Background and aims Discontinuation of long-term suppression of HBV replication with nucleos(t)ide analogues (NUCs) can result in durable immune control of hepatitis B virus (HBV) replication in HBeAg negative patients. We have assessed the effect of NUC discontinuation in HBeAg negative patients in a prospective, multicenter, randomized trial (the Stop-NUC study).
Einleitung: Chronische Hepatitis B ist der weltweit wichtigste Faktor bei der Entstehung hepatozellulärer Karzinome. Daten zu Epidemiologie und Verlauf HBV-assoziierter HCCs liegen bevorzugt aus Ländern mit hoher HBV-Prävalenz vor. In der vorliegenden Untersuchung werden die HBV-assoziiertem HCC aus einem großen deutschen Kollektiv charakterisiert.
Primary biliary cholangitis (PBC) is a chronic, cholestatic liver disease that can lead to end-stage liver disease, provoking not alone physical symptoms.
Autoimmune hepatitis (AIH) is a rare chronic liver disease. Impaired health-related quality of life (HRQL) contributes to the overall disease burden. At current, only limited data related to the impact of treatment response on HRQL are available.The aim of the study was to determine the impact of biochemical remission on HRQL.Patients with AIH were prospectively enrolled between July 2018 and June 2019. A liver disease-specific tool, the chronic liver disease questionnaire (CLDQ) and the generic EQ-5D-5L were used to quantify HRQL. Treatment response was assessed biochemically by measurement of immunoglobulin G, ALT and AST. The cohort was divided into two groups according to their biochemical remission status in either complete vs. incomplete remission. Clinical as well as laboratory parameters and comorbidities were analysed using univariable and multivariable analysis to identify predictors of poor HRQL.A total of 116 AIH patients were included (median age: 55; 77.6% female), of which 9.5% had liver cirrhosis. In this cohort, 38 (38.4%) showed a complete and 61 (61.6%) an incomplete biochemical remission at study entry. The HRQL was significantly higher in patients with a complete as compared to an incomplete biochemical remission (CLDQ overall score: 5.66 ± 1.15 vs. 5.10 ± 1.35; p = 0.03). In contrast, the generic EQ-5D-5L UI-value was not different between the groups. Multivariable analysis identified AST (p = 0.02) and an incomplete biochemical remission (p = 0.04) as independent predictors of reduced HRQL (CLDQ total value).Patients with a complete biochemical remission had a significantly higher HRQL. Liver-related quality of life in patients living with AIH is dependent on the response to immunosuppressive treatment.
Background Patients with chronic liver disease often suffer from unspecific symptoms and report severe impairment in the quality of life. The underlying mechanisms are multifactorial and include disease-specific but also liver related causes. The current analysis evaluated the association of hepatocellular apoptosis in non-viral chronic liver disease and health-related quality of life (HRQL). Furthermore we examined factors, which influence patient's physical and mental well-being. Methods A total of 150 patients with non-infectious chronic liver disease were included between January 2014 and June 2015. The German version of the Chronic Liver Disease Questionnaire (CLDQ-D), a liver disease specific instrument to assess HRQL, was employed. Hepatocellular apoptosis was determined by measuring Cytokeratin 18 (CK18, M30 Apoptosense ELISA). Results Female gender (5.24 vs. 5.54, p = 0.04), diabetes mellitus type II (4.75 vs. 5.46, p<0.001) and daily drug intake (5.24 vs. 6.01, p = 0.003) were associated with a significant impairment in HRQL. HRQL was not significantly different between the examined liver diseases. Levels of CK18 were the highest in patients with NASH compared to all other disease entities (p<0.001). Interestingly, CK18 