RESUMO
Objective To investigate the role of PKCθ signal pathway on regulation of L-selectin (CD62L) expression in human activated γδT cells. Methods Human peripheral blood mononuclear cells (PBMC) were stimulated with Mycobacterium tuberculosis antigen (Mtb-Ag) and cultured for 6-8 d to generate Mtb-Ag activated T cells(MtbAT) as γδT cells enrichment T cell line. The MtbAT were stimulated with PMA or PMA + IMN for 3, 6, 12 and 24 h, respectively, or MtbAT cultured for 8 d were stimulated with Mtb-Ag, or PMA, with or without PKC0 inhibitor Rottlerin for 4 h. After the treated cells stained with fluo-rescent labeled monoclonal antibodies, the expression of CD62L on γδT cells were measured by flow cytome-try (FCM ). Results The expression of CD62L on γδT cells cultured for 6-8 d were 75.0%-87.0%. Decrease of CD62L from the surface of γδT cells by 3 h to 12 h after exposure to PMA (42.3% to 23.5% ), but CD62L expression increased to 53.2% when γδT cells were exposed to PMA for 24 h. The expression of CD62L of γδT cells decreased to 52.1% and 39.3% respectively when γδT cells were exposed to PMA + IMN for 3 h and 6 h. After treated with PMA + IMN for 12 h and 24 h, the expression of CD62L were 52.9% and 35. 3% respectively. The CD62L expression of γδT cells treated with PMA and Rottlerin (47.9%) were higher than that treated with PMA alone (31.8%). After Mtb-Ag restimulated MtbAT for 4 h, the CD62L level of γδT cells decreased from 70.0% to 54.8%, Rottlerin could inhibite Mtb-Ag down regulation CD62L level of γδT cells (63.1%). Conclusion The CD62L expression of γδT cells could be ingibited partly by the inhibitor of PKCθ signal pathway may regulate L-selectin (CD62L) expression of activated human γδT cells.
RESUMO
Objective To explore the roles of PKCθ(protein kinase Cθ)signaling pathway on the activation,proliferation and TH1/TH2 cytokines production of the γδT lymphocytes stimulated by Mycobacterium tuberculosis antigen(Mtb-Ag) in vitro.Methods Peripheral blood mononuclear cells(PBMC)were pretreated with or without Rottlerin at 5.0 μmol/L,and stimulated with Mtb-Ag and cultured in rhIL-2 containing medium.After different time of culture,activation molecules and cytokines production of γδT cells were measured by flow cytometry.The proliferation proportion and the percentage of each generation of γδT cells were determined by carboxylfluoreseein diacetate succinmidyl ester(CFSE)labeling technique and flow cytometry.Results After 3d of stimulation with Mtb-Ag,the expression rates of CD69 and CD25 of γδT cells were 46.2%and 45.6%,respectively.Pretreatment of 5.0 μmol/L Rottlerin markedly inhibited the both expressions of CD69 and CD25 in γδT cells(P＜0.01).After stimulation and expansion in vitro for 5,10,and 15 d,the percentages of the γδT cell were 9.6%,54.6%and 82.4%,respectively.There was a few γδT cells in propagation on the 5th day of culture,and almost all γδT cell divisions were above 6 generations on the 10th and 15th day of culture.Pretreatment of the Rotflerin significantly suppressed the γδT cell proliferation,but after 10 d culture,there were still a few parts of γδT cells in propagation.Meanwhile,after 7,14,and 21d of culture,upon stimulation with PMA+Ionomycin,the IFN-γ producing-γδT cells were about 80%at all times.But only after 21d culture,IL-4-producing γδT cells was 2.6%.,The percentage of IFN-γ producing γδT cells markedly reduced in Rottlerin group(P＜0.01).IL-4 secretion of the γδT cells was almost completely blocked.Conclusion PKCO signal pathway involves in activation,proliferation and differentiation of the γδT lymphocytes stimulated by Mtb-Ag.