Prostaglandin E₂: Immune Regulation To T Lymphocytes And Effect On Mouse Model Of Graft-versus-host Disease After Allo-Hematopoietic Stem Cell Transplantation

ObjectTo study the effect of different concentration of PGE₂ on proliferation of peripheral blood T lymphocytes.To analyze the effect of PGE₂ on productive change of the specific cytokines interferon-γ(IFNγ) and interleukin-4(IL-4) from different T lymphocyte subgroups and to evaluate immune regulation of PGE₂ to development direction of Th1 and Th2 lymphocytes.To investigate the role of PGE₂ on mouse model of acute graft versus host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation(allo-HSCT).MethodsPeripheral blood(PB) mononuclear cells(MNCs) were stimulated by anti-human CD3 monoclonal antibody(mAb) and anti-human CD28 monoclonal antibody(mAb) and cultured at a concentration of 1×10⁵ cells/well in 96-well plate,and incubated with complete medium(RPMI1640 with 10%FCS) at 37℃in 5%CO₂ in the presence of the different concentration of PGE₂,or without PGE₂.After 96h of incubation,cells were pulsed with BrdU for 24h and harvested.The proliferation was tested according to the manufacture's protocol.The cultured PB MNCs were activated by addition of anti-human CD3 mAb and anti-human CD28 mAb in the presence of 14μmol/L PGE₂,or without PGE₂.24h,48h, 72h and 120h after culture,the supernatants were collected for measurement of IL-4 and IFN-γlevels detected by ELISA.The PB MNCs were cultured in the presence of 14μmol/L PGE₂.After 20h culture, 50ng/ml phorbol-12-myriatate-13-acetate(PMA),1μg/ml ionomycin and 2μg/ml monensin were added respectively to the cells and incubated for another 4-6h.Then the cells were collected and the production of IL-4/IFN-γ,of CD4⁺ and CD8⁺ T cells were analyzed.After female BALB/c(H-2^d) mice received lethal total body irradiation(TBI),bone marrow(BM) cells and spleen cells from male C57BL/6(H-2^b) mice were transfused into the recipient mice via the tail vein,then were injected PGE₂ intravenously at day 0,4,8,12 or not.Hematopoietic reconstruction,pathological changes of aGVHD and survival time were observed.ResultThe PB MNCs proliferated in the presence of anti-human CD3 mAb and anti-human CD28 mAb.When the concentrations of PGE₂ were 7μmol/L,14μmol/L and 28μmol/L,the inhibitory rates of T cells proliferation were(18.3429±15.8648)%, (54.3114±19.1912)%and(91.2057±4.5962)%respectively.With the increased concentration of PGE₂,the inhibitory rate significantly increased(P=0.001).There was significant positive correlation between inhibitory rate of T cells and PGE₂ concentriton(correlation coefficient=0.889,P =0.000).The IFN-γconcentration was increased continually with prolonging cuture time in control group(P=0.046),but the difference between the IFN-γconcentration of 120h and 72h supernatant in test group had no statistical significance(P=0.917).The IFN-γconcentrations of 24h supernatant in test and control group were(537.63±440.65) pg/ml and(5215.47±6073.39) pg/ml respectively(P=0.016).Those of 48h supernatant were(6244.33±5578.32) pg/ml and(43046.00±31216.73) pg/ml respectively(P=0.010).Those were(9592.87±7787.19) pg/ml and (69876.33±32381.93) pg/ml respectively(P=0.004).Those of 120h supernatant of the test group and the control group were(10456.66±12077.94) pg/ml and (88934.33±36166.85) pg/ml respectively(P=0.004).The IFN-γconcentrations producted at different time in test group were significantly lower compared with those in control group.The IL-4 concentrations producted at different time had no significant change in test group.The concentrations at 24h,48h,72h and 120h were(21.80±6.84) pg/ml,(16.51±8.15) pg/ml,(11.85±9.99) pg/ml and(15.88±13.24) pg/ml(P = 0.400),While those at 24h,48h,72h and 120h in control group were(144.14±119.85)pg/ml, (36.83±21.30)pg/ml,(21.17±16.51)pg/ml and(14.82±12.89)pg/ml.The IL-4 concentration in 24h in control group was significantly higher than that at 48h,72h and 120h in control group(P=0.007,0.003 and 0.002).After cutured for 24h the IL-4 concentration in test group was significantly lower than that in control group(P =0.037),but for 48h,72h and 120h,there was no statistical(P=0.068,0.343 and 0.870).The proportion of CD4⁺IFN-γ⁺T cells was 23.81(16.16-48.73)%in test group and 24.04(12.03-50.77)%in control group(P=0.767).The proportion of CD4⁺IL-4⁺T cells 3.98(1.61-38.19)%in test group was slightly higher than 2.74(1.82-9.72)%in control group(P=0.051).The ratios of CD4⁺IL-4⁺T cells to CD4⁺IFN-γ⁺ T cells in test group and in control group were 0.21094(0.069-0.784) and 0.13239(0.045-0.511) respectively,and the ratio in test group was significantly higher than that in control group(P=0.011).The proportion of CD8⁺IFN-γ⁺ T cells was 44.48(22.52-60.68)%in test group and 39.02(20.63-62.93)%in control group(P=0.441).The proportion of CD8⁺IL-4⁺T cells was 19.04(4.21-50.69)%in test group and 9.46(0.48-48.02)%in control group, the proportion in test group was significantly higher than that in control group (P=0.015).The ratios of CD8⁺IL-4⁺ T cells to CD8⁺IFN-γ⁺ T cells in test group and in control group were 0.42806(0.159-0.949) and 0.27775(0.017-0.918),and the ratio in test group was significantly higher than that in control group(P=0.011).The examination of mice receiving allo-HSCT indicated that mice developed body weight loss at a median of 15(13-18)d and 17.5(14-19)d after transplantation in group A and group B respectively,but the difference had no statistical significance(P =0.096).GVHD scores in group A and group B were(6.80±2.17) and(5.00±2.97) respectively,but the difference was not significant(P=0.247).The difference of the 50d survival between group A and group B was not statistical significance(P=0.398).ConclusionPGE₂ inhibits the proliferation of T lymphocytes,and it inhibitory rate was increases with the increase of PGE₂ concentration.PGE₂ influences production of Th1 and Th2 cytokines and suppresses the level of IFN-γafter 24h coculture,and significantly influences peak appearance of IFN-γproduced by T lymphocyte.PGE₂ can continue to inhibit the production of IFN-γ.The effect of PGE₂ on IL-4 producted by T lymphocyte appeares at 24h of culture,but its continuous effect was not significant.PGE₂ enhances the ratio of CD4⁺IL-4⁺T lymphocytes to CD4⁺IFN-γ⁺T lymphocytes and the ratio of CD8⁺IL-4⁺T lymphocytes to CD8⁺IFN-γ⁺T lymphocytes, and regulates T cells toward Th2 cell development.PGE₂ has a tendency to delay the occurrence of aGVHD in mice allo-HSCT,but does not ameliorate the manifestation and severity of aGVHD and survival.