Studies On The Role Of Dendritic Cells In The Mechanism Of Maternal-fetal Tolerance

Chapter 1 Studies on dendritic cell subset in the menstrual cycle,normal pregnancy and spontaneous abortionAim: To estimate the roles of MDC (myeloid dendritic cell) and PDC (plasmacytoid dendritic cell) in the establishment and maintenance of maternal-fetal tolerance by detecting the populations of peripheral blood MDC and PDC and the ratio of MDC/PDC in menstrual cycle, normal pregnancy and spontaneous abortion, which providing some basic theory for clinical diseases.Methods: Peripheral blood was collected from 30 pregnant women in the follicular and luteal phases of menstrual cycle; Peripheral blood was collected from 30 normal pregnant women at the first (8~10wk) , second (20~22wk) and third (36~38wk) trimester, with healthy non-pregnant women in the follicular phase served as control group; Peripheral blood was collected from 30 patients with spontaneous abortion, with normal early pregnant women served as control group. Mononuclear cells were isolated from peripheral blood and stained with four-color immunofluorescence, the percentages of MDC and PDC and ratio of MDC/PDC in peripheral blood mononuclear cell (PBMC) were detected by using flow cytometry. Radioimmunoassay was used to detect the levels of estradiol and progesterone in PBMC in phases of the menstrual cycle, spontaneous abortion and normal early pregnancy.Results: Compared with women in the follicular phase of the menstrual cycle, thepercentage of MDC and the ratio of MDC/PDC in the luteal phase were significantly lower (P<0.01) , the percentage of PDC in the luteal phases did not significantly differ(P>0.05), the levels of estradiol and progesterone in the luteal phase were significantly higher (P<0.0\) .Compared with healthy non-pregnant women in the follicular phase, the percentages of MDC and PDC and the ratio of MDC/PDC at the first trimester did not significantly differ (P>0.05) , the percentages of MDC and PDC and the ratio of MDC/PDC at the second, third trimester were significantly lower (P<0.01) . Compared with the first trimester of normal pregnancy, the percentages of MDC and PDC and the ratio of MDC/PDC at the second, third trimester were significantly lower (P<0.01) . Furthermore, the percentages of MDC and PDC and the ratio of MDC/PDC on day 42 after delivery were significantly higher than those at the third trimester (P<0.01) , but compared with healthy non-pregnant women, the percentage of MDC were still lower(PO.05) , the percentage of PDC and the ratio of MDC/PDC did not significantly differ (P>0.05) . Compared with healthy non-pregnancy women in the follicular phase, the percentages of MDC and PDC in umbilical cord blood were significantly lower(PO.01) ,while the ratio of MDC/PDC in umbilical cord blood did not significantly differ (P>0.05) .Compared with healthy early pregnant women, the percentage of MDC in peripheral blood of patients with spontaneous abortion was significantly higher(P<0.01), the ratio of MDC/PDC was higher (/><0.05) , while the percentage of PDC was not statistically significant (P>0.05) , the levels of estradiol and progesterone in peripheral blood of patients with spontaneous abortion were significantly lower (P<0.01) .Conclusion: Our results show that the depressed percentage of MDC and ratio of MDC/PDC in the luteal phase of menstrual cycle may be associated with immune suppression necessary for an embryo's nidation. The significantly lower percentage of MDC and PDC and ratio of MDC/PDC at the second, first trimester during normal pregnancy may be involved in establishment and maintenance the immunotolerance between the mother and fetus. The increased percentage of MDC and ratio of MDC/PDC at spontaneous abortion women may lead to immune rejection of fetus bymother. Dendritic cell subsets may be controlled by estradiol and progesterone.Chapter 2 Effects of 17p~estradiol and progesterone on the maturation and immune function of dendritic cell from humanperipheral bloodAim: To evaluate the effects of 17p-estradiol CE2) and progesterone (P4) on the maturation and immune function of dendritic cell derived from human peripheral blood.Methods: Monoclear cells were isolated from human peripheral blood, peripheral blood monocyte-derived DC were cultured at the presence of E2 (E2 group) and P4 (P4 group) at doses of 10"7mol/L and 10"6mol/L. The growths were observed with light microscope every day, the morphologic changes were observed on day 7 after culturing with scanning electronic microscope. The immunophenotypes of DC in control group, E2 and P4 groups were analyzed by flow cytometry. The levels of IL-10 and 1L-12 production in culture supernatant of control group, E2 and P4 groups were examined by ELISA assay. The capabilities of the stimulatory activity of the DC on allogeneic T cells in mixed reaction were tested by incorporation of 3H-TdR in control group, E2 and P4 groups.Results: Compared with control group, DC of E2 group displayed less dendritic pseudopod, expressed low levels of MHC-IL CD4(K CD80 and CD86, produced the decreased levels of IL-12 and the increased levels of IL-10, and exhibited weak activity in stimulating the proliferation of allogeneic T cells (P<0.01). DC of P4 group showed less dendritic pseudopod and expressed low levels of MHC-IK CD40> CD80 and CD86, the level of IL-12 decreased and the level of IL-10 increased in P4 group, the function of stimulating the proliferation of allogeneic T cells in P4 group were significantly weaker (P<0.01) .Conclusion: Our results indicate that E2 and P4 exert negative effects on the maturation and immune function of dendritic cell derived from human peripheral blood. During normal pregnancy, an increase of E2 and P4 may inhibit the maturationand immune function of dendritic cell, which may contribute to the inhibition of maternal immune response to fetal alloantigen and may induce maternal-fatal immunotolerance.Chapter 3 Roles of dendritic cell treated with 17p-estradiol andprogesterone in immune tolerance induction in skin allograftAim: To investigate the roles of bone marrow-derived dendritic cell of donor murine treated with E2 and P4 in immune tolerance induction in skin allograft, and to explore relationship between dendritic cell and CD4+CD25+ regulatory T cells (CD4+CD25+ T ) and the mechanism of immune tolerance.Methods: Bone marrow-derived dendritic cell of C57 murine as donor were cultured respectively treated with E2 (E2 group) and P4 (P4 group) at doses of and 10"6 mol/L for 7 days. Balb/c murine as recipient received respectively one injection of dendritice cells of E2 group^ P4 group > mature dendritic cell group and immature dendritic cell group through the lateral tain vein, skin transplantation was performed in the absence of immunosupression after 7 days, murine which received PBS through the lateral tain vein were served as control group. Time of skin survival was observed after transplantation. Flow cytometry were used to analyze the percentage of CD4+CD25+ T cells in peripheral respectively before and after transplantationoResults: Our results showed that time of skin survival of control group was (10.1 ±1.6) days. Compared with control group, time of skin survival of mature dendritic cell group was (7.2 ±0.7) days and shortened 2.9 day s (P<0.01), time of skin survival of immature dendritic cell group was (16.6 ±1.1) days and prolonged 6.5 days (P<0.01) . Compared with immature dendritic cell and control group, the time of skin survival in E2 and P4 group were significantly longer (PO.01) , especially, time of skin survival still prolonged 10.6 and 8.1 days respectively after skin rejection in immature dendritic group. The percentage of CD4+CD25+ regulatory T cells in E2 and P4 group were significantly higher than immature dendritic cell group and controlgroup (P<0.01) .Conclusion: In skin allograft model, dendritice cells of E2 and P4 group could prolong the allograft survival time, suggesting that negative effect of E2 and P4on the maturation and immune function of dendritic cell and the enhancement of CD4+CD25+ T cells may be associated with the immune tolerance effect for allograft.