Effect Of Estrogen On The Proliferation Of Lymphocytes And The Antigen-Specific Regulatory Function Of CD4~+CD25~+ Treg Induced By Estrogen

Objective:To investigate the CD4⁺CD25⁺Treg induced by gradient concertration estrogen and the related mechanism.Methods:(1) Spleen cells from C57BL/6 mouse(as responder cell) were incubated with the Mitoncycin-C treated BALB/c splenocytes(as antigen-presenting cells) and stimulated with gradient concertration estrogen(1:10 serially diluted,10^-5M～10^-9M, OM as control),the proliferation of responder cell was analyzec by AlamarBlue ^TM; percentage of CD4⁺CD25⁺ subsets of responder cells was detected by Facs;FoxP3 mRNA by RT-PCR.(2) CFSE labeled spleen cells from C57BL/6 mouse were incubated with estrogen(10^-7M) or propylpyrazoletriol(10^-7M) stimulated with anti-CD3 anti-body and anti-CD28 anti-body,the Proliferation index was analyzed by MODFIT ^TM CELL PROLIFERATION MODEL^TM software;CD4⁺CD25⁺ subsets of responder cells was detected by Facs.(3) Spleen cells from C57BL/6 mouse were incubated with estrogen(10^-7M) and were stimulated with anti-CD3 anti-body and anti-CD28 anti-body,TNF-α,IL-10,TGF-βmRNA genes transcription of responder cells were analyzed by semi-quantitative RT-PCR.Results:(1) Low dose of estrogen can promote the proliferation of responder cells,but the proliferation decreased when the concentraton of estrogen increased.Especially at pregnant level(10^-7M),there was a significant peak in both the percentage of CD4⁺CD25⁺Treg and expression of FoxP3 mRNA gene transcription. (2) The proliferation of responder cells was significantly inhibited by both pregnancy levels of estrogen(10^-7 M) and propylpyrazoletriol(10^-7 M),intriguingly, the percentage of CD4⁺CD25⁺ was up-regulation in both group.(3) The pregnancy levels of estrogen(10^-7 M) promoted the transcription of IL-10,TGF-βmRNA,but inhibited the transcription of TNF-αmRNA.Conclusions:(1) The effects of estrogen on T cell activation was related to the concentration. Physical level of estrogen stimulates proliferation of responder cells,but there was a significant peak in both the percentage of CD4⁺CD25⁺Treg and FoxP3 mRNA gene transcription at pregnant level of estrogen(10^-7M),(2) The proliferation of responder cells was significantly inhibited by both pregnant levels of estrogen(10^-7M) and propylpyrazoletriol(10^-7M),intriguingly, the percentage of CD4⁺CD25⁺ was up-regulation in both group.(3) Pregnant levels of estrogen breaks the balance of Th1--type cytokines and Th2-type cytokines by promoting the expressions of Th2-type cytokines,and thus exerts the immunosuppressive function. Part 2.The Antigen-Specific Regulatory Function Of CD4⁺CD25⁺Treg Induced By EstrogenObjective:To investigate the CD4⁺CD25⁺Treg induced by pregnancy level of Estrogen in vitro/in vivo and its antigen-specific regulatory function.Methods:(1) Spleen cells from C57BL/6 mouse(as responder cells) were incubated with CFSE labeled and Mitoncycin-C treated BALB/c splenocytes(as antigen-presenting cells) and stimulated by pregnant level of estrogen(10^-7M) for 3 days at 37℃in 5% CO₂.Cells were harvested,and the un-CFSE labeled CD4⁺CD25⁺Treg were sorted by Flow Cytomete.(2) CD4⁺CD25⁺Treg from Spleen cells of pregnant C57BL/6 mice injected with MMC treated BALB/C splenocytes through the tail vein(1×10⁶,200μl) for 5 days were sorted.(3) The different source of CD4⁺CD25⁺Treg cells from C57BL/6 mice were incubated with MMC treated BALB/C splenocytes(as donor-specific APCs) or MMC treated C3H/He splenocytes separately(as a third-party APCs) at a ration of 1:2:2,freshly splencoytes form C57BL/6 mice as control.Detected the proliferation of responder cells were detected by AlamarBlue^TM.(4) The skin transplantation model was set up by grafting skin from BALB/c or C3H/he mice to C57BL/6 mice.The different source of CD4⁺CD25⁺Treg cells were injected to the recipients through the tail vein.Result:(1) The pregnant level of estrogen promoted the percentage of CD4⁺CD25⁺ Treg whether in vivo and in vitro,but the in vivo induction had a better effect,the expression rate was(7.13±0.56)%vs.(12.13±1.76)%. (2) Sorting the CD4⁺CD25⁺ Treg by Flow Cytomete was an effective and reliability method.(3) The CD4⁺CD25⁺ Treg induced by pregnant level of estrogen in vitro had the antigen-specific characteristics,such as suppressing the activation and proliferation of responder cells,especially form the donor antigens.The proliferation rates of reactor cells stimulated by donor antigens and third-party antigens were(0.28±0.08) vs(0.59±0.16),p<0.05.This kind of CD4⁺CD25⁺ Treg significantly prolonged the paternal skin grafs' survival time.(5) The Nrp1⁺T cells sorted from pregnant C57BL/6 mice also had the antigen-specific characteristics,The proliferation rates of reactor cells stimulated by donor antigens and third-party antigens were(0.25±0.06)vs(0.61±0.11),p<0.01. This kind of CD4⁺CD25⁺ Treg significantly prolonged the paternal skin grafs' survival time.Conclusions:Both of the CD4⁺CD25⁺ Treg induced by pregnant levels of estrogen in vivo or in vitro had the antigen-specific characteristics,the regulatory function of CD4⁺CD25⁺ Treg induced in vitro was more effective in vitro induced.