ABSTRACT
Epstein-Barr virus-induced gene 3 (EBI3) is a subunit of the composite cytokines IL-27 and IL-35. Both have beneficial functions or effects in models of infectious and autoimmune diseases. This suggests that administration of EBI3 could be therapeutically useful by binding free p28 and p35 to generate IL-27 and IL-35. IL-27- and IL-35-independent functions of EBI3 could compromise its therapeutic uses. We therefore assessed the effects of EBI3 on cytokine receptor-expressing cells. We observed that EBI3 activates STAT3 and induces the proliferation of the IL-6-dependent B9 mouse plasmacytoma cell line. Analyses using blocking mAbs and Ba/F3 transfectants expressing gp130 indicate that EBI3 activity was linked to its capacity to mediate IL-6 trans-signaling, albeit less efficiently than soluble IL-6Rα. In line with this interpretation, co-immunoprecipitation and SPR experiments indicated that EBI3 binds IL-6. An important pro-inflammatory function of IL-6 trans-signaling is to activate blood vessel endothelial cells. We observed that EBI3 in combination with IL-6 could induce the expression of chemokines by human venal endothelial cells. Our results indicate that EBI3 can promote pro-inflammatory IL-6 functions by mediating trans-signaling. These unexpected observations suggest that use of EBI3 as a therapeutic biologic for autoimmune diseases will likely require co-administration of soluble gp130 to prevent the side effects associated with IL-6 trans-signaling. Together with previous studies that demonstrated activation of IL-6R by p28 (IL-30), new findings further suggest a complex interrelation between IL-27 and IL-6.
Subject(s)
Interleukin-6/metabolism , Minor Histocompatibility Antigens/metabolism , Receptors, Cytokine/metabolism , Signal Transduction , Animals , Antibodies, Monoclonal/chemistry , Cell Line, Tumor , Cell Proliferation , Chemokines/metabolism , Cytokine Receptor gp130/metabolism , Female , HEK293 Cells , Human Umbilical Vein Endothelial Cells , Humans , Inflammation , Interleukins/metabolism , Mice , Mice, Inbred C57BL , Phosphorylation , Plasmacytoma/metabolism , Protein Binding , Receptors, Interleukin-6/metabolism , Recombinant Fusion Proteins/metabolismABSTRACT
Ionic liquid self-assembled monolayers (SAM) were designed and applied for binding streptavidin, promoting affinity biosensing and enzyme activity on gold surfaces of sensors. The synthesis of 1-((+)-biotin)pentanamido)propyl)-3-(12-mercaptododecyl)-imidazolium bromide, a biotinylated ionic liquid (IL-biotin), which self-assembles on gold film, afforded streptavidin sensing with surface plasmon resonance (SPR). The IL-biotin-SAM efficiently formed a full streptavidin monolayer. The synthesis of 1-(carboxymethyl)-3-(mercaptododecyl)-imidazoliumbromide, a carboxylated IL (IL-COOH), was used to immobilize anti-IgG to create an affinity biosensor. The IL-COOH demonstrated efficient detection of IgG in the nanomolar concentration range, similar to the alkylthiols SAM and PEG. In addition, the IL-COOH demonstrated low fouling in crude serum, to a level equivalent to PEG. The IL-COOH was further modified with N,N'-bis (carboxymethyl)-l-lysine hydrate to bind copper ions and then, chelate histidine-tagged biomolecules. Human dihydrofolate reductase (hDHFR) was chelated to the modified IL-COOH. By monitoring enzyme activity in situ on the SPR sensor, it was revealed that the IL-COOH SAM improved the activity of hDHFR by 24% in comparison to classical SAM. Thereby, IL-SAM has been synthesized and successfully applied to three important biosensing schemes, demonstrating the advantages of this new class of monolayers.
