A G Protein-coupled Receptor Responsive to Bile Acids

Abstract

So far some nuclear receptors for bile acids have been identified. However, no cell surface receptor for bile acids has yet been reported. We found that a novel G protein-coupled receptor, TGR5, is responsive to bile acids as a cell-surface receptor. Bile acids specifically induced receptor internalization, the activation of extracellular signal-regulated kinase mitogen-activated protein kinase, the increase of guanosine 5′-O-3-thio-triphosphate binding in membrane fractions, and intracellular cAMP production in Chinese hamster ovary cells expressing TGR5. Our quantitative analyses for TGR5 mRNA showed that it was abundantly expressed in monocytes/macrophages in human and rabbit. Treatment with bile acids was found to suppress the functions of rabbit alveolar macrophages including phagocytosis and lipopolysaccharide-stimulated cytokine productions. We prepared a monocytic cell line expressing TGR5 by transfecting a TGR5 cDNA into THP-1 cells that did not express TGR5 originally. Treatment with bile acids suppressed the cytokine productions in the THP-1 cells expressing TGR5, whereas it did not influence those in the original THP-1 cells, suggesting that TGR5 is implicated in the suppression of macrophage functions by bile acids. So far some nuclear receptors for bile acids have been identified. However, no cell surface receptor for bile acids has yet been reported. We found that a novel G protein-coupled receptor, TGR5, is responsive to bile acids as a cell-surface receptor. Bile acids specifically induced receptor internalization, the activation of extracellular signal-regulated kinase mitogen-activated protein kinase, the increase of guanosine 5′-O-3-thio-triphosphate binding in membrane fractions, and intracellular cAMP production in Chinese hamster ovary cells expressing TGR5. Our quantitative analyses for TGR5 mRNA showed that it was abundantly expressed in monocytes/macrophages in human and rabbit. Treatment with bile acids was found to suppress the functions of rabbit alveolar macrophages including phagocytosis and lipopolysaccharide-stimulated cytokine productions. We prepared a monocytic cell line expressing TGR5 by transfecting a TGR5 cDNA into THP-1 cells that did not express TGR5 originally. Treatment with bile acids suppressed the cytokine productions in the THP-1 cells expressing TGR5, whereas it did not influence those in the original THP-1 cells, suggesting that TGR5 is implicated in the suppression of macrophage functions by bile acids. Bile acids are not simply byproducts of cholesterol metabolism but play essential roles in the absorption of dietary lipids and in the regulation of bile acid synthesis (1Russell D.W. Setchell K.D.R. Biochemistry. 1992; 31: 4737-4749Google Scholar). Farnesoid X receptor and pregnane X receptor have been recently identified as specific nuclear receptors for bile acids (2Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. Hull M.V. Lustig K.D. Mangelsdorf D.J. Shan B. Science. 1999; 284: 1362-1365Google Scholar, 3Parks D.J. Blanchard S.G. Bledsoe R.K. Chandra G. Consler T.G. Kliewer S.A. Stimmel J.B. Willson T.M. Zavacki A.M. Moore D.D. Lehmann J.M. Science. 1999; 284: 1365-1368Google Scholar, 4Jones S.A. Moore L.B. Shenk J.L. Wisely G.B. Hamilton G.A. McKee D.D. Tomkinson N.C. LeCluyse E.L. Lambert M.H. Willson T.M. Kliewer S.A. Moore J.T. Mol. Endocrinol. 2000; 14: 27-39Google Scholar, 5Staudinger J.L. Goodwin B. Jones S.A. Hawkins-Brown D. MacKenzie K.I. LaTour A. Liu Y. Klaassen C.D. Brown K.K. Reinhard J. Willson T.M. Koller B.H. Kliewer S.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3369-3374Google Scholar). Through the activation of farnesoid X receptor bile acids repress the expression of cholesterol 7α-hydroxylase, the rate-limiting enzyme in bile acid synthesis (2Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. Hull M.V. Lustig K.D. Mangelsdorf D.J. Shan B. Science. 