuppressive protocol was the same as the model protocol.(3) After kidney transplantation, dynamic monitoring of blood routine, rapamycinconcentration, CD4+CD25+T regulatory cells; Observation the survival duration oftransplanted kidney; transplanted kidney biopsy; transplanted kidney ultrasound andCT scanning.Results1 All of blood group testing are negative using normal monoclonal antibodymethod and agglutination inhibition test (testing saliva).So, normal monoclonalantibody method and agglutination inhibition test can not detect the blood group ofrhesus. Anti-A and anti-B agglutinins testing method can detect the blood group ofrhesus. There are human-likeA 3 cases (7%), B 25cases (58.14%), AB 15 cases(34.88%). There is not a case of human-like O being found.2 The average duration of left nephrectomy and kidney transplantation is no more than two hours. After blood reperfusion, transplanted kidneys recovered function soonand began to work. There is much urine during operation. The figure of ultrasounddetecting transplanted kidney showed abundant blood supply. The survival durationof transplanted kidney is 5, 5, 6, 7days, respectively. Acute rejection occurred in alltransplanted kidney. There is not complication of hematoma, infection, urinary fistulaor ureteral obstruction. However, one rhesus lost transplanted kidney because of renalartery embolization 5 days after surgery. Another rhesus died of intestinal obstruction7 days after surgery. Others survive well after graft kidney nephrectomy. Comparedwith normal kidney, graft kidney became swelling obviously. There are manyinflammatory cells infiltration and focal necrosis in slices of graft biopsy.3 UVC irradiation inducing PBMC apoptosis is superior to X-ray irradiation andCo60γirradiation. So we made a more depth study on UVC irradiation inducingPBMC apoptosis. Major factors effecting PBMC apoptotic rate were found as follows:irradiation distance, irradiation duration, volume of culture medium in dish ofdiameter 6cm, FBS concentration of medium. Apoptotic rate of UVC irradiation at20cm is significantly higher than that at 30cm(P＜0.001). Apoptotic rate of UVCirradiation 60 minutes is significantly higher than that of 40 and 50 minutes(P＜0.01),Necrosis rate of UVC irradiation 60 minutes is not more than 10%. Apoptotic rate ofD6cm dish containing 4ml culture medium is significantly higher than that of D6cmdish containing 5ml culture medium(P＜0.01), but necrosis rate of D6cm dishcontaining 4ml culture medium is significantly less than that of D6cm dish containing3ml culture medium(P＜0.01). Apoptotic rate of using 10%FBS culture medium issignificantly higher than that of using 15% or 20%FBS culture medium (P＜0.005).4 Donor apoptotic cells transfusion inducing allograft renal transplantationtolerance:4 (1) The number of PBMC isolated from 50ml peripheral blood of rhesus is 8×10~7-8.5×10~7, the total gotten cells number is 7.2×10~7-7.5×10~7 after UVCirradiation~ The apoptotic rate of it is about 40-60%, and the necrosis rate is about5-10%.4 (2) Transplanted kidney survival duration: control group: 5 (allograft lost becauseof renal artery embolization), 6, 14 days, apoptotic group: 7(died of intestinalobstruction 7 days after surgery), 19, 30 days.4 (3) Apoptotic group: transplanted kidney ultrasound result of monkey 99523showed less blood flow 6days after surgery, size of kidney 53×25×35mm, acuterejection confirmed by allograft biopsy. The monkey died of intestinal obstruction 7days after surgery. The blood supply of the transplanted kidney in monkey 97387,00209 is abundant, the size and structure of transplanted kidney were normal, sizeswere 41×28×32mm, 52×24×38mm respectively. There were a few of lymphocytes inslices of allograft biopsy.Control group: The ultrasound result of monkey 99581 showed transplanted kidneyswell obviously and size 55×25×40mm five days after surgery. Pathologicalexamination of graft kidney showed many inflammatory cells infiltration and focalnecrosis in slices. The allograft size of monkey 98447 is 43×18.3×37mm. There wasno blood flow confirmed by CT scanning in the graft kidney. The graft kidneywithout renal artery pulsatility was black during graft nephrectomy. It indicates thatthere is renal artery embolization. The slice of the graft showed ischemia necrosis.Transplanted kidney ultrasound result of monkey 00473 showed abundant blood flow7days after surgery, size 53x41x39mm, resistant index 0.54, acute rejectionconfirmed by allograft biopsy. Abundant blood flow was confirmed by ultrasoundand there were many lymphocyte cells infiltration in biopsy slice.4 (4) The monitoring of CD4+CD25+T cells: Before surgery, the proportion ofCD4+CD25+T cells in blood of monkey 97387 and 00209 is 3.49%,3.19%, respectively. The rate increased to 11.79% and 13.58%, respectively, being about 3-4times as before surgery. In control group, compared with that before surgery, the rateof CD4+CD25+T cells of monkey 00473 is stable after surgery.Conclusions1 Compared with monoclonal antibodies against A and B method, agglutinationinhibition test (testing saliva) method and anti-A and anti-B agglutinins test methodbeing used to test rhesus blood group, results indicated that anti-A and anti-Bagglutinins test method is more sensitive and effective. The ABO phenotypefrequencies of rhesus monkey were as follows: A-7%, B-58.14%, AB-34.88%. Thereis not a case of human-like O being found. Monoclonal antibodies against A and Bmethod, agglutination inhibition test (testing saliva) method can not detect bloodgroup of rhesus monkey.2 Compared with old model, the new model keeping a original kidney left hasmany advantages as follows: (1)Keeping a original kidney left is more consistent withclinical conditions, because in clinics, all of original kidneys is not resected. So it isof not only benefit to reducing trauma, but also benefit to recovery after surgery. (2)Itis of not only benefit to maintaining the stability of the body environment, but alsobenefit to reducing the difficult degree of peri-operative care.(3)The recipient willsurvive long-term instead of death after rejection kidney was resected. It not onlysimulates the clinical condition, but also reduces the unnecessary death of recipientmonkey. It is accordance with animal ethics.(4)It is of benefit to long-term followingobservation of immunology and is a good model of researching tolerance.3 UVC irradiation inducing PBMC apoptosis is more sensitive and effective thanX-ray irradiation and Co60γirradiation. The best conditions of UVC irradiationinducing PBMC apoptosis are using 10%FBS culture medium, D6cm dish containing4ml culture medium, irradiation distance 20cm, irradiation duration 60minutes. 4 The graft survival duration in apoptotic group is longer than that in control group,which indicates that there is partial tolerance, however, acute rejection occurred inboth apoptotic group and control group, which indicates that apoptotic cells do notinduce complete tolerance.5 Transfusion apoptotic cells can induce CD4+CD25+T regulatory cell to increasewhich may be a cause of graft survival prolonging.