Differential Expression Of Cytokines Induced By Anisomycin From T Cells And Its Function

Background：Anisomycin, as one of antibiotics, which is used as a proteinsynthesis inhibitor in memory research, of course, has a long history. Dependent onour previous finding no matter in vitro and in vivo, anisomycin could definitelyinhibit the proliferation and activation of T lymphocytes, while the expression levelsof cytokines, such as IL-2, IL-4and IFN-γ secreted by T lymphocytes were alldecreased, these results show that anisomycin may play an important role in immuneresponse medicated by T lymphocytes in mice. However, the effect about migration,chemotaxis and apoptosis and also other biological behaviors on T cells induced byanisomycin has not been reported recently. In the study we first systematicallyfocused on the differential expressions of numerous cytokines in T cells stimulated byanisomycin in order to investigate the mechanism by which T lymphocytes areinhibited with anisomycin.Objective: A large number of cytokines differently secreted by T lymphocyteswere screened out through the treatment of anisomycin, and then the effect ofdifferentially expressed cytokines MMP9and IGFBP6on apoptosis of T lymphocytesinduced by anisomycin were explored.Methods: T cells were isolated from mouse lymph nodes usingimmuno-magnetic beads-negative cell sorting and treated with anisomycin. Cytokines,with the differentially stable expression, were screened out of144cytokines used anantibody microarray technique. Total RNA was isolated by Trizol, RT-PCR and qPCRwere employed to determine alterations of ccl9, cxcl9, il-17e, igfbp6, ccl24and mmp9mRNA levels. ELISA was used to determine the level of IGFBP6andMMP9（Pro） again from the culture supernatant of the differently treated T cells. At last, we chose MMP9and IGFBP6cytokines with the stably differentialexpression for the following research, T cells were pretreated with SP600125for1hour before co-cultured with anisomycin，and the cell apoptosis was detected by FlowCytometry. The mRNA levels of igfbp6, mmp9and jnk2were detected by RT-PCRand qPCR, and the secretion level of MMP9, IGFBP6as well as other T cellcorrelated cytokines in the cell supernatant were measured by ELISA. And theexpressions of MMP9, IGFBP6, JNK and p-JNK proteins were measured by WesternBlot to investigate the correlation between MMP9(Pro) and IGFBP6with T cellapoptosis via JNK signal pathway activated by anisomycin.Results: Anisomycin down-regulated the expressions of47cytokines andup-regulated the expressions of14cytokines secreted from T cells. The expressions ofCCL9, CXCL9, CCL24and MMP9（Pro）cytokines were significantly inhibited byanisomycin, but the levels of IL-17E and IGFBP6were increased. RT-PCR, qPCR,ELISA and Western Blot results all showed that the expression of MMP9weredecreased and the expression of IGFBP6were increased by anisomycin, respectively.But SP600125could inordinately reverse the phenomenon. These results reveal thatMMP9and IGFBP6may medicate T cell apoptosis induced by anisomycin via JNKsignal pathway. In addition, our ELISA experiments showed that the secretion levelsof IL-1β, IL-6and IL-10, TGF-β1could be up-regulated and down-regulated byanisomycin respectively, but the levels of IL-6, IL-10and TGF-β1would be reversedby SP600125to some extent.Conclusions: Anisomycin can change the levels of54cytokines in all of144cytokines, of which the expression levels of CCL9,CXCL9,CCL24and MMP9（Pro）are significantly down-regulated and the expression levels of IL-17E and IGFBP6areup-regulated. Anisomycin may induce MMP9（Pro）and IGFBP6to participate in theT cell apoptosis regulated by JNK signal pathway.