exhibited significant correlations with obesity (p<0.001) and hyperlipidemia (p<0.001). In patients with cirrhosis levels of CK18 correlated with the MELD score (r = 0.18, p = 0.03) and were significantly higher compared to patients without existing cirrhosis (265.5 U/l vs. 186.9U/l, p = 0.047). Additionally, CK18 showed a significant correlation with the presence and the degree of hepatic fibrosis (p = 0.003) and inflammation (p<0.001) in liver histology. Finally, there was a small negative association between CLDQ and CK18 (r = -0.16, p = 0.048). Conclusion Different parameters are influencing HRQL and CK18 levels in chronic non-viral liver disease and the amount of hepatocellular apoptosis correlates with the impairment in HRQL in chronic non-viral liver diseases. These findings support the role of liver-protective therapies for the improvement of the quality of life in chronic liver disease.
Bile acids induce hepatocyte injury by enhancing death receptor-mediated apoptosis. In this study, bile acid effects on TRAIL-mediated apoptosis were examined to gain insight into bile acid potentiation of death receptor signaling. TRAIL-induced apoptosis of HuH-7 cells, stably transfected with a bile acid transporter, was enhanced by bile acids. Caspase 8 and 10 activation, bid cleavage, cytosolic cytochrome c, and caspase 3 activation by TRAIL were all increased by the bile acid glycochenodeoxycholate (GCDCA). GCDCA (100 μm) did not alter expression of TRAIL-R1/DR4, TRAIL-R2/DR5, procaspase 8, cFLIP-L, cFLIP-s, Bax, Bcl-xL, or Bax. However, both caspase 8 and caspase 10 recruitment and processing within the TRAIL death-inducing signaling complex (DISC) were greater in GCDCA-treated cells whereas recruitment of cFLIP long and short was reduced. GCDCA stimulated phosphorylation of both cFLIP isoforms, which was associated with decreased binding to GST-FADD. The protein kinase C antagonist chelerythrine prevented bile acid-stimulated cFLIP-L and -s phosphorylation, restored cFLIP binding to GST-FADD, and attenuated bile acid potentiation of TRAIL-induced apoptosis. These results provide new insights into the mechanisms of bile acid cytotoxicity and the proapoptotic effects of cFLIP phosphorylation in TRAIL signaling. Bile acids induce hepatocyte injury by enhancing death receptor-mediated apoptosis. In this study, bile acid effects on TRAIL-mediated apoptosis were examined to gain insight into bile acid potentiation of death receptor signaling. TRAIL-induced apoptosis of HuH-7 cells, stably transfected with a bile acid transporter, was enhanced by bile acids. Caspase 8 and 10 activation, bid cleavage, cytosolic cytochrome c, and caspase 3 activation by TRAIL were all increased by the bile acid glycochenodeoxycholate (GCDCA). GCDCA (100 μm) did not alter expression of TRAIL-R1/DR4, TRAIL-R2/DR5, procaspase 8, cFLIP-L, cFLIP-s, Bax, Bcl-xL, or Bax. However, both caspase 8 and caspase 10 recruitment and processing within the TRAIL death-inducing signaling complex (DISC) were greater in GCDCA-treated cells whereas recruitment of cFLIP long and short was reduced. GCDCA stimulated phosphorylation of both cFLIP isoforms, which was associated with decreased binding to GST-FADD. The protein kinase C antagonist chelerythrine prevented bile acid-stimulated cFLIP-L and -s phosphorylation, restored cFLIP binding to GST-FADD, and attenuated bile acid potentiation of TRAIL-induced apoptosis. These results provide new insights into the mechanisms of bile acid cytotoxicity and the proapoptotic effects of cFLIP phosphorylation in TRAIL signaling. tumor necrosis factor-related apoptosis-inducing ligand receptor chenodeoxycholic acid cellular FLICE inhibitory protein 4′,6-diamidino-2-phenylindole dihydrochloride deoxycholic acid death-inducing signaling complex death receptor Fas-associated death domain protein glycochenodeoxycholic