| Kidney transplantation is the most effective method to treat renal failure. Successful kidney transplantation can effectively improve quality of patients’lives and prolong the survival time. Rejection and infection, which are caused by imbalance of immunity system, are trick questions for kidney transplant recipients. With developments of surgical technique and the application of tissue typing and immunosuppression, the rate of rejection response decreased gradually in the past decades. However, the rate of infection has remained high. As a consequence, researchers focus on how to accurately evaluate the immunity status of kidney transplantation recipients, take medicine correctly, keep balance of immunity system and make graft survived.The red blood cells are mainly believed to the a transporter of oxygen, whereas recently it has been found that it has immune function, too, which make it most abundantnatural immunity cells since the number of red blood cells is maximum in humansand animals. Because of the lack of nucleus,traditional views regard mature red blood cells have no effect on immune response. Red blood cell immune system was raised by Siegel in 1981 firstly. Then many immune-related markers, which have abilities of recognition, adhesion, concentration, attacking antigen and removing circulation immune complex, have been identified on its surface. In vitro experiments found leukocyte phagocytic function were significantly increased with complement and red blood cells. Moreover, red blood cells can prevent tumor cells in vivo diffusion. These findings suggest that red blood cells have the function of immune regulation. Red blood cell is an important basis of immune balance and stability and constitute a complete immune regulation network together with lymphocytes and other immune cells. Markers on red blood cells’surface are the genetic base of immunity function and red blood cells can combine with antigens via CD35 and present them to the macrophage. Besides, it can regulate the expression of cell cytokines by second antigen presentation. The red blood cells are involved in nonspecific immunity regulation through production of enhancement factors of natural kill cells (NK cells) and specific TNF-induced factors as well as the regulation of cell cytokines production. Although the exact mechanisms are still unveiled, the CD58 and CD59 expressed on red blood cells are ligands for CD2 on lymphocyte which can activate the production of cytokine. So determination of immunity markers on red blood cells could be used to evaluate the immunitv function of red blood cells. Recently, many researches showed that many diseases, such as hepatitis, benign or malignant tumor, autoimmune disease and diabetes, were related with abnormity of red blood cells. However, little research has been made in immune function of red blood cells in kidney transplantation. Further study about the immunity of red blood cells in kidney transplantation recipients is needed. The flow cytometry was used to investigate the expression of CD35/CD58/CD59. Transwell and co-culture assays were used to detect interaction of red blood cells with T and B lymphocytes. The objective of this study was to observe the dynamic expression of immune markers of red blood cells during the renal transplantation periods, regulation of lymphocytes in vitro, and comprehended the understanding of red blood cell immunity after transplantation, as well as provided a better evidence for the clinical therapy.1. The expression of CD35/CD58/CD59 on red blood cells in renal transplantation recipients.Fifty cases of recipient with stable renal function and 65 cases with ESRD (CRF group) and fifty cases of healthy control were enrolled in this study. We also made histopathology,11 cases were defined as renal allograft function delay recovery group(DGF group),16 cases were defined as acute rejection reaction group (AR group). The expression of CD35/CD58/CD59 in red blood cells were determined by FCM to investigate relationship of immunity of red blood cells and ESRD.The results showed that the mean fluorescence intensity (MFI) of CD35, CD58 and CD59 expression on RBC of the cases in CRF group were (25.26+7.9%), (89.94+13.8%) and (75.38+16.49), respectively, which were significantly lower than that of control group(P<0.05). While the MFI of CD35 (27.04+6.0), CD59 (87.62+20.90) expressed on RBC of the case of stable recipients were higher than that of CRF group but lower than that of control group (P<0.05). The expression levels of CD59 in stable group was significantly higher than that of CRF group (P<0.05). The levels of CD58 in stable group showed no significant differencewith other groups (P>0.05). CD35 levels in DGF group(22.46+5.01) and AR group(24.51+6.58) were different, which were significantly lower than control group(P<0.05).The levels of CD58 in DGF(73.24+11.11) and AR group(77.42+16.60) were different, which were significantly lower than control group (P<0.05). The levels of CD59 in DGF(58.66+15.82) and AR group(77.17+28.49) were different, which were significantly lower than control group (P<0.05). The levels of CD59 in DGF was lower than that of AR group (P<0.05). These results indicated that the renaldysfunction of CRF decrease the expression of immune molecules on the RBCs, and the immune function of RBC has been winkled. As the renal function elevate after the kidney transplantation, the expression of immune molecules were up regulated, but except the CD58 has no significant difference from the health control, the CD35 and CD59 expression on the stale recipient were still lower than that of the health control. When the DGF occurred on the recipients, the extent of ischemic reperfusion injury (IRI) were severe, and more reactive oxygen species(ROS) were presented in the blood flow, these condition could cause significant effect on the RBC immune function, especial the CD58 and