Progesterone Exerts Nongenomic Effects on Na+/H+ Exchange as to Immunomodulate Phytohemagglutinin (PHA)-induced Cytokine Secretion and Proliferation in Human Peripheral T Cells
碩士 === 國立陽明大學 === 生理學研究所 === 91 === Progesterone is an endogenous immunomodulator, and can suppress T cell activation during pregnancy. When analyzed under a genome time scale, the classic steroid receptor pathway does not have any effect on ion fluxes. Our previous findings, progesterone antagoni...
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ndltd-TW-091YM0001160082015-10-13T13:39:19Z http://ndltd.ncl.edu.tw/handle/29682444359849278642 Progesterone Exerts Nongenomic Effects on Na+/H+ Exchange as to Immunomodulate Phytohemagglutinin (PHA)-induced Cytokine Secretion and Proliferation in Human Peripheral T Cells 助孕酮透過非基因性抑制鈉氫交換蛋白免疫調節人類T細胞對PHA刺激所引發的細胞激素分泌和增生反應 Mei-Chi Shie 謝美琦 碩士 國立陽明大學 生理學研究所 91 Progesterone is an endogenous immunomodulator, and can suppress T cell activation during pregnancy. When analyzed under a genome time scale, the classic steroid receptor pathway does not have any effect on ion fluxes. Our previous findings, progesterone antagonist, RU486, could block the intracellular calcium increases and pH (pHi) decrease in human peripheral T cells by progesterone. The acidification was blocked by Na+/H+ exchange inhibitor, 3-methylsulphonyl-4-piperidinobenzoyl, guanidine hydrochloride (HOE694); but not by 5-(N,N- dimethyl)-amiloride (DMA). Therefore, the aim of this study was to investigate whether progesterone exerted nongenomic effects through the inhibition of Na+/H+ exchange (NHE) activities and how it suppressed cytokine secretion and proliferation in human peripheral T cells. T cells from human peripheral blood and leukemia cell line-Jurkat were used to determine the pHi by the fluorescent dye, BCECF. The changes in intracellular calcium ([Ca2+]i) by fluorescent dye, Fura-2. In this study, the expression of NHE1 mRNA was detected by RT-PCR. IL-10 and IFN-γ were determined by ELISA. The proliferation was determined by [3H]-thymidine uptake into DNA or 3-[4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT) reduction. The new findings indicated that 1) progesterone prevented PMA-induced alkalinization, but PMA did not affect the acidification induced by progesterone. 2) The mRNA expression of NHE was reached peak at 4 h after PHA stimulation. 3) The recovery capability of NHE from acidification following application of a NH3 pulse by addition NH4Cl was suppressed by progesterone (Ki=13.48 μM), but the inactive steroid, 20α-hydroxyprogesterone (20α-OHP), could not affect NHE. 4) Progesterone also resulted an elevation of [Ca2+]i, a decrease of pHi and a suppression of NHE activation(K i =8.93 μM) in Jurkat T cells. 5) Both progesterone and its antagonist, RU486 suppressed T cell proliferation. RU486 did not reverse the suppressive effects by progesterone on PHA-induced [3H]-thymidine uptake. However, DMA could mimic the action of progesterone to suppress PHA-induced [3H]-thymidine uptake. 20α-OHP did not suppress PHA-induced [3H]-thymidine uptake. 6) Progesterone exhibited less effective on suppression PHA-induced [3H]-thymidine uptake when addition of PHA with progesterone at same time than 72 h apart. 7) Progesterone attenuated PHA-induced MTT reduction, but it attenuated [3H]-thymidine uptake with much more extent. In conclusion, the nongenomic effect on inhibition of NHE activity by progesterone did lead to suppress PHA-induced T cell proliferation. These facts indicated that the nongenomic effect on inhibition of NHE activity by progesterone might direct an immunomodulation on human T cells. Eileen Jea Chien 賈愛華 2003 學位論文 ; thesis 94 zh-TW |