acid glycodeoxycholic acid horseradish peroxidase sodium-dependent taurocholate co-transporting polypeptide phosphate-buffered saline taurochenodeoxycholic acid taurodeoxycholic acid glutathione S-transferase protein kinase C green fluorescent protein Bile acids, amphipathic molecules synthesized from cholesterol within hepatocytes, are secreted across the hepatocyte canalicular membrane into bile. Bile acids accumulate within the liver in cholestasis, a pathologic condition characterized by impaired bile acid canalicular secretion. Elevated bile acid concentrations within the liver promote liver injury and the development of liver cirrhosis and liver failure (1Greim H. Trulzsch D. Czygan P. Rudick J. Hutterer F. Schaffner F. Popper H. Gastroenterology. 1972; 63: 846-850Abstract Full Text PDF PubMed Google Scholar, 2Popper H. Annu. Rev. Med. 1968; 19: 39-56Crossref PubMed Scopus (86) Google Scholar, 3Popper H. Schaffner F. Hum. Pathol. 1970; 1: 1-24Crossref PubMed Scopus (123) Google Scholar). For example, children with the progressive familial intrahepatic cholestasis (PFIC) subtype 2 syndrome lack the canalicular transport protein for bile acid secretion, and develop a progressive liver disease caused by the inability to excrete bile acids from hepatocytes (4Strautnieks S.S. Bull L.N. Knisely A.S. Kocoshis S.A. Dahl N. Arnell H. Sokal E. Dahan K. Childs S. Ling V. Tanner M.S. Kagalwalla A.F. Nemeth A. Pawlowska J. Baker A. Mieli-Vergani G. Freimer N.B. Gardiner R.M. Thompson R.J. Nat. 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Thus, bile acid modulation of death receptor signaling plays a critical role in cholestatic liver injury. Further insights into how toxic bile acids promote death receptor cytotoxic signaling would, therefore, be of scientific and potentially therapeutic relevance. Once aggregated, both death receptors Fas and DR5/TRAIL-R2 signal cell death by inducing a death-inducing signaling complex (DISC) composed of the cytoplasmic adapter protein FADD (Fas-associated death domain) and initiator caspases, procaspase 8 and 10 (11–13). Procaspases are recruited to the DISC via homotypic interactions between death effector domains (DED) of both FADD and the initiator caspases. Once recruited to the DISC, these procaspases undergo autocatalytic processing within the DISC (14Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6182) Google Scholar, 15Shi Y. Mol. Cell. 2002; 9: 459-470Abstract Full Text Full Text PDF PubMed Scopus (1465) Google Scholar). Procaspase 8, which exists as p55 and p53 isoforms, is first cleaved to p43/41 forms releasing a C-terminal p10 subunit and then subsequently processed to produce p23, p18, and p10 subunits. The p18 and p10 subunits combine to form a heterotetramer, the active form of this protease (14Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6182) Google Scholar, 15Shi Y. Mol. Cell. 2002; 9: 459-470Abstract Full Text Full Text PDF PubMed Scopus (1465) Google Scholar). Likewise, procaspase 10, which exists as a p54 and two p59 isoforms with active protease domains, is initially cleaved to p47/43 forms releasing a C-terminal p12 subunit, and then processed to produce the active p25 and p22/17, and p12 subunits (13Kischkel F.C. Lawrence D.A. Tinel A. LeBlanc H. Virmani A. Schow P. Gazdar A. Blenis J. Arnott D. Ashkenazi A. J. Biol. Chem. 2001; 276: 46639-46646Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar). In hepatocytes, which undergo death receptor-mediated apoptosis by the so-called type-II signaling pathway (16Scaffidi C. Fulda S. Srinivasan A. Friesen C., Li, F. Tomaselli K.J. Debatin K.M. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 1675-1687Crossref PubMed Scopus (2633) Google Scholar), activated caspase 8 cleaves Bid, a proapoptotic BH3 domain-only protein (17Li H. Zhu H., Xu, C.J. