Study Of The Relationship Between Fcgr3B Gene Copy Number Variation And The Acute Rejection After Renal Transplantation And Its Possible Mechanism

IntroductionFrom the 60's when the renal transplantation were carried out arroud the world, it has became the best choice for the renal substistution theropy for end stadge renal disease patients because of the better living quality and less restriction when it compare to dialysis. After various kinds of immunosuppressive drugs have been applicated in clinic, the long and short allograft living was promoted significantly. But the mechanism of the acute allograftrejection is very complicated, many factor was involved in it and impact the allograft living. For example, the panel reactive antibody(PRA), cold ischemia time, HLA mismatch and the MHC-?molecular related antibody(MICA) can all impact the acute allograft rejection. But it is obscure that when the factors mentioned above are equal, the happening of acute allograft rejection and the allograft living is still quite different. So finding the latent factors which influence the acute rejection is the purpose of researchers in recent years.The relationship between gene polymorphism and renal allograft acute rejection was the hotspot in recent years. But most studies were focus on the single nucleotide polymorphism(SNP), for example the platelet specific antigen gene, chemotaxis gene etc. The discovery of gene copy number variation(CNV) give us a new view of the gene polymorphism. The chromosome difference between people was no longer only a few basyls, but may be a long range of nucleotides which could involve a complete gene. Recently, more and more CNV had been found, and many of them have an intimate relation with some diseases. For example, the nervous system gene CNV of EFNA5, GLUR7, CACNG2 and AKAP5, the immune gene CNV of the CCL3,CCL4. A research published on the Nature in 2006 indicated that Fcgr3B gene CNV was associated with the SLE and lupus nephritis. The pathogenesis between autoimmune diseases and acute allograft rejection is resemble, beside that the neutorphil which express the Fc??B was found to appear in many acute allograft rejection biopsy. All these relations make us to presume that the Fcgr3B CNV may play an important role in the acute renal allograft rejection. In our study, we discussed the relation between CNV of Fcgr3B and acute renal allograft rejection, then we tried to find the mechanism of it.Part?The relation between Fcgr3B gene copy number polymorphism and acute renal allograft rejectionObjective Recently, the ESRD patients increased 7%-8% every year. The best treatment for ESRD is renal transplantation. Accompanied by the new immunosuppressive theropy, the rate of acute rejection decreased obviously, and the short term of allograft living increased, but 10 year living rate is still low. Acute rejection is the biggest barrier of succesed transplantation. In recent years, many research indicate that people with low copy number of Fcgr3B gene have a higher liability of SLE and vasculitis. Beacause the immune background was resemble between autoimmune disease and acute rejection, we decide to find out the relation between the CNV of Fcgr3B and acute renal allograft rejection. Methods Extract the DNA from the peripheral blood of 1027 renal allograft recipients and 450 health controls. Detect the Fcgr3B gene copy number by real-time PCR (stander curve method), and verified the results by Southern Blot. Compare the Fcgr3B gene copy number between renal allograft recipients and health controls, renal allograte recipients with stable allograft function and who went acute rejection, acute rejection recipients with different pathological type.Results In Han Chinese, the distribution of Fcgr3B gene copy number was similar to Caucasoid. The frequency of 0,1,2,3 copy in renal allograft recipients was 5.1%, 50.2%,38.1% and 5.6% respectively. And in health controls was 2.4%,39%,47.3% and 6.9% respectively. Because the most copy number was 2, we divided the renal allograft recipients and health controls into low copy number group (<2 copy) and high copy number group(?2 copy). The frequency of low copy number in health controls was lower than renal allograft recipients significantly (P<0.0001,x2=22.921,95% confidence interval:1.378-2.158). The frequency of 0,1,2,3 copy in renal allograft recipients with stable allograft function was 4.3%,48.9%,40.0%,5.6%, in acute rejection recipients was 7.9%,55.0%,31.4%,5.2% respectively. The frequency of low copy number in acute rejection recipients was significantly higher than recipients with stable allograft function. (P=0.009, x2=6.842,95% confidence interval:0.495-0.905). The acute rejection recipients were divided into acute cellular mediated rejection (ACMR), acute humoral mediated rejection(C4d+)(AHMR) and bordline change. For AHMR, the proportion of low copy number recipients and high copy number recipients was 19.4% and 21.2%(P=0.752)Conclusion In Han Chinese, people with low copy number of Fcgr3B was prone to ESRD and have high risk of acute rejection after renal transplantation Part?The influence of Fcgr3B copy number variation to the neutrophil of renal allograft