| ObjectImmunoglobulin-like transcripts (ILTs) belong to a kind of receptor family discovered recently, which are differentially expressed on myeloid and lymphoid cell subsets. Most of them play an important role to convert immune active T and NK cells into Treg and tolerant NK cells respectively and so on by interacting with their ligands. As a sort of immune suppression, cyclosporin A (CsA) is frequently used to prevent graft-versus-host disease and treat autoimmune disease. Previous studies have confirmed that inhibitory receptors ILT3and ILT4induced immune tolerance on the exogenous organ, and the rate of transplantation success could be improved by upregulating them. There were some researches about the regulation of CsA on human immunologic reaction, but the effect of CsA on the ILT3and ILT4expression on lymphocytes was less reported. Using CsA with different concentration to process nature killer (NKL) cells in vitro experiment, we detected the expression of inhibitory receptors ILT3and ILT4on NKL cells, cytotoxicity against tumors and proliferation of NKL cells as well. At the same time, ILT3and ILT4mRNA in peripheral blood lymphocytes of renal transplant recipients before and after operation were serially measured. Comparing the difference of ILT3/4expression between stable patients and those suffering from rejection, and relationship between ILT3/4expression and rejection after renal transplantation was analyzed in order to explore the application value of ILT3/4on the diagnosis of rejection. The research may have important implications to investigate the mechanism of CsA and provide references for rational use of immunosuppressant agents. It is also significant to make the early diagnosis of rejection after kidney transplantation.Methods一. Experiment in vitro:1. NKL cell line was dealed with CsA(5,10or15mg/L) for12h,24h and36h respectively, then real-time quantitative PCR and flow cytometry were used to detect the alteration of ILT3and ILT4expression from mRNA and protein level before and after medication respectively;2. The expression of HLA-G in BGC-823and JEG-3cells lines were measured at the same time. NKL cells treated with or without CsA(15mg/L) for36h were co-incubated with BGC-823or JEG-3cells, and then the cytolytic activity of NKL cells against BGC-823or JEG-3cells was determined with MTT assay;3. Meanwhile, effect of CsA with distinct concentration on proliferation of NKL cells was also measured with MTT Method.二. Clinical trial:experimental grouping, including the experimental group, healthy control group and the preoperative control group. For recipients undergoing renal transplantation from the Organ Transplant Center of Qilu Hospital, peripheral blood samples were obtained, which were divided into rejection group and stable group after treatment based on postoperative recovery. Randomly selected healthy workers of Shandong University Qilu Hospital were as healthy control group. Real-time fluorescent quantitative PCR were used to detect ILT3and ILT4mRNA expression changes of peripheral blood lymphocytes (including before and the D1, W1, W3, M1, M2and M3after operation; before and D1, D3, D7after rejection). Meanwhile, detected the concentration of CsA in blood and creatinine、urea nitrogen in serum and so on, and analysed the value of ILT3and ILT4in diagnosing rejection. Informed consent was obtained from each research individual. Approval to conduct this study was granted by the Medical Ethics Committee of Qilu Hospital. Results1. CsA up-regulated the expression of ILT3/4on NKL cells, and the degrees of up-regulation were related to reaction time and drug concentration. Both RNA and protein levels of ILT3and ILT4were raised clearly on NKL cells dealed with CsA (15mg/L) for36h;2. The ability of NKL cells coped with CsA (15mg/L) for36h to kill the same target cells decreased markedly. And cytotoxicity of NKL cells treated without or with CsA (15mg/L) on BGC-823cells were higher than that on JEG-3cells;3. CsA with different concentration inhibited the proliferation of NKL cells in varying degrees, and the proliferation inhibition rates were enhanced with increasing concentration during the same period. Furthermore, as time went by the growth inhibition rates of NKL cells by CsA with same concentration were gradually strengthened;4. ILT3/4mRNA expression level varied among different individuals of experimental group and control group, and there was a large respective divergence. Overall, the difference of ILT3/4mRNA between pre operation group and healthy control group had no sense in statistics;5. Stable group after operation:The level of ILT3and ILT4mRNA in peripheral blood lymphocytes began to rise on the seventh day after operation and consistently elevated thereafter to the highest level within the first month. It declined gradually afterward and kept at a higher level than pre-transplant operation (1.69to1.73and2.06to2.12times of preoperative level respectively).6. Rejection group after operation:There was no significant difference of ILT3/4mRNA expression level in the same period between stable group after operation and rejection group postoperation before rejection; At the episodes of rejection, the level of ILT3/4expression decreased significantly, which was lower than that of preoperative level and stable level before rejection. Subsequently, ILT3/4expression increased in different degree after using a large number of immunosuppressive agents. Conclusions1. CsA could up-regulate the expression of ILT3/4on NKL cells, and thus influenced the cytotoxicity against tumor cells and proliferation of NKL cells, which indicated that ILT3/4might participate in the regulation of NKL cells proliferation and interaction between ILT3/4and HLA-G affect cytotoxicity of NKL cells. Otherwise, the upregulation degrees of ILT3/4were related to drug concentration and so on. The results provided certain referential value for rational application of CsA clinically;2. ILT3and ILT4expression of lymphocytes in renal transplant recipients who recovered well increased compared with preoperative. However, the level of ILT3/4expression decreased significantly at the episodes of rejection, which was lower than that of preoperative level and stable level before rejection.In summary, the inhibitory receptors ILT3and ILT4might play the important role in the occurrence and development of cancer and rejection after transplantation. Therefore, ILT3and ILT4were expected to become noninvasive detection indexes in the clinical diagnosis and observation of some tumors and rejection after organ transplantation. |
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