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3798) Google Scholar, 18Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3085) Google Scholar). The cleaved or truncated form of Bid (tBid) translocates to mitochondria and induces cytochrome c release into the cytosol, which then binds to apoptosis-activating factor-1 (Apaf-1), resulting in activation of caspase 9, followed by activation of effector caspases, caspase 3, 6, and 7 (14Thornberry N.A. Lazebnik Y. 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Hornung F. Lenardo M.J. Hayden M.R. Roy S. Nicholson D.W. Cell Death Differ. 1998; 5: 271-288Crossref PubMed Scopus (280) Google Scholar) regulates both recruitment and processing of procaspases within the DISC (29Krueger A. Baumann S. Krammer P.H. Kirchhoff S. Mol. Cell. Biol. 2001; 21: 8247-8254Crossref PubMed Scopus (491) Google Scholar, 30Burns T.F. El-Deiry W.S. J. Biol. Chem. 2001; 276: 37879-37886Abstract Full Text Full Text PDF PubMed Google Scholar, 31Thome M. Tschopp J. Nat. Rev. Immunol. 2001; 1: 50-58Crossref PubMed Scopus (352) Google Scholar). On the mRNA level, several cFLIP splice variants exist; however, only two protein forms, cFLIP-L (55 kDa) and cFLIP-s (26 kDa), have been identified (21Irmler M. Thome M. Hahne M. Schneider P. Hofmann K. Steiner V. Bodmer J.L. Schroter M. Burns K. Mattmann C. Rimoldi D. French L.E. Tschopp J. Nature. 1997; 388: 190-195Crossref PubMed Scopus (2231) Google Scholar, 24Shu H.B. Halpin D.R. Goeddel D.V. Immunity. 1997; 6: 751-763Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 28Rasper D.M. Vaillancourt J.P. Hadano S. Houtzager V.M. Seiden I. Keen S.L. Tawa P. Xanthoudakis S. Nasir J. Martindale D. Koop B.F. Peterson E.P. Thornberry N.A. Huang J. MacPherson D.P. Black S.C. Hornung F. Lenardo M.J. Hayden M.R. Roy S. Nicholson D.W. Cell Death Differ. 1998; 5: 271-288Crossref PubMed Scopus (280) Google Scholar, 32Scaffidi C. Schmitz I. Krammer P.H. Peter M.E. J. Biol. Chem. 1999; 274: 1541-1548Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar). cFLIP-L is structurally similar to procaspase 8 in that it contains two death effector domains and a caspase-like domain. However, the caspase domain lacks the critical active site cysteine residue essential for catalytic activity. The short form of cFLIP, cFLIP-s, is also composed of two death effector domains, a structure resembling the N-terminal half of procaspase 8, but lacks the entire caspase domain (29Krueger A. Baumann S. Krammer P.H. Kirchhoff S. Mol. Cell. Biol. 2001; 21: 8247-8254Crossref PubMed Scopus (491) Google Scholar). Both cFLIP-L and cFLIP-s bind to FADD within the DISC via DED-DED homotypic interactions. cFLIP-s directly inhibits caspase 8 activation within the DISC. Interestingly, cFLIP-L is first cleaved within the DISC in a caspase dependent manner to a p43 polypeptide (33Krueger A. Schmitz I. Baumann S. Krammer P.H. Kirchhoff S. J. Biol. Chem. 2001; 276: 20633-20640Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar); this cleaved form of cFLIP-L inhibits complete processing of caspase 8 to its active subunits (29Krueger A. Baumann S. Krammer P.H. Kirchhoff S. Mol. Cell. Biol. 2001; 21: 8247-8254Crossref PubMed Scopus (491) Google Scholar, 32Scaffidi C. Schmitz I. Krammer P.H. Peter M.E. J. Biol. Chem. 1999; 274: 1541-1548Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar, 33Krueger A. Schmitz I. Baumann S. Krammer P.H. Kirchhoff S. J. Biol. Chem. 2001; 276: 20633-20640Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar). cFLIP isoforms, which are expressed by hepatocytes (34Qiao L. Studer E. Leach K. McKinstry R. Gupta S. Decker R. Kukreja R. Valerie K. Nagarkatti P., El Deiry W. Molkentin J. Schmidt-Ullrich R. Fisher P.B. Grant S. Hylemon P.B. Dent P. Mol. Biol. Cell. 2001; 12: 2629-2645Crossref PubMed Scopus (205) Google Scholar, 35Desbarats J. Newell M.K. Nat. Med. 2000; 6: 