1999; 284: 1362-1365Google Scholar,3Parks D.J. Blanchard S.G. Bledsoe R.K. Chandra G. Consler T.G. Kliewer S.A. Stimmel J.B. Willson T.M. Zavacki A.M. Moore D.D. Lehmann J.M. Science. 1999; 284: 1365-1368Google Scholar). The activation of pregnane X receptor by bile acids results in both the repression of cholesterol 7α-hydroxylase and the transcriptional induction of cytochrome P450 3a, the bile acid-metabolizing enzyme (4Jones S.A. Moore L.B. Shenk J.L. Wisely G.B. Hamilton G.A. McKee D.D. Tomkinson N.C. LeCluyse E.L. Lambert M.H. Willson T.M. Kliewer S.A. Moore J.T. Mol. Endocrinol. 2000; 14: 27-39Google Scholar,5Staudinger J.L. Goodwin B. Jones S.A. Hawkins-Brown D. MacKenzie K.I. LaTour A. Liu Y. Klaassen C.D. Brown K.K. Reinhard J. Willson T.M. Koller B.H. Kliewer S.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3369-3374Google Scholar). However, no cell surface receptor for bile acids has yet been identified. In hepatobiliary diseases including obstructive jaundice, viral hepatitis, and primary biliary cirrhosis, the mean serum concentration of bile acids exceeds 100 μm (range, 70–400 μm), whereas normally this remains below 10 μm (6Keane R.M. Gadacz T.R. Munster A.M. Birmingham W. Winchurch R.A. Surgery. 1984; 95: 439-443Google Scholar). At such high concentrations, bile acids are known to exhibit immunosuppressive effects on cell-mediated immunity and macrophage functions (6Keane R.M. Gadacz T.R. Munster A.M. Birmingham W. Winchurch R.A. Surgery. 1984; 95: 439-443Google Scholar, 7Kimmings A.N. van Deventer S.J.H. Obertop H. Rauws E.A.J. Gouma D.J. J. Am. Coll. Surg. 1995; 181: 567-581Google Scholar, 8Drivas G. James O. Wardle N. Br. Med. J. 1976; 26: 1568-1569Google Scholar). The phagocytic capacity of the reticuloendothelial system including Kupffer cells is depressed in cholestasis or obstructive jaundice (8Drivas G. James O. Wardle N. Br. Med. J. 1976; 26: 1568-1569Google Scholar). Cholestatic jaundice frequently causes infectious complications and endotoxemia, which are closely related to elevated serum bile acid levels (7Kimmings A.N. van Deventer S.J.H. Obertop H. Rauws E.A.J. Gouma D.J. J. Am. Coll. Surg. 1995; 181: 567-581Google Scholar, 9Pain J.A. Cahill C.J. Bailey M.E. Br. J. Surg. 1985; 72: 942-945Google Scholar). Furthermore, bile acids including deoxycholic acid (DCA) 1The abbreviations used are: DCA, deoxycholic acid; CDCA, chenodeoxycholic acid; LPS, lipopolysaccharide; IL, interleukin; TNFα, tumor necrosis factor α; GPCR, G protein-coupled receptor; CHO cells, Chinese hamster ovary cells; TGR5-GFP, a fusion protein of human TGR5 and green fluorescent protein; TLCA, taurine-conjugated lithocholic acid; CHO-TGR5 cells, CHO cells expressing human TGR5; THP-TGR5 cells, THP-1 cells expressing human TGR5; MAP kinase, mitogen-activated protein kinase; AMs, adherent alveolar macrophage cells; LCA, lithocholic acid; CA, cholic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GTPγS, guanosine 5′-3-O-(thio)triphosphate 1The abbreviations used are: DCA, deoxycholic acid; CDCA, chenodeoxycholic acid; LPS, lipopolysaccharide; IL, interleukin; TNFα, tumor necrosis factor α; GPCR, G protein-coupled receptor; CHO cells, Chinese hamster ovary cells; TGR5-GFP, a fusion protein of human TGR5 and green fluorescent protein; TLCA, taurine-conjugated lithocholic acid; CHO-TGR5 cells, CHO cells expressing human TGR5; THP-TGR5 cells, THP-1 cells expressing human TGR5; MAP kinase, mitogen-activated protein kinase; AMs, adherent alveolar macrophage cells; LCA, lithocholic acid; CA, cholic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GTPγS, guanosine 5′-3-O-(thio)triphosphate and chenodeoxycholic acid (CDCA) have been demonstrated to have inhibitory activities on the lipopolysaccharide (LPS)-induced production of cytokines in macrophages, including interleukin (IL)-1, IL-6, and tumor necrosis factor α (TNFα) (10Greve J.W. Gouma D.J. Buurman W.A. Hepatology. 1989; 10: 454-458Google Scholar, 11Calmus Y. Guechot J. Podevin P. Bonnefis M.T. Giboudeau J. Poupon R. Hepatology. 