Toll-like receptors (TLRs) are found on the membranes of pattern recognition receptors and not only play important roles in activating immune responses but are also involved in the pathogenesis of inflammatory disease, injury and cancer. Furthermore, TLRs are also able to recognize endogenous alarmins released by damaged tissue and necrosis and/or apoptotic cells and are present in numerous autoimmune diseases. Therefore, the release of endogenous TLR ligands plays an important role in initiating and driving inflammatory diseases. Increasing data suggest a role for TLR signaling in rheumatoid arthritis, which is an autoimmune disease. Although their involvement is not comprehensively understood, the TLRs signaling transducers may provide potential therapeutic targets.
Glucocorticoid-induced tumor-necrosis factor receptor(GITR)and its ligand,GITRL,play significant roles in regulating immune responses.It is clear that human soluble GITRL(hsGITRL)transduces signal activity through multiple oligomerization states.To develop human soluble trimeric GITRL protein as a potential therapeutic target,we explored the link of the isoleucine-zipper(ILZ)motif to the N-terminus of the human soluble GITRL with two leucine sequences.hsGITRL,with the ILZ motif(ILZ-hsGITRL),was firstly expressed in Escherichia coli,which exhibited a predominant trimer when identified by Sephadex G-100 filtration and non-reducing SDS–polyacrylamide gel electrophoresis(SDS-PAGE).The significantly higher biological activity of the ILZ-hsGITRL compared with hsGITRL was confirmed by CD41 T proliferation,interferon-c(IFN-c)secretion and binding activity assay.To reveal and compare the underlying mechanisms,the level of extracellular signal-regulated kinase-1/2(ERK1/2)phosphorylation was examined,indicating that ILZ-hsGITRL induced more persistent and stronger ERK1/2 activation than hsGITRL.In conclusion,the incorporation of an ILZ motif could markedly improve the costimulation of hsGITRL.