920-923Crossref PubMed Scopus (209) Google Scholar), are therefore, potent negative regulators of death receptor cytotoxic signaling. A potential mechanism by which toxic bile acids may promote death receptor cytotoxic signaling is by modulating the composition of the DISC. Thus, the overall objective of this study was to examine the effects of bile acids on the TRAIL DISC. We employed HuH-7 cells stably transfected with the sodium-dependent transporting polypeptide to ensure bile acid transport and GCDCA as the cytotoxic bile acid as its concentrations are increased in cholestasis. TRAIL-mediated death receptor signaling was examined as a relevant model of death receptor signaling modulated by bile acids (7Higuchi H. Bronk S.F. Takikawa Y. Werneburg N. Takimoto R., El- Deiry W. Gores G.J. J. Biol. Chem. 2001; 276: 38610-38618Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). The results demonstrate that bile acids sensitize cells to TRAIL-induced apoptosis by stimulating cFLIP phosphorylation, which reduces its translocation to the DISC thereby facilitating activation of caspases 8 and 10. Reagents were purchased from the following suppliers: DCA, GDCA, TDCA, CDCA, GCDCA, TCDCA, ursodeoxycholic acid (UDCA);, chelerythrine, anti-FLAG M2 mouse monoclonal antibody (IgG1), and Sepharose-CL 4B were obtained from Sigma Chemicals Co.; DAPI was from Molecular Probes Inc. (Eugene, OR); rabbit polyclonal anti-caspase 8, mouse monoclonal (IgG2b) anti-cytochrome c, rabbit polyclonal anti-caspase 3, HRP-conjugated anti-mouse IgG1 and HRP-conjugated anti-mouse Ig κ-chain were from PharMingen (San Diego, CA); rabbit polyclonal anti-DR4, goat polyclonal anti-DR5, mouse monoclonal (IgG1) anti-DR5, and rat monoclonal (IgG2a) anti-cFLIP were from Alexis (San Diego, CA); mouse monoclonal (IgG1-κ) anti-caspase 10 was from MBL (Nagoya, Japan); mouse monoclonal (IgG1) anti-FADD was from Transduction Laboratories (San Diego, CA); goat polyclonal anti-Bid was from R&D systems (Minneapolis, MN); mouse monoclonal (IgG2a) anti-Bcl-xL was purchased from Exalpha Biologicals (Boston, MA); mouse monoclonal (IgG1) anti-Bcl-2, rabbit polyclonal anti-Bax, and goat polyclonal anti-actin were from Santa Cruz Biotechnology (Santa Cruz, CA); HRP-conjugated anti-goat Ig, rabbit Ig, and mouse Ig were from Biosource (Camarillo, CA); protein G-Sepharose was from Zymed Laboratories, Inc. (San Francisco, CA); Sepharose-coupled glutathione was obtained from Amersham Biosciences; Alexa Fluor 633-conjugated donkey anti-goat IgG was obtained from Molecular Probes Inc. HuH-7 cells, a human hepatocellular carcinoma cell line stably transfected with the sodium-dependent taurocholate co-transporting polypeptide (Ntcp), were employed for this study (7Higuchi H. Bronk S.F. Takikawa Y. Werneburg N. Takimoto R., El- Deiry W. Gores G.J. J. Biol. Chem. 2001; 276: 38610-38618Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Established clones (HuH-BAT for HuH-Bile Acid Transporting) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin (100,000 units/liter), streptomycin (100 mg/liter), gentamycin (100 mg/liter), and G418 (1,200 mg/liter). The extracellular portion of human TRAIL (amino acids 95–281) was subcloned into the pFLAG expression plasmid (Sigma Chemicals Co.) between XhoI andXbaI restriction sites. The N-terminal FLAG epitope-tagged TRAIL was expressed in Escherichia coli and purified over an anti-FLAG monoclonal antibody (M2)-agarose column (Sigma Chemicals Co.) (36Schneider P. Methods Enzymol. 2000; 322: 325-345Crossref PubMed Google Scholar). Purity of the recombinant protein was determined by SDS-PAGE and silver nitrate staining. Apoptosis was quantitated by assessing the characteristic nuclear changes of apoptosis (i.e. chromatin condensation and nuclear fragmentation) using the nuclear binding dye 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) and fluorescence microscopy (37Kwo P. Patel T. Bronk S.F. Gores G.J. Am. J. Physiol. 