Influence of stimulation by LPS and CD40 ligandization in vitro on the TLR4-MD2 expression and IL-12 production in DCs modified by sCD40 gene

AIM: To study the influence of stimulation by LPS and CD40 ligandization in vitro on the TLR4-MD2 expression and IL-12 production in dendritic cells (DCs) modified by sCD40 gene and provide the experimental clues of inducing dornor-specific immune tolerance.METHODS: Plasmid pEGFP-N1/sCD40 and pEGFP-N1 was transfected into DC2.4 cell line with lipofectamine.After 6 h of treatment with LPS and anti-CD40mAb,the expression of TLR4-MD2 on DCs was determined with flow cytometry (FCM) and RT-PCR.IL-12p70 protein was detected by ELISA.RESULTS: LPS treatment of DCs down-regulated surface expression of TLR4-MD2,LPS treatment along with anti-CD40mAb significantly up-regulated TLR4-MD2 surface expression.CD40 ligandization did not affect TLR4-MD2 mRNA expression in DCs but partly increased its low level induced by LPS and markly enhanced IL-12p70 secretion after LPS stimulation.DCs modified by sCD40 gene inhibited the above effect.CONCLUSION: After treatment with LPS and anti-CD40mAb,DCs modified by sCD40 gene down-regulate surface expression of TLR4-MD2 and IL-12p70 secretion decreases significantly,which might be linked with the interruption of TLR4-MD2 transportation from cytoplasm.