Activation of T cell death-associated gene 8 regulates the cytokine production of T cells and macrophages in vitro

• Published in: European journal of pharmacology 2012-05, Vol.683 (1-3), p.325-331
• Main Authors: Onozawa, Yoshiko, Fujita, Yoshifumi, Kuwabara, Harumi, Nagasaki, Miyuki, Komai, Tomoaki, Oda, Tomiichiro
• Format: Article
• Language: English
• Subjects: Acidification; adenosine; agonists; AMP; Animal models; animal ovaries; Animals; Anti-Inflammatory Agents, Non-Steroidal - pharmacology; Antibodies; Cell activation; Cells, Cultured; CHO Cells; Cricetinae; Cricetulus; Cyclic AMP; Cytokine; Cytokines; Cytokines - metabolism; disease models; Drug Discovery; Female; G protein-coupled receptors; G-protein-coupled receptor; genes; GPR65; HEK293 Cells; Humans; Inflammation; Interleukin 10; Interleukin 2; Interleukin 6; Knockout mice; Lipopolysaccharides; Lymphocytes T; Macrophages; Macrophages - drug effects; Macrophages - immunology; Macrophages - metabolism; Male; Mice; Mice, Knockout; Ovarian cancer; ovarian neoplasms; Peritoneum; pharmacology; Protons; Pyridazines - pharmacology; Receptors, G-Protein-Coupled - agonists; Receptors, G-Protein-Coupled - genetics; Receptors, G-Protein-Coupled - metabolism; Recombinant Proteins - agonists; Recombinant Proteins - metabolism; screening; Small Molecule Libraries - pharmacology; Spleen - cytology; Spleen - drug effects; Spleen - immunology; Spleen - metabolism; Splenocytes; T cell death-associated gene 8; T-lymphocytes; T-Lymphocytes - drug effects; T-Lymphocytes - immunology; T-Lymphocytes - metabolism; Thiadiazines - pharmacology; Tumor necrosis factor- alpha
• ISSN: 0014-2999; 1879-0712
• DOI: 10.1016/j.ejphar.2012.03.007

An orphan G-protein-coupled receptor, T cell death-associated gene 8 (TDAG8) which has been reported to be a proton sensor, inhibits the production of pro-inflammatory cytokines induced by extracellular acidification. Recently, we have found that TDAG8 knockout mice showed significant exacerbation in various immune-mediated inflammation disease models. To elucidate the role of TDAG8, we screened an in-house library to find compounds which have a profile as a TDAG8 agonist using a cyclic adenosine 5′-monophosphate assay. Among the screening hits, we focused on (3-[(2,4-dichlorobenzyl)thio]-1,6-dimethyl-5,6-dihydro-1H-pyridazino[4,5-e][1,3,4]thiadiazin-5-one) (named BTB09089). BTB09089 did not act on other proton sensing G-protein-coupled receptors such as G-protein-coupled receptor 4 (GPR4) nor ovarian cancer G-protein-coupled receptor 1 (OGR1). Moreover, BTB09089 increased cAMP level in the splenocytes from wild-type littermates but not from TDAG8-deficient mice. Thus, BTB09089 was found to be a TDAG8 specific agonist. We then investigated the effects of BTB09089 on T cells and macrophages in vitro. In splenocytes, BTB09089 suppressed the production of IL-2 stimulated with anti-CD3 and anti-CD28 antibodies. In peritoneal exuded macrophages induced by thioglycollate, BTB09089 suppressed the production of TNF-α and IL-6 while it increased that of IL-10 when stimulated with lipopolysaccharide. These effects were observed in cells from wild type mice, but not those from TDAG8 knockout mice. These results indicate that activation of TDAG8 attenuates immune-mediated inflammation by regulating the cytokine production of T cells and macrophages.