1992; 16: 719-723Google Scholar). However, the precise mechanisms involved have remained unclear. Here we show that a novel G protein-coupled receptor (GPCR), TGR5, is responsive to bile acids and discuss the possibility that bile acids suppress macrophage functions via TGR5. Expression vectors with human TGR5 cDNA (pAKKO-TGR5) and rat Gi cDNA (pAKKO-Gi) were, respectively, constructed by inserting their coding regions into pAKKO-111H (12Hinuma S. Hosoya M. Ogi K. Tanaka H. Nagai Y. Onda H. Biochim. Biophys. Acta. 1994; 1219: 251-259Google Scholar). Chinese hamster ovary (CHO) dhfr− cells stably transfected with only pAKKO-111H (mock CHO cells) were cultured in a medium and used as host cells. TGR5, luciferase, and Gi were transiently expressed in the host cells by co-transfection using a LipofectAMINE 2000 (Invitrogen). After culture overnight, the cells were incubated with test compounds for 4 h. Luciferase activity was measured with a PicaGene LT.2.0 (Toyo Ink). The expression vector with a fusion protein of human TGR5 and green fluorescent protein (TGR5-GFP), pAKKO-TGR5-GFP, was constructed by the insertion of a fused DNA so that the human TGR5- and GFP-coding regions were connected in tandem. Mock CHO cells seeded onto chambered coverglasses (Nalgene) were transfected with pAKKO-TGR5-GFP and cultured overnight. After treatment with 50 μm taurine-conjugated lithocholic acid (TLCA) for 30 min, the cells were examined under a confocal fluorescence microscope. CHO cells expressing human TGR5 (CHO-TGR5) cells were established by transfecting pAKKO-TGR5 into CHO dhfr− cells (12Hinuma S. Hosoya M. Ogi K. Tanaka H. Nagai Y. Onda H. Biochim. Biophys. Acta. 1994; 1219: 251-259Google Scholar). THP-1 cells expressing human TGR5 (THP-TGR5) cells were established by transfecting pcDNA 3.1 (Invitrogen) inserted with human TGR5 cDNA and selecting neomycin-resistant cells. CHO-TGR5 and mock CHO cells were cultured in a medium containing 0.5% dialyzed fetal bovine serum and then additionally cultured overnight in a medium containing 0.1% bovine serum albumin. The cells were preincubated with fresh medium for 3 h and then exposed to TLCA at 2 μm. Western blotting was performed with a PhosphoPlus p44/42 MAP kinase (Thr-202/Tyr-204) antibody kit (Cell Signaling Technology). Membrane fractions prepared from CHO-TGR5 and mock CHO cells as described elsewhere (13Ohtaki T. Ogi K. Masuda Y. Mitsuoka K. Fujiyoshi Y. Kitada C. Sawada H. Onda H. Fujino M. J. Biol. Chem. 1998; 273: 15464-15473Google Scholar) were suspended at 500 μg/ml in a binding buffer (pH 7.4) containing 50 mm Tris, 150 mm NaCl, 5 mm MgCl2, 1 mm EGTA, 30 μm GDP, and 0.05% CHAPS. The membrane fractions (196 μl) were mixed with TLCA (2 μl of dimethyl sulfoxide solution) and 100 nm [35S]GTPγS (Amersham Biosciences) (2 μl). After incubation at 25 °C for 60 min, the reaction mixtures were diluted with 1.8 ml of a chilled washing buffer, which was a modified binding buffer without GDP, and then filtered through nitrocellulose filters (Schleicher & Schuell). The filters were washed with 1.8 ml of the washing buffer, dried, and subjected to a liquid scintillation counter to measure [35S]GTPγS bound to the membrane fractions. CHO-TGR5 cells (2 × 104) were incubated with the samples for 20 min in the presence of 0.2 mm 3-isobutyl-1-methylxanthine (Sigma). Rabbit adherent alveolar macrophage cells (AMs) (2 × 105 cells) were treated with TLCA (200 μm) for 4 min in the presence of 1 mm3-isobutyl-1-methylxanthine. THP-TGR5 or THP-1 cells (1 × 105 cells) were treated with bile acids (50 μm) for 20 min in the presence of 1 mm3-isobutyl-1-methylxanthine. The amount of cAMP was determined with a cAMP-Screen System (Applied Biosystems). Poly(A)+RNAs from human tissues and a human blood fraction multiple tissue cDNA panel were purchased from Clontech. After a 48-h culture, AMs in the culture plates were washed twice with fresh medium. Total RNAs were extracted from the adherent cells or rabbit tissues using an Isogen (Nippongene). Random-primed cDNAs were synthesized and then subjected to quantitative reverse