| Objective:To explore the changes of IL-9 and associated cytokines in peripheral blood from patients with Graves'disease(GD),and analyze the relationship between IL-9 levels and GD.TGF-βand IL-4 could stimulate CD8~+T cells to differentiate into IL-9 producing CD8~+T cells.The phenotype of the IL-9~+CD8~+T cells was explored.Methods:(1) Isolated peripheral blood mononuclear cells(PBMCs) were stimulated for 5 hours with PMA and Ionomycin.Then IL-9 gene was amplificated by PCR and the amplication was cloned into pMD18-T vector.The pMD18-T vector was transformed into E.coli DH5αand then identified by PCR,restriction digestion and DNA sequencing.Real-time fluorescent quantitative PCR assay for detection human IL-9 mRNA was established by the recombinant plasmids,which served as the standard plasmids.(2) The samples including 23 patients with GD and 21 healthy subjects were collected from Jiangsu University affiliated people's hospital.The expression of IL-9,IL-10 and IL-4 in PBMCs and the concentration of IL-9 in plasma were detected by quantitative real-time polymerase chain reaction(QRT-PCR) and enzyme linked immunosorbent assay(ELISA). CD4~+IL-9~+T cell and CD8~+IL-9~+T cell frequency after induction of PBMCs by TFGβ1 and IL-4 in vitro were also detected by flow cytometric analysis.(3) PBMCs from healthy subjects were activated by plate bound anti-CD3 mAb(OKT-3) in the present of TGF-βand IL-4,the IL-9~+ cell frequency in CD8~+T was analyzed by flow cytometry after 5 days.The expression of several cytokines was detected by QRT-PCR and the phenotype of the IL-9~+ CD8~+T cells was analyzed by flow cytometric.Results:(1) The real-time fluorescent quantitative PCR assay for detection human IL-9 mRNA expression was established.The coefficient of correlation of the standard curve of this method was 0.995-0.998,the linearity ranged from 10~2copies/μl to 10~7copies/μl and melting curve analysis showed single peak.(2) The expression of IL-9,IL-10 and IL-4 mRNA in PBMCs was more abundant in patients with Graves'disease than in healthy controls (P＜0.05).The relatively expression of IL-9 in GD was 1.83(IQR 1.01-4.3),it was greater than in controls which was 0.30(IQR 0.04-1.80) (p＜0.05);The expression of IL-10 in GD was 0.92(IQR 0.28-1.60),it was more than in controls which was 0.03(IQR 0.02-0.20)(p＜0.05);The relatively expression of IL-4 in GD was 28.70(IQR14.64-54.65),it was greater than in controls which was 4.47(IQR 0.70-17.81)(p＜0.05)..(3) IL-9 mRNA expression was positively correlated to IL-10 levels (R=0.6861,P＜0.001),but no correlated to IL-4 expression. (4) The concentration of plasma IL-9 was more abundant in Graves' disease patients(n=23,250±205pg/ml) than in healthy controls(n=20, 120±50 pg/ml)(P＜0.05).(5)IL-9~+CD4~+T cell frequencies were increased from patients(n=14, 2.13±0.88%) compared with those from healthy controls(n=12,1.15±0.49%) after induction of PBMCS by TFGβ1 and IL-4 in vitro(P＜0.05). IL-9~+CD8~+T cell frequencies were higher form patients(n=14,3.0±0.57%) compared with those from healthy controls(n=12,1.01±0.40%) after induction.(6)TGF-βand IL-4 could stimulate CD8~+ T cells to differentiate into IL-9 producing cells in plate bound-anti-CD3 mAb(pbCD3) activated PBMCs.The expression GATA-3 and C-MAF is increased in those cells, and the IL-9-producing CD8~+ T cells expression Granzyme-B(100%) and IFN-γ(46%).Conclusions:(1) Our observations suggest that increased IL-9 might be involved in the pathogenesis of Graves'disease.(2) These data show that the existence of a human IL-9 producing CD8~+T subset.This cell subset display Granzyme-B and IFN-γ.
|
PDF Full Text Request