Adhesion Molecule Lsectin A Negative Regulator Of T Cell Function In Liver Injury Research

Recently we have cloned a novel C-type lectin LSECtin, which is a member of a family comprising CD23, DC-SIGN and DC-SIGNR. The LSECtin gene family plays an impotent role in immunity system. LSECtin protein can bind activated T cells. A tissue chip data show that LSECtin is a membrane protein and mainly express on LSEC and Kupffer in liver. In this study, we want to determine the role of LSECtin in liver immunology.To identify LSECtin ligand on activated T cells, we performed an immunoblot analysis and an immunoprecipitation analysis on lysates of surface-biotinylated activated Jurkat cells with LSECtin-Fc. Then a 140kD and an 80kD ligand specifically interacted with LSECtin-Fc. Mass Spectrometric analysis suggested that CD44 was a potential LSECtin ligand. The interaction between LSECtin and CD44 was confirmed with immunoprecipitation and ELISA-based binding assay. Binding of LSECtin to activated T cells was inhibited with the treatment with N-linked glycosylation inhibitor on the contrary the treatment with O-linked glycosylation inhibitor. The mutant LSECtin with Ca²⁺ site 2 mutated in carbohydrate recognition domain (CRD) can not bind CD44 transfected cells. These results indicated that the interaction between LSECtin and CD44 is infected with protein glycosylation and carbohydrate recognition domain.The proliferative response of PBL to stimulation with anti-CD3 mAb was measured in a BrdU ELISA assay. LSECtin binding decreased the member of PBL in vitro. LSECtin binding increased the apoptosis rate of activated T cells. Detection of cytokines in the culture medium by ELISA, the expression of IL2, IFN-γin activated T cells were reduced by treated with LSECtin-Fc. These results indicated that LSECtin is a molecule which can negatively regulate T cell immunity in vitro. The transgenic RNAi mice were product through conventional pronuclear injection of a shRNA expressing cassette. The mice which LSECtin expression level was knock-down can also create with a modified hydrodynamic transfection method to transfect siRNA in vivo. The hepatic cell damage was more pronounced serious in LSECtin KD (knock down) mice compared with the LSECtin WT (wide type) mice after ConA injection. Detection the cytokine mRNA in the liver of LSECtin WT and LSECtin KD mice was measured in a realtime-PCR assay. The level of IL-2, IL-4, IL-6, IFN-γFas and FasL were increased in LSECtin KD mice compared with the LSECtin WT mice after ConA injection. The mice treated with LSECtin expression plasmid injection in vivo can resist the liver damage induced by ConA.In conclusion, as a new cell adhesion molecule in liver, LSECtin binds activated T cell in a CD44 dependent manner, and can negatively regulate T cell immunity in vitro and in vivo, thereby represents an important mechanism for regulation of the pathology in liver diseases and the immune tolerance in liver.