1995; 268: G613-G621PubMed Google Scholar). Cells were lysed by incubation on ice for 30 min in lysis buffer containing 20 mm Tris-HCl (pH 7.5), 1% Triton X-100, 150 mm NaCl, 10% glycerol, 1 mm Na3VO4, 50 mm NaF, 100 mm phenylmethylsulfonyl fluoride, and a commercial protease inhibitor mixture (Complete Protease Inhibitor Mixture; Roche Molecular Biochemicals). After insoluble debris was pelleted by centrifugation at 14,000 × g for 15 min at 4 °C, the supernatants were collected. Samples were resolved by 12.5% SDS-PAGE, transferred to nitrocellulose membrane, and blotted with appropriate primary antibodies at a dilution of 1:1,000. Peroxidase-conjugated secondary antibodies were incubated at a dilution of 1:2,000 to 1:10,000. Bound antibody was visualized using the chemiluminescent substrate (ECL; Amersham Biosciences) and exposed to Kodak X-OMAT film. Cytosolic extracts for cytochromec immunoblot assay were obtained as described by Leistet al. (38Leist M. Volbracht C. Fava E. Nicotera P. Mol. Pharmacol. 1998; 54: 789-801Crossref PubMed Scopus (129) Google Scholar). Briefly, at the desired time points, the culture medium was exchanged with permeabilization buffer (210 mm d-mannitol, 70 mm sucrose, 10 mm HEPES, 5 mm succinate, 0.2 mmEGTA, 0.15% bovine serum albumin, 80 μg/ml digitonin, pH 7.2). The cells were incubated in this buffer for 5 min on ice. The permeabilization buffer was then removed and centrifuged for 10 min at 13,000 × g. Supernatants representing the cytosolic extract were employed for the immunoblot analysis. HuH-BAT cells were treated with FLAG-TRAIL (200 ng/ml) plus anti-FLAG M2 antibody (2 μg/ml) in the presence or absence of GCDCA (50 μm). At desired time points, cells were washed with cold PBS and lysed by incubation on ice for 30 min in the same lysis buffer described in immunoblot analysis. Insoluble debris was removed by centrifugation at 14,000 × g for 15 min at 4 °C. For time 0, 1 ml of cell lysate from untreated cells was supplemented with 0.5 μg of FLAG-TRAIL and 1 μg of anti-FLAG M2 antibody. After the protein concentration in the extracts was determined by Bradford assay, cell lysates containing 3 mg of protein in 0.5 ml of lysis buffer were precleared by incubation with 30 μl of Sepharose-CL 4B for 3 h at 4 °C, and then aliquots of protein G-Sepharose (30 μl) were added for an additional 2 h at 4 °C. Immune complexes were pelleted by centrifugation for 5 min at 14,000 ×g, washed five times with lysis buffer, and released from the beads by boiling for 5 min in SDS sample buffer. Samples were subjected to SDS-PAGE, transferred to nitrocellulose membrane, and sequentially probed with antibodies for DR4, DR5, caspase 8, caspase 10, FADD, and cFLIP. The pGex4T-2 construct containing GST fused to full-length FADD was kindly provided by Dr. M. Peter (University of Chicago). E. coli strain DH5α cells transformed with this construct were grown overnight in the presence of 1 mm isopropyl-d-thiogalactoside. GST-FADD was bound to glutathione-Sepharose beads according to the manufacturer's protocol and eluted by incubation with 10 mm glutathione in 50 mm Tris-HCl (pH 9.0). The eluted GST-FADD was dialyzed against PBS overnight. After HuH-BAT cells were treated with GCDCA (100 μm) for 3 h, cell lysates were prepared by freezing and thawing cells in PBS containing the same protease and phosphatase inhibitors described above for the lysis buffer. Aliquots containing 3 mg of protein were incubated for 3 h at 4 °C with purified GST-FADD. Glutathione-Sepharose was added for an additional 2 h at 4 °C. FADD-bound proteins were then pelleted by centrifugation for 5 min at 600 × g, washed five times with PBS buffer containing the protease and phosphatase inhibitors described above for the lysis