The Effect of Adenosine on TLR Signaling Pathways in Human Monocytic THP-1 Cells.

Adenosine is an important metabolite that serves as a potent regulator of inflammation and mediates various biological functions in different cell types. Adenosine inhibits the proinflammatory actions of inflammatory and immune cells via interaction with its receptors, particularly A2A receptors. Adenosine receptors belong to the seven-transmembrane G-protein coupled receptors and via the different G proteins transfer signals through different effectors including adenyl cyclase, PKA, PKC, PI3K, and MAP kinases. The mechanisms by which the adenosine regulates immune responses and how adenosine receptor pathways interact with other signaling pathways are currently unknown. Toll-like receptors (TLRs) of the innate immune cells recognize conserved microbial structures, such as bacterial lipopolysaccharide and viral double-stranded RNA, and activate signaling pathways that result in innate immune responses against microbial infections. Fcγ receptors of the innate cells play a critical role in the clearance of pathogens, regulation of inflammation and co-ordination of the immune response. We seek to understand the interaction between adenosine (via adenosine receptors) and TLR- and Fcγ R- mediated signaling pathways. We have initiated study on the effect of adenosine and lipopolysaccharide (LPS) on expression of TLR2, TLR4, FcγRI and Fcγ RII receptors in the human monocytic cell line THP-1. We incubated cells with 100μM adenosine for three hours at 37°C and assayed the expression of receptors using flow cytometry. Our results suggest that adenosine increases the expression of TLR2 and TLR4 in both undifferentiated cells and the cells induced to become macrophages by phorbol ester. Incubation with adenosine for 24 hours further increases the expression of TLR2 and TLR4 in both undifferentiated and differentiated THP-1 cells. Similarly, incubation with LPS for three hours increases the expression TLR2 and TLR4 in both undifferentiated and differentiated THP-1 cells. In contrast, the expression level of FcγRI and FcγRII receptors do not change in the presence of either adenosine or LPS. These observations suggest that adenosine specifically enhances expression of TLRs but not Fcγ receptors. To further understand the interaction between adenosine and TLR pathways, we are continuously investigating the effect of adenosine on expression and the protein modification (e.g. phosphorylation) of TLR2, TLR4, and the molecules in the TLR signaling cascades including MyD88, IRAK and MAP kinases using real-time RT-PCR and western blotting.