Experimental Research Of Exogenous MICA Molecule On Biological Functions Of Endothelial Cell And Killing Effect Of NK Cell In Vitro

Major histocompatibility complex class I-related chain A (MICA) antibodies are associated with acute rejection and poor renal-allograft survival, which has become focus of basic and clinical research of transplant immunology. Acute rejection was occurred in renal-allograft recipients with good HLA matching and anti-MICA antibodies were detected in serum and rejected allografts. Anti-MICA antibodies also existed in serum of patients waiting for renal transplantation, which indicated that the patients produced specific antibodies against MICA antigen before transplantation. There was close relationship between MICA molecules with chronic renal-allograft dysfunction. MICA protein is expressed on the surface of vascular endothelial cells, but not on T lymphocyte. Therefore, MICA molecule might cause injury of allograft vessles directly and result in renal-allograft dysfunction. It has been reported that soluble MICA (sMICA) can reduce immunologic injury of allograft in heart transplant recipients and protect allograft function. It was demonstrated that MICA antigen can make T and B lymphocytes proliferation.MICA is ligand of NKG2D receptor on NK cell and NKG2D is an activating receptor. But whether MICA antigen can influence endothelial cells, whether sMIICA can protect renal allograft and whether NK cell be activated to kill endothelial cell have not been reported. Therfore, this study will conclude three parts: (1) Observe the impact of biological characteristics and secreting function of endothelial cell after we add MICA antigen to the media for cultivation; (2) Moderating effects of exogenous antigen for MICA expression in endothelial cell and sMICA in superant; (3) Coculture NK cell and endothelial cell expressing MICA protein and observe the cytotoxicity of NK cell against endothelial cell. PartⅠ: Impact of MICA antigen to biological functions of endothelial cellsObjective To investigate the impact of major histocompatibility complex class I-related chain A (MICA) antigen to biological characteristics and secreting function of endothelial cell.Methods HUVECs were divided into three experimental groups(A5,A10,A25 ), which were stimulated with exogenous recombinant MICA protein at 5ng/ml, 10ng/ml, 25ng/ml respectively. Group A0 was added equivalent volume PBS as control. The apoptosis and cell cycle was measured by flow cytometry (FCM). The proliferation of endothelial cell was detected by MTT assay. The level of metabolic product of PGI2 (6-keto-PGF1α),endothelin (ET-1),tissue plasminogen activator (t-PA) and its inhibitor (PAI) in supernatant of all groups were detected.Results All experimental groups were higher values of A570 than control group. Both group A5 and A10 were significantly higher than the control group and group A25 (P < 0.05), but there were no significant differences between group A5 and A10 (P > 0.05). The proliferation rate at 48h was the highest in identical dosage. A570 value of experimental groups at 48h was 0.458, 0.446 and 0.389 respectively. The proliferation rate at 72h and 96h were persistently decreased as extending time. There were significant differences (P < 0.05). The level of 6-keto-PGF1αof group A10, A25 was 144.6 and 132.8pg/ml, which was lower than that of group A0 (226.5pg/ml) and A5 (232.6pg/ml). The level of ET-1 of group A10, A25 was 23.6 and 25.8pg/ml while the level of ET-1 of group A0, A5 was 11.8 and 10.4pg/ml. The differences were statistically significant (P < 0.05), but there was no difference between group A0 with A5, group A0 with A5 (P > 0.05). The level of t-PA of experimental groups were 161.2, 154.2 and 157.8ng/ml and the level of PAI were 221.6, 248.5 and 252.4ng/ml. Compared with control group, the level of t-PA in three experimental groups rised markedly and the level of PAI decreased obviously. These differences had statistical significances (P < 0.05). But there was no significant difference among the experimental groups (P > 0.05). There were no cell apoptosis and changes of cell cycle in all experimental groups.Conclusions The small dosage of MICA antigen can stimulate endothelial cell persistent proliferation. Meanwhile, MICA antigen can make endothelial cell injured and cause the coagulation function enhanced, fibrinolytic function declined.PartⅡ: Impact of exogenous antigen for expression of MICA gene in endothelial cellObjective To investigate the expression of MICA gene in endothelial cell stimulated with exogenous antigen.Methods The endothelial cells were divided into control group and three experimental groups, which were stimulated with exogenous recombinant MICA antigen (group A) and heat shock protein (HSP, group B) at 5ng/ml, 10ng/ml and 25ng/ml. The expression of MICA mRNA was detected by real-time fluorescence quantitative PCR. The expression of MICA protein was measured by Western blot. The expression of MICA total protein on the cell surface was detected by using flow cytometry (FCM). The levels of soluble MICA (sMICA) in supernatant of all groups were detected by ELISA.Results The expression of MICA mRNA and MICA protein were increased significantly after MICA stimulation (P < 0.05). The expression of MICA mRNA of group A10 were remarkably higher than the group A25, but had no differences with group A5. The expression of MICA membrane protein of group A10 were higher than that of group A25 (P < 0.05), but had no differences with group A5. The level of sMICA in three experimental groups were 35.8,27.4,21.8pg/ml respectively,which decreased obviously comparing with that of control group( 352.5pg/ml). These differences had statistical significances (P < 0.05). But there was no significant difference among the experimental groups (P > 0.05). However, the expression of MICA gene and sMICA level didn't change after HSP stimulation.Conclusions The exogenous MICA antigen up-regulate the expression of MICA mRNA and protein, especially the membrane protein on the cell surface significant increase, but sMICA in supernatant decreased.PartⅢ: Killing effect of NK Cell to Endothelial Cell mediated by MICA MoleculeObjective To observe the role of MICA molecule in the process of NK cells killing endothelial cells.Methods NK cells were selected by using immumomagnetic beads (CD56 positive isolation kit) according to the manufacturer's recommended conditions. It was divided into four groups. Group A was NK cells and HUVECs of group A0 (see PartⅡ), group B was NK cells and HUVECs of group A10. NK cells and HUVECs expressing MICA molecule were cocultured for 10h. All the cells were stained by fluorescein AO/EB and the dead cells were measured under fluorescence microscope. Killing effect of NK cells was calculated by the number of dead cells among total cells. Level of IFN-γand perforin in cell superant were detected by ELISA.Results The purity of NK cells selected by using immumomagnetic beads was 88.5%. The killing effect of NK cells to group Bwas 35.5%, which was higher than that of group A, 12.6%. There were significant differences between two groups (P < 0.05). Level of IFN-γand perforin in group B were higher than that of group A and the differences were statistically significant (P < 0.05).Conclusion Expression of MICA molecule on the surface of HUVECs can raise the cytotoxicity of NK cells to HUVECs. IFN-γand perforin might be bioactive substance playing killing effect.