Inhibitory effect of the mitogen activated protein kinase specific inhibitor PD98059 on Mtb-Ag-activated γδΤ cells.

Objective: This study aimed to observe the inhibitory effects of mitogen activated protein kinase (MAPK/ERK) specific inhibitor PD98059 on Mtb-Ag activated γδΤ cells and to investigate the role of MAPK in MAPK/ERK pathway of γδΤ cells activated by Mtb-Ag.

Methods: Healthy human peripheral blood mononuclear cell (PBMC) was isolated from the peripheral blood, and then stimulated by phorbol esters (PMA), ionomycin (IM) and Mtb-Ag. PBMC of the experimental group was pretreated by PD98059 of 0, 1, 10 or 100 μmol/L while the control group was given no pretreatment. Flow cytometry was applied to detect the expression of CD69 in γδΤ cells at 0 h, 6 h, 12 h, 24 h, 48 h and 72 h after treatment respectively. The influence of PD98059 on the amplification of γδΤ cells was also detected.

Results: After stimulated by PMA and IM for 6 hours, the expression of CD69 in γδΤ reached the peak of (99.3±1.09)%, while in the Mtb-Ag stimulation group, it reached the highest level of 75.2% at 24 hours. The level of CD69 in two group was significant difference between the two groups at each time respectively (p<0.05). Pretreated with PD98059 of 0, 1, 10, 100 μmol/L, the level of CD 69 expression in Mtb-Ag stimulated γδΤ was 79±0.8%, 75±0.7%, 54±0.5% and 17±0.2% respectively; In PMA+IM stimulated group, CD69 expression were all more than 98% in different concentration of PD98059. After treated with PD98059 of 0, 1, 10, 100 μmol/L, total cell number increased from 1.5×10 to (10.3±2.5)×10, (9.5±2.1)×10, (5.8±1.8)×10 and (2.1±0.5)×10 respectively. Number of γδΤ cells reached to (6.2±0.9)×10, (5.02±0.8)×10, (2.05±0.5)×10 and (0.41±0.1)×10 respectively.

Conclusion: γδΤ cells were specifically activated by Mtb-Ag, and the activation was depended on MAPK/ERK pathway. The activation effects of Mtb-Ag on γδΤ cells can be obviously inhibited by PD98059.