transcription-PCR analysis using an ABI Prism 7700 sequence detector (14Fujii R. Fukusumi S. Hosoya M. Kawamata Y. Habara Y. Hinuma S. Sekiguchi M. Kitada C. Kurokawa T. Nishimura O. Onda H. Sumino Y. Fujino M. Regul. Pept. 1999; 83: 1-10Google Scholar). AMs were obtained by the lavage of lungs of female New Zealand White rabbits weighing 2.5–3.0 kg (Kitayama LABES), purified through gradient centrifugation with a Ficoll-Paque Plus (Amersham Pharmacia), and then suspended in Dulbecco's modified Eagle's medium containing 2% fetal bovine serum, nonessential amino acids, and antibiotics. The viability of the cells was more than 95% as determined by trypan blue-exclusion tests. The cells were comprised of more than 90% macrophages as determined by phagocytic tests and morphological criteria. Rabbit AMs thereby obtained were cultured overnight and used for experiments. After pretreatment with bile acids (100 μm) for 16 h, AMs were incubated with heat-inactivated yeast cells in the presence of fresh rabbit serum for 40 min, and then the AMs containing yeast cells were counted under a microscope. In the assay for cytokine secretion, AMs were preincubated with bile acids for 1 h and then treated with 1 ng/ml LPS (Escherichia coli O111:B4, Wako) in the presence of bile acids for 12 h. THP-TGR5 or THP-1 cells were treated as in AMs with the exception of LPS concentration at 50 ng/ml. TNFα concentrations (which could be neutralized by the anti-TNFα antibody) in the supernatants were measured by bioassay using L929 cells (15Evans T.J. Mol. Biotechnol. 2000; 15: Scholar). In for in the we found a human DNA sequence coding for a novel on this we a cDNA the GPCR, as TGR5, from human We TGR5 cDNAs in TGR5 and amino acid respectively, with that in and the known TGR5 at and amino acid with and N. J.A. J. 2001; We to for TGR5 as an we a to for by S. Y. R. Kawamata Y. Hosoya M. Fukusumi S. Kitada C. Y. T. H. Sekiguchi M. Kurokawa T. Nishimura O. Onda H. Fujino M. 1998; Scholar, S. Onda H. Fujino M. J. Mol. Med. 1999; in this we a We a fused to and expression vectors of human TGR5 and rat G protein α Gi the of into CHO cells. We then more than compounds by activities induced in to intracellular cAMP production and specific to bile acids including TLCA, lithocholic acid DCA, and at 25 μm. In we that TGR5 from not only human but the examined to compounds in this assay not suggesting that TGR5 functions as a receptor for bile acids in TGR5 was to be a on sequence we expressed in CHO cells and then examined J.L. Sci. 2000; Scholar, Y. K. G. W. Y. Biol. 2000; Scholar). In the of a was at the membrane but into the in to TLCA that the of TLCA and TGR5 in the we prepared membrane fractions from CHO-TGR5 and examined [35S]GTPγS binding to fractions levels of the binding were at 1 μm the at μm TLCA in a However, such in [35S]GTPγS binding were not in the membrane fractions of mock CHO cells. signal-regulated kinase MAP kinase is in the of Y. K. G. W. Y. Biol. 2000; Scholar, J. G. J. J. M. S. M.H. 1999; Scholar). Treatment with TLCA extracellular signal-regulated kinase MAP kinase activity in CHO-TGR5 cells but not in mock CHO cells 2 In we found that TLCA, LCA, DCA, CDCA, and cholic acid induced the production of cAMP in CHO-TGR5 cells 3 at the concentrations of and bile acids did not the production of cAMP in mock CHO cells not We examined cholesterol and related compounds in cAMP production in CHO-TGR5 cells 3 The activities to increase in with and not only but and were acid and cholesterol were only but showed results that the as as the acid are for the to exhibit activity on TGR5. acid and which are for farnesoid X receptor, pregnane X receptor, and X receptor, D.J. Blanchard S.G. Bledsoe R.K. Chandra G. Consler T.G. Kliewer S.A. Stimmel J.B. Willson T.M. Zavacki A.M. Moore D.D. Lehmann J.M. Science. 1999; 284: 1365-1368Google Scholar, 4Jones S.A. Moore L.B. Shenk J.L. Wisely G.B. Hamilton G.A. McKee D.D. Tomkinson N.C. LeCluyse E.L. Lambert M.H. Willson T.M. Kliewer S.A. Moore J.T. Mol. Endocrinol. 