buffer, and released from the beads by boiling for 5 min in SDS sample buffer. Recovery of procaspase 8, procaspase 10, and cFLIP was assessed by immunoblot analysis. A human cDNA for cFLIP was a gift from Dr. P. Dent (Virginia Commonwealth University, Richmond, VA). cDNA encoding amino acids 1–202 was subcloned into green fluorescent protein (GFP)-expression plasmid pEGFP-N1 (Clontech, Palo Alto, CA) at the Kpn-I site. The plasmid pEGFP-cFLIP was transfected into the HuH-BAT cells using LipofectAMINE Plus (Invitrogen, Carlsbad, CA). Forty-eight hours later, the cells were treated with FLAG-TRAIL plus M2 antibodies in the presence or absence of GCDCA (100 μm). The cFLIP-GFP fluorescence was continuously observed by employing fluorescence microscopy (TE200 Nikon Inverted Fluorescent Microscope, Nikon, Tokyo, Japan), and the cellular distribution of green fluorescence was visualized at 30-min intervals. To visualize co-localization of the cFLIP-GFP and DR5, immunofluorescence for DR5 immunoreactivity was performed as described previously (7Higuchi H. Bronk S.F. Takikawa Y. Werneburg N. Takimoto R., El- Deiry W. Gores G.J. J. Biol. Chem. 2001; 276: 38610-38618Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). In brief, cells were fixed with 3% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100, and incubated with goat anti-DR5 primary antisera (1:300 dilution) for 2 h at 37 °C. After washing with PBS, the cells were incubated with Alexa Fluor 633-conjugated anti-goat IgG (Molecular Probes, 10 mg/ml) for 1 h at 37 °C. Fluorescence was visualized by laser scanning confocal microscopy (Axiovert 100 m-LSM 510, Carl Zeiss Inc., Thornwood, NY). Excitation and emission wavelengths for GFP were 488 and 505 nm, respectively, and for Alexa Fluor 633 the excitation and emission wavelengths were 633 and 650 nm, respectively. HuH-BAT cells in 10-cm plates were incubated with γ-32P (200 μCi/ml) in the presence or absence of DC or GDCA (100 μm) for 3 h. Cells were lysed with the lysis buffer described under "Immunoblot Analysis." After the protein concentration in the extracts was determined, cell lysates containing 4 mg of protein in 0.5 ml of lysis buffer were precleared by incubation with 30 μl of Sepharose-CL 4B for 3 h at 4 °C. cFLIP protein was immunoprecipitated by incubation with rat monoclonal (IgG2a) anti-cFLIP (Alexis) antibody (5 μg) and protein G-Sepharose (30 μl) for an additional 2 h at 4 °C. Immune complexes were pelleted by centrifugation for 5 min at 14,000 ×g, washed five times with lysis buffer, and released from the beads by boiling for 5 min in SDS sample buffer. Samples were resolved by SDS-PAGE, and transferred to nitrocellulose membranes. Radioactivity was determined by autoradiography at −70 °C overnight. All data represent at least three independent experiments and are expressed as the mean ± S.D. unless otherwise indicated. Differences between groups were compared using analysis of variance for repeated measures and a post-hoc Bonferroni test to correct for multiple comparisons. Initially, the effects of various bile acids on TRAIL-induced apoptosis was carefully characterized using the bile acid transporting HuH-BAT cells. Cellular apoptosis was minimal (<15%) following treatment of the cells with the unconjugated and conjugated forms of DCA and CDCA (50 μm) for 4 h (Fig. 1, A and B). TRAIL itself (100 ng/ml) induced only slight apoptosis over the same time period, 27 ± 3% apoptosis. In contrast, all of the bile acids (50 μm) studied markedly sensitized cells to TRAIL-mediated apoptosis. Of the bile acids examined, unconjugated and glycine conjugates of DCA and CDCA were the most potent in enhancing TRAIL-induced apoptosis, and increased TRAIL-mediated apoptosis ∼4-fold (Fig. 1, A and B). The ability of bile acids to enhance TRAIL-mediated apoptosis was concentration-dependent (Fig. 