recipientsObjective The protein production of Fcgr3B—Fc?R?B is a kind of low affility IgG receptor, it anchors to the cell membrane via glycosyl phosphatidyl inositol(GPI),but with out any part inside the cell. Fc?R?B only expresses in human being, and mainly on the neutrophils. The main function of Fc?R?B was the clearance of immune-complex. The CNV of Fcgr3B may change the phenotype inevitably. CNV can impact the phenotype by dose effect, stereospecific blockade and influence the regulatory element. The objective of this study is to find out the influence of Fcgr3B copy number variation to the neutrophil of renal allograft recipients.Methods Randomly pick up 15 renal allograft recipients with stable allograft function in low copy number group and high copy number group respectively according to the frequency of the copy number. Among the 15 recipients in each group,4 cases were 0 copy,11 cases were 1 copy,9 cases were 2 copy,5 cases were 3 copy and 1 cases was 4 copy. We extract neutrophil from each case by neutrophil separating medium. Fc?R?B receptor expression was detected by Flow cytometry, fluorescence intensity represents the quantity. Then we examed the phagocytosis capacity of neutrophil in different copy number group in use of the sterile latex beads. Breath burst (H2O2 production) was also detected by stimulate the neutrophil with the soluble immune-complex. Transwell assey was used to exam the chemotaxis capacity of neutrophil in different copy number group. Considering the gene expression may be influence by neighboring CNV, we detected the Fcgr3B, Fcgr2A, Fcgr2B gene expression.Results The purity and survival rate of the neutrophil in our experiment both were over 95%. Fc?R?B receptor expression was significantly higher in high copy number group than the low copy number group (Mean fluorescence intensity of CD 16b FITC were 5.61±0.54?12.59±1.33 (Mean±SEM) respectively, P?0.0001). But the phagocytosis of the sterile latex beads, breath burst and chemotaxis capacity between the two groups were not significantly different. For the mRNA expression, we find that the Fcgr3B mRNA expression was associated with the Fcgr3B gene copy number, the Fcgr3B mRNA expression in high copy number group was significantly higher than low copy number group(P<0.0001). On the contrary, the Fcgr2A mRNA expression was significantly lower in the high copy number group(P=0.0004). No difference of the Fcgr2B mRNA expression was found(P=0.1026).Conclusion Fcgr3B gene copy number was positive correlation with the mRNA and FcyRIIIB receptor expression. The capacity of phagocytosis, breath burst, chemotaxis were not affected.Part?Study of the latent mechanism of the association between Fcgr3B gene copy number variation and acute renal allograft rejectionObjective In our previous study, we found that renal allograft recipient with low Fcgr3B gene copy number was prone to develop acute rejection. As the biggest quantity of the innate immune cells, neutrophil was found to infiltrate into the allograft during the acute rejection episode. More and more researchers found that neutrophil had a tight relationship with the allograft acute rejection. Neutrophil expresses a lot of cytokines and chemokines, which chemotax and active T cells and B cells. At the aid of the neutrophil, the acute rejection accelerates. In order to find the mechanism which associates the CNV and acute rejection, we decide to focus on the relation between the neutrophil and T, B lymphocytes during acute rejection. Find out the why low copy number of Fcgr3B had a high rate of acute rejection.Methods Study the cases used in Part?, we use the IFN-?+TNF-?stimulated neutrophils of different copy number group for 12 hours, detect the T, B cell related chemokines IP-10, Mig, I-TAC and BLys gene expression by real- time PCR. At the mean time, the CXCL1, CXCL2, MAC-1 and IL-8 gene expression were also detected in low and high copy number recipients. We also hydrolyze the Fc??B receptor from the membrane of the neutrophil by PI-PLC and cross-link of the Fc??B receptor by CD 16b antibody. After this, we detected the mRNA expression of the 3 T cell related gene mentioned above before and after the treatments.Results All chemokins and cytokines were up-grated after 12 hours stimulation by IFN-?+TNF-?in different copy number recipients. Among them, IP-10, Mig, I-TAC mRNA expression were significantly higher in low copy number recipients than high copy number ones after stimulation(P=0.0211,0.0321,0.0183 respectively). But no significant differencese were found about BLys, CXCL1, CXCL2, MAC-1 AND IL-8 mRNA expression in two copy number group. After hydrolization of the Fc??B receptor, the IP-10, Mig, I-TAC mRNA expression were increasing accompanied by the increasing of the PI-PLC. But after cross-linking of the Fc??B receptor, the IP-10, Mig, I-TAC mRNA expression were decreased significantly in some gene.Conclusion The CNV of Fcgr3B impact the gene expression of the T cell related chemokines(IP-10, Mig and I-TAC). These gene expression in Low copy number recipients were significantly higher than high copy number ones, so that the low copy number recipients were more prone to acute allograft rejection.