2000; 14: 27-39Google Scholar, T.R. Mangelsdorf D.J. showed activity to TGR5. we CHO cell expressing TLCA induced a to TGR5 but not to or not results that TGR5 functions as a specific cell surface receptor for bile of cAMP production in CHO-TGR5 cells by bile acids. analyses for cAMP production induced by bile acids. The the of bile acids. of cAMP production activities in bile acids and in related CHO-TGR5 cells were treated with the compounds at 2 μm. the mean of in cAMP production in at 10 μm. acid; In the expression levels of TGR5 mRNA were high in and rabbit but in rat and not We tissue in human and rabbit by reverse levels of TGR5 mRNA were in human and whereas levels were found in tissues including and fetal In human TGR5 mRNA was in the 4 rabbit the of TGR5 mRNA was in the We a high of TGR5 mRNA in AMs, that at of the cells expressing TGR5 is a We used rabbit AMs in the experiments. increase of intracellular cAMP results in the suppression of cytokine production in macrophages T. C. T. T. S. J. Tanaka H. N. Nagai H. Scholar). In has been to as the LPS receptor R.A. Science. Scholar). as demonstrated bile acids were to macrophage functions via TGR5, we examined this TLCA was found to increase cAMP production in AMs TLCA, and suppressed phagocytic activity in AMs 5 Furthermore, TLCA the induction of cytokine TNFα, IL-6, and in AMs with LPS 5 TNFα was with LCA, DCA, and and their or inhibitory activities with the cAMP production activities on CHO-TGR5 cells. the effects of bile acids were through TGR5, we established a human monocytic cell line expressing TGR5 by transfecting an expression vector of human TGR5 into THP-1 cells. The original THP-1 cells expressed TGR5 TLCA, LCA, and induced cAMP production in THP-TGR5 cells, whereas TLCA did not so in THP-1 cells TNFα was by bile acids including TLCA, LCA, DCA, and in THP-TGR5 cells but not in THP-1 cells the inhibitory activities of bile acids on TNFα from THP-TGR5 cells those in rabbit by bile acids via TGR5 in THP-1 cells expressing TGR5. increase in cAMP production in THP-TGR5 or THP-1 cells by bile acids. suppression of TNFα in THP-TGR5 cells by bile acids. of bile acids on TNFα in THP-1 cells. THP-TGR5 or THP-1 cells were treated as in 5 with the exception of LPS concentration at 50 ng/ml. the mean with We have a novel GPCR, TGR5, on the of sequence of the TGR5 was found to be to which has been recently by S. S. T. H. S. Scholar). However, the and functions of this receptor have been In this we have demonstrated that TGR5 functions as a cell surface receptor responsive to bile acids as nuclear receptors for bile acids have been we this is the on the of a responsive to bile acids. We have found that the primary and to bile acids are in TGR5 and suggesting that TGR5 has some We to a binding of to the membrane fractions of CHO-TGR5 but showed high binding to and cell membrane fractions not We that compounds with high to TGR5 be to the binding of a to TGR5 in However, of we demonstrated that TGR5 functions as a cell surface receptor responsive to bile acids on the of of using a fusion protein of TGR5 and we found that the fusion protein was at the membrane of CHO cells, and bile acids induced the of the fusion protein from the cell membrane to the Furthermore, we demonstrated that [35S]GTPγS binding were specifically induced in the membrane fractions prepared from CHO-TGR5 by the of and is specifically induced in G to results that TGR5 is specifically by with the results of and [35S]GTPγS TGR5 is to be responsive to bile acids. The treatment of bile acids specifically induced the activation of extracellular signal-regulated kinase MAP kinase and intracellular cAMP production in CHO cells expressing TGR5. However, we could not in intracellular in CHO cells expressing TGR5, suggesting that TGR5 to but not to or have not only nuclear receptors but cell surface receptors T. T. K. Y. T. Scholar). However, that the nuclear and cell surface bile acid receptors or of bile acids showed activity on TGR5. However, are to the nuclear receptors in the of a specific bile acids as In the of the bile acids were for TGR5 than for the nuclear receptors 10 the tissue of TGR5 mRNA from those of the nuclear high levels of TGR5 mRNA were in the and whereas the nuclear receptors are expressed in the and (2Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. Hull M.V. Lustig K.D. Mangelsdorf D.J. Shan B. Science. 