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碩士 === 國立陽明大學 === 生理學研究所 === 91 === Progesterone is an endogenous immunomodulator, and can suppress T cell activation during pregnancy. When analyzed under a genome time scale, the classic steroid receptor pathway does not have any effect on ion fluxes. Our previous findings, progesterone antagonist, RU486, could block the intracellular calcium increases and pH (pHi) decrease in human peripheral T cells by progesterone. The acidification was blocked by Na+/H+ exchange inhibitor, 3-methylsulphonyl-4-piperidinobenzoyl, guanidine hydrochloride (HOE694); but not by 5-(N,N- dimethyl)-amiloride (DMA). Therefore, the aim of this study was to investigate whether progesterone exerted nongenomic effects through the inhibition of Na+/H+ exchange (NHE) activities and how it suppressed cytokine secretion and proliferation in human peripheral T cells. T cells from human peripheral blood and leukemia cell line-Jurkat were used to determine the pHi by the fluorescent dye, BCECF. The changes in intracellular calcium ([Ca2+]i) by fluorescent dye, Fura-2. In this study, the expression of NHE1 mRNA was detected by RT-PCR. IL-10 and IFN-γ were determined by ELISA. The proliferation was determined by [3H]-thymidine uptake into DNA or 3-[4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT) reduction. The new findings indicated that 1) progesterone prevented PMA-induced alkalinization, but PMA did not affect the acidification induced by progesterone. 2) The mRNA expression of NHE was reached peak at 4 h after PHA stimulation. 3) The recovery capability of NHE from acidification following application of a NH3 pulse by addition NH4Cl was suppressed by progesterone (Ki=13.48 μM), but the inactive steroid, 20α-hydroxyprogesterone (20α-OHP), could not affect NHE. 4) Progesterone also resulted an elevation of [Ca2+]i, a decrease of pHi and a suppression of NHE activation(K i =8.93 μM) in Jurkat T cells. 5) Both progesterone and its antagonist, RU486 suppressed T cell proliferation. RU486 did not reverse the suppressive effects by progesterone on PHA-induced [3H]-thymidine uptake. However, DMA could mimic the action of progesterone to suppress PHA-induced [3H]-thymidine uptake. 20α-OHP did not suppress PHA-induced [3H]-thymidine uptake. 6) Progesterone exhibited less effective on suppression PHA-induced [3H]-thymidine uptake when addition of PHA with progesterone at same time than 72 h apart. 7) Progesterone attenuated PHA-induced MTT reduction, but it attenuated [3H]-thymidine uptake with much more extent. In conclusion, the nongenomic effect on inhibition of NHE activity by progesterone did lead to suppress PHA-induced T cell proliferation. These facts indicated that the nongenomic effect on inhibition of NHE activity by progesterone might direct an immunomodulation on human T cells.
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author2 |
Eileen Jea Chien |
author_facet |
Eileen Jea Chien Mei-Chi Shie 謝美琦 |
author |
Mei-Chi Shie 謝美琦 |
spellingShingle |
Mei-Chi Shie 謝美琦 Progesterone Exerts Nongenomic Effects on Na+/H+ Exchange as to Immunomodulate Phytohemagglutinin (PHA)-induced Cytokine Secretion and Proliferation in Human Peripheral T Cells |
author_sort |
Mei-Chi Shie |
title |
Progesterone Exerts Nongenomic Effects on Na+/H+ Exchange as to Immunomodulate Phytohemagglutinin (PHA)-induced Cytokine Secretion and Proliferation in Human Peripheral T Cells |
title_short |
Progesterone Exerts Nongenomic Effects on Na+/H+ Exchange as to Immunomodulate Phytohemagglutinin (PHA)-induced Cytokine Secretion and Proliferation in Human Peripheral T Cells |
title_full |
Progesterone Exerts Nongenomic Effects on Na+/H+ Exchange as to Immunomodulate Phytohemagglutinin (PHA)-induced Cytokine Secretion and Proliferation in Human Peripheral T Cells |
title_fullStr |
Progesterone Exerts Nongenomic Effects on Na+/H+ Exchange as to Immunomodulate Phytohemagglutinin (PHA)-induced Cytokine Secretion and Proliferation in Human Peripheral T Cells |
title_full_unstemmed |
Progesterone Exerts Nongenomic Effects on Na+/H+ Exchange as to Immunomodulate Phytohemagglutinin (PHA)-induced Cytokine Secretion and Proliferation in Human Peripheral T Cells |
title_sort |
progesterone exerts nongenomic effects on na+/h+ exchange as to immunomodulate phytohemagglutinin (pha)-induced cytokine secretion and proliferation in human peripheral t cells |
publishDate |
2003 |
url |
http://ndltd.ncl.edu.tw/handle/29682444359849278642 |
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