2). Both GCDCA and GDCA at a concentration of 50 μm enhanced TRAIL-mediated apoptosis as the concentration of TRAIL was increased (0–1,600 ng/ml), with maximum cell killing at TRAIL concentrations of ≥400 ng/ml (Fig. 2,A and C). Likewise, cells were increasingly sensitized to TRAIL (50 ng/ml) cytotoxicity as the concentration of bile acid was increased (0–200 μm); maximum cell killing was observed with a bile acid concentration of 100 μm for GCDCA and a concentration 50 μm for GDCA (Fig. 2,B and D). At all the conditions tested, cellular apoptosis was synergistically increased by the combination of the bile acid and TRAIL. These data demonstrate that bile acids sensitize cells to TRAIL-mediated apoptosis. Because GCDCA is one of the major bile acids in human bile (39Hofmann A.F. Hepatology. 1990; 12 (; discussion 22S-25S.): 17S-22SPubMed Google Scholar), we choose GCDCA for subsequent experiments.Figure 2Glycine-conjugated bile acids potently sensitize cells to TRAIL-mediated apoptosis. HuH-BAT cells were incubated with indicated concentrations of FLAG-TRAIL plus anti-FLAG M2 antibody (2 μg/ml) or M2 antibody alone in the presence or absence of either GCDCA (panels A and B) or GDCA (panels C and D) for 12 h. Apoptosis was evaluated by DAPI staining and fluorescence microscopy. All data were expressed as mean ± S.D. from three individual experiments.p < 0.01 for no bile acid group versuseither GCDCA (panel A) or GDCA-treated group at all the TRAIL concentrations tested. p < 0.01 for TRAIL-treated group versus no TRAIL group at all the bile acid concentrations tested (GCDCA in panel B, GDCA inpanel D).View Large Image Figure ViewerDownload Hi-res image Download (PPT) TRAIL-mediated apoptosis in HuH-BAT cells, a hepatocyte-derived cell line, occurs via the Type II pathway (Bid cleavage, cytochrome c release, and subsequent activation of caspase 3). Bile acids, therefore, could sensitize cells to TRAIL cytotoxicity by either enhancing initiator caspase activation and Bid cleavage and/or promoting mitochondrial dysfunction with cytochromec release. To distinguish between these two possibilities, immunoblot analysis for apoptotic effector proteins upstream (caspase 8/10, tBid) and downstream of mitochondria (cytosolic cytochromec and caspase 3) was examined. GCDCA (50 μm) alone did not activate this signaling cascade. After treatment of HuH-BAT cells with TRAIL (100 ng/ml) for 0–8 h, immunoblot analysis of cell lysates demonstrated processed polypeptides for both initiator caspases, caspase 8 and 10, appearance of
Hepatocyte apoptosis by death receptors, hepatic inflammation, and fibrosis are prominent features of liver diseases. However, the link between these processes remains unclear. Our aim was to ascertain whether engulfment of apoptotic bodies by Kupffer cells promotes hepatic inflammation and fibrosis. Isolated murine Kupffer cells efficiently engulfed apoptotic bodies generated from UV–treated mouse hepatocytes. Engulfment of the apoptotic bodies, but not latex beads, stimulated Kupffer cell generation of death ligands, including Fas ligand, and tumor necrosis factor α (TNF–α). Both apoptotic body phagocytosis and death ligand generation were attenuated by gadolinium chloride, a Kupffer cell toxicant. Kupffer cells isolated from 3–day bile duct–ligated (BDL) mice were phenotypically similar to apoptotic body–“fed” Kupffer cells with enhanced death ligand expression; inhibition of hepatocyte apoptosis with a caspase inhibitor prevented this Kupffer cell activation. Consistent with a role for Kupffer cells in liver inflammation and fibrosis, gadolinium chloride attenuated neutrophil infiltration and markers for stellate cell activation. In conclusion, these findings support a model of cholestatic liver injury where Kupffer cell engulfment of apoptotic bodies promotes inflammation and fibrogenesis.
