| Objective: naive CD4~+T cells differentiate into Th0 cells after stimulated by antigen. Th0 cells differentiate into Th1, Th2, Th17 and Treg cells with the the presence of different cytokines. They play different biological effects. Treg cells have immunosuppressive effects and their main function is to maintain immune tolerance and immune homeostasis. Treg cells are closely relative with RA. With the presence of TGF-β1, na?ve CD4~+T cells differentiate into Treg cells. FOXP3 is the specific transcription factors to detect the differentiation of Treg cells from CD4~+T cells.PGE2 and LTB4 are both metabolites of arachidonic acid and important inflammatory mediators in the development of RA. Currently, RA was often treated with non-steroidal anti-inflammatory drugs which inhibited the generation of PGE2 by inhibiting Cycloxygenase. Some research said LTB4 receptor antagonist could treat RA in some extent. Previous studies in our laboratory confirmed that PGE2 could inhibit the differentiation of Treg from Th0 cells through EP2 and EP4 receptor. LTB4 could inhibit the differe -ntiation of Treg cells from Th0 cells. In collagen-induced arthritis mouse model, LTB4 could also develop the suppression of the differentiation of Treg cells. Both of PGE2 and LTB4 participate in the regulation of immune system. However,no one have reported that whether PGE2 or LTB4 could regulate the differentiation of human Treg cells from CD4~+T cells. Therefore this subject made human peripheral blood CD4~+T cells as target, investigated the effects of PGE2 and LTB4 on the differentiation of Treg cells, while to reveal the immune regulation of PGE2 and LTB4 in RA.Methods:1 Effect of PGE2 on the differentiation of Treg cells(1) CD4~+T cells in peripheral blood were sorted by immunomagnetic beads and their purity was detected by flow cytometry; cells were seeded in pre-coated 24-well plate by anti-CD3 Ab, stimulated with anti-CD28 Ab and TGF-β1, induced to Treg cells from CD4+~T cells in vitro, and harvested at different time points and investigated the time-dependent expression of FOXP3 mRNA; cells were seeded in pre-coated 24-well plate by anti-CD3 Ab, stimulated with anti-CD28 Ab, TGF-β1 and IL-2, induced to Treg cells from CD4+~T cells in vitro, cells were harvested after 7 days, and the proportion of CD4~+CD25~+ FOXP3~+cells were detected by flow cytometry.(2) Effect of PGE2 on the differentiation of Treg cells. Effect of PGE2 on the expression of FOXP3 mRNA: cells were divided into four groups: control group and PGE2(0.1μM, 1μM, 10μM) groups, the expression of FOXP3 mRNA was detected by real-time RT-PCR after incubated 36 hours; effect of 10μM PGE2 on the differentiation of Treg cells at different time points, cells were divided into two groups: control group and PGE2(10μM) group, cells were harvested at different time and their expression of FOXP3 mRNA was detected by real-time RT-PCR; effect of PGE2 on the amount of CD4+CD25+ FOXP3+ cells, cells were divided into four groups: control group and PGE2(0.1μM, 1μM, 10μM) groups, the proportion of CD4~+CD25~+ FOXP3~+ cells was detected by flow cytometry after incubated 7 days.2 Effect of LTB4 on the differentiation of Treg cellsEffect of LTB4 on the the expression of FOXP3 mRNA: cells were divided into four groups: control group and LTB4(0.01μM, 0.1μM, 1μM) groups, the expression of FOXP3 mRNA was detected by real-time RT-PCR after incubated 36 hours; effect of LTB4 on the amount of CD4~+CD25~+ FOXP3~+ cells: cells were divided into four groups: control group and LTB4(0.01μM, 0.1μM, 1μM) groups, the proportion of CD4~+ CD25~+FOXP3~+ cells was detected by flow cytometry after incubated 7 days.SPSS13.0 software was used for statistical analysis. All data were represented as mean±standard deviation. One-way ANOVA was performed to determined whether an overall statistically significant change existed before using LSD test. A value of P<0.05 was considered statistically significant. Results:1 Effect of PGE2 on the differentiation of Treg cells.(1) The purity of the Magnetic separated CD4~+T cells was more than 97% by Flow cytometry analysis; in the presence of TGF-β1 with anti-CD3/CD28 Ab, the expression of FOXP3 mRNA peaked at 36 hours; in the presence of TGF-β1 and IL-2 with anti-CD3/CD28 Ab, (72.9533±17.2270)% of cells were CD4~+CD25~+ FOXP3~+ cells after incubated 7 days. It turned out that we induced Treg cells from CD4~+T cells successfully in vitro.(2) PGE2 dose-dependently inhibited the expression of FOXP3 mRNA at 36 hours, PGE2 (0.1μM, 1μM, 10μM) groups (0.8544±0.0745, 0.6972±0.1047, 0.2220±0.0377) were lower than control group (1.0000±0.0000) (P<0.05); 10μM PGE2 inhibited the expression of FOXP3 mRNA at different time points, and it had the strongest inhibition at 7th day. 10μM PGE2(5d, 7d) groups (4.1127±0.5213, 3.6510±0.5057) were lower than control group (10.0970±1.7363, 21.0310±4.1888) (P<0.05); the proportion of CD4+CD25+ FOXP3+ cells was detected by flow cytometry after incubated 7 days with different treatments. PGE2(0.1μM, 1μM, 10μM)groups could decreased the percentage of Treg cells, and were dose-dependently PGE2(1μM, 10μM) groups (41.4933±9.5664, 23.9367±5.3066)% were lower than control group(72.9533±17.2270)%. ( P<0.05).The present study indicated that PGE2 inhibited the differentiation of Treg cells from CD4~+T cells. 2 Effect of LTB4 on the differentiation of Treg cellsTreg cells were induced from CD4~+T cells and treated with different concentrations of LTB4, when LTB4(0.01μM, 0.1μM, 1μM) groups (0.9757±0.2335, 1.0058±0.1900, 1.0779±0.2348) compared with control group (1.0000±0.0000), there was no significant difference at 36 hours (P>0.05); the proportion of CD4~+CD25~+FOXP3~+ cells was detected by flow cytometry after incubated 7 days with different concentrations of LTB4, and we found that different concentrations of LTB4 (0.01μM, 0.1μM, 1μM)groups (78.7000±12.9752,76.5200±9.9611,77.8133±13.8570)% had no obvious effect on the proportion of CD4~+CD25~+ FOXP3~+ cells quantity when compared with control group (72.9533±17.2270)% (P>0.05).The present study indicated that LTB4 has no obvious effect on the differentiation of Treg cells from CD4~+T cells.Conclusions:1 Treg cells were successfully induced from CD4~+T cells separated from human peripheral blood by Immunomagnetic beads. This research suggested that PGE2 inhibited the differentiation of Treg cells from CD4~+T cells, and was dose-dependently.2 It didn't detected that LTB4 had regulation on the differentiation of Treg cells in human.
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