1999; 284: 1362-1365Google Scholar, 3Parks D.J. Blanchard S.G. Bledsoe R.K. Chandra G. Consler T.G. Kliewer S.A. Stimmel J.B. Willson T.M. Zavacki A.M. Moore D.D. Lehmann J.M. Science. 1999; 284: 1365-1368Google Scholar, 4Jones S.A. Moore L.B. Shenk J.L. Wisely G.B. Hamilton G.A. McKee D.D. Tomkinson N.C. LeCluyse E.L. Lambert M.H. Willson T.M. Kliewer S.A. Moore J.T. Mol. Endocrinol. 2000; 14: 27-39Google Scholar, 5Staudinger J.L. Goodwin B. Jones S.A. Hawkins-Brown D. MacKenzie K.I. LaTour A. Liu Y. Klaassen C.D. Brown K.K. Reinhard J. Willson T.M. Koller B.H. Kliewer S.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3369-3374Google Scholar). immunosuppressive effects of bile acids have been (6Keane R.M. Gadacz T.R. Munster A.M. Birmingham W. Winchurch R.A. Surgery. 1984; 95: 439-443Google Scholar, 7Kimmings A.N. van Deventer S.J.H. Obertop H. Rauws E.A.J. Gouma D.J. J. Am. Coll. Surg. 1995; 181: 567-581Google Scholar, 8Drivas G. James O. Wardle N. Br. Med. J. 1976; 26: 1568-1569Google Scholar, 9Pain J.A. Cahill C.J. Bailey M.E. Br. J. Surg. 1985; 72: 942-945Google Scholar, J.W. Gouma D.J. Buurman W.A. Hepatology. 1989; 10: 454-458Google Scholar, 11Calmus Y. Guechot J. Podevin P. Bonnefis M.T. Giboudeau J. Poupon R. Hepatology. 1992; 16: 719-723Google the precise mechanisms have remained unclear. The phagocytic capacity of the macrophages including Kupffer cells is depressed in cholestasis or obstructive jaundice (8Drivas G. James O. Wardle N. Br. Med. J. 1976; 26: 1568-1569Google Scholar). Furthermore, bile acids including and have been to suppress production of cytokines in macrophages, including IL-6, and TNFα (10Greve J.W. Gouma D.J. Buurman W.A. Hepatology. 1989; 10: 454-458Google Scholar, 11Calmus Y. Guechot J. Podevin P. Bonnefis M.T. Giboudeau J. Poupon R. Hepatology. 1992; 16: 719-723Google Scholar). for the is that bile acids to cell However, we that cell were more than 90% the treatment of rabbit AMs with bile acids to μm. has been that cell of are not by the incubation with μm DCA, CDCA, and acid (6Keane R.M. Gadacz T.R. Munster A.M. Birmingham W. Winchurch R.A. Surgery. 1984; 95: 439-443Google Scholar). it is that the immunosuppressive functions of bile acids are the results of to cell (10Greve J.W. Gouma D.J. Buurman W.A. Hepatology. 1989; 10: 454-458Google Scholar) that bile acids such as and TNFα in human (10Greve J.W. Gouma D.J. Buurman W.A. Hepatology. 1989; 10: 454-458Google Scholar). have demonstrated that bile acids not as measured in a that the of bile acids is not a of bile acids and In bile acids induced cAMP production in rabbit AMs and THP-TGR5 cells. has been known that an increase of intracellular cAMP results in the suppression of cytokine production in macrophages T. C. T. T. S. J. Tanaka H. N. Nagai H. Scholar). We showed that TGR5 was abundantly expressed in monocytes/macrophages and that bile acids including LCA, DCA, and TNFα in rabbit In bile acids suppressed TNFα in THP-TGR5 cells but not in THP-1 cells. results that the suppression of macrophage functions by bile acids is at via TGR5 through an increase of cAMP However, we could not that the suppression of macrophage functions was via TGR5 by of experiments. the functions of TGR5, we to for TGR5 to the TGR5 but we to RNAs TGR5 is by a sequence so that it was to We but of were to suppress the expression of TGR5. We that to this with high be results that TGR5 a in the regulation of macrophage functions by bile acids, we not the possibility that TGR5 has TGR5 mRNA is not only in tissues but in Our that TGR5 is responsive to bile acids an in the functions of TGR5 in We Y. O. and H. Onda for and H. and A. for