CD59 were lower than the stable recipient, the CD59 expression of the DGF group were even lower than that of AR group, which indicated that the ROS produced when IRI occurred has significant effect on the expression of immune molecules on RBC. When the AR occur, the inflammation was localized on the graft regional, so the changes of the expression of immune molecules were little.2. The correlational study of Red blood cell immune function and lymphocyte subsets and the effect of cytomegalovirus infection on CD35/CD58/CD59 expression in renal transplantation.Peripheral blood specimens were sequentially collected from thirty cases of recipients with stable graft function at the day before transplantation, day 3, day 7 and day 14 post transplantation. If there is more than one PP65 antigen positive in every 200 thousand white blood cells, the recipients of kidney transplantation were diagnosed CMV infection. CD35/CD58/CD59 were determined in sixty one cases recipients with CMV active infection to evaluate the relationship between the status of immunity and immunity function of red blood cells. The expression of CD35/CD58/CD59 of red blood cells, the lymphocyte subset of T cells, B cells and NK cells were determined by flow cytometry.The study found that CD35 expression was positively correlated with CD59 and CD4+T cell percentage, and negative correlated with B cell percentage, there were statistically significant differences.The levels of CD35 at the day before transplantation, day 3, day 7 and day 14 post transplantation were (27.00+7.5), (28.04+7.2), (25.70+6.83) and (24.19+6.0), which were lower than that in control group (P<0.05). The levels of CD59 were lower than that of control group post transplant too (P<0.05). While the levels of CD58 at four time points showed no significant difference compared with control group (P>0.05). The absolute numbers of CD3+T lymphocyte (773.5+301) at the third day after transplant were lower than that of control group at four stages (P<0.05). The expression levels of CD3+T cells significantly decreased at the third day after transplantation when compared with that of one day before transplantation (2288+723)(P<0.05). The absolute number of NK cells at the third day decreased to the lowest levels (108.3+40.1) when compared with control group, and the time point at one day before transplantation and the fourth day after transplantation, the difference is statically significant(P<0.05).The absolute numbers of B cells increased extensively at the seventh day and fourteenth day after transplantation (P<0.05) and even higher than the control and that at before transplantation. The expression levels of CD35/CD58/CD59 in CMV group were significantly lower than those of control group (P<0.05).The expression level of CD59 in CMV group was also lower than that of latent infection (P<0.05).The levels of CD35/CD59 expression in low-active infection group were lower than high-active infection group.3. The interaction of red blood cells with T and B lymphocytes.Density gradient centrifugation was used to separated red blood cells and mononuclear cells, red blood cells were stimulated with immunosuppressor and then culture for 48h, there had no prominent change in each group. Further co-culture experiments showed that the proportion of B cells in stable red blood cell group was significantly higher than that in the rejection group, and the expression of CD 19 was significantly up-regulated. We further analyzed B cell subsets and found that these increasing B cells were IL-10 producing B cells, which were also named as regulatory B cell (Breg).To clarity the relationship between red blood cells and Breg, we use Macs purified B cells and T cells, and found red blood cells alone could not promote B cells proliferation. Interestingly, the addition of T cell could markedly induce proliferation of B cells, these results indicated that T cells mediated the interaction between red blood cells and B cells. We then in-depth study their cell regulatory mechanisms, ELISA results showed that IL-10 and TGF-β concentration in stable red blood cell co-culture supernatant were higher than that in rejection co-culture supernatant. IL-10 and TGF-β neutralization could partly inhibit the promotion of stable red blood cell co-culture supernatant on IL-10 producing B cells.Conclusion:The renal dysfunction of CRF decrease the expression of immune molecules CD35/CD58/CD59 on the RBCs, indicating that the immune function of RBC was winkled by the renal failure. As the renal function elevate after the kidney transplantation, the expression of CD35/CD58/CD59 were up regulated, but except the CD58 has no significant difference from the health control, the CD35 and CD59 expression on the stale recipient were still lower than that of the health control, maybe partly caused by the immune suppression agents. The lymphocyte immunity and RBC immunity were both influenced by the transplantation, and with the renal function recovered, both of them could be elevated. When the recipients suffer from DGF, AR and CMV infection, the expression of CD35/CD58/CD59 were down regulated on RBCs, indicated that these process could lower the RBC immunity function and associated with the disease process.Immunesupression agents did not affect the expression of red blood cell surface molecules within short period.The RBCs obtain from stable recipients could enhance proliferation of the B cell in PBMCobtain from health control, this process were through the cytokine secreted by T cell which were activated by the RBCs. The determination of CD35/CD58/CD59 of red blood cells by flow cytometry could be used to evaluate immune function of transplant recipients, and help to early diagnosis and prevent rejection,infection and other adverse reaction. It also has a clinical significance in guiding the rational use of drugs. |
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