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Hepatocellular thyroid hormone efflux is a novel mechanism for regulation of thyroid hormone action
288A
725
AASLD ABSTRACTS
ADENOVIRUS-MEDIATEDEXPRESSION OF CINC/GRO (IL-8) IN RAT LIVER INDUCES A NEUTROPHILIC HEPATITIS. 1| Maher. MK Scott. MC Burton. Liver Center Laboratory and Department of Medicine, University of Califomia, San Francisco. Interleukin-8 (IL-8)is a potent nentrophil chemoattractant produced in the liver by hepatocytes and Kupffer cells. It has recently been implicated in the pathogenesis of alcoholic hepatitis, based on a positive correlation between liver/plasma IL-8levels and hepatic injury. Despite the association between IL-8and nentrophilic liver injury, it remains uncertain whether liver-derived IL-8 actually provokes anentrophilic hepatitis in vivo. The objective of this study was to determine the pathogenic potential of liver-derived IL-8, by overexpressing its rat analogue CINC/gro in rat liver via adenovirusmediated gene delivery. Methods: A 291-bp cDNA encoding CINC/gro was cloned and inserted into an EIA-deleted Ad5 expression vector (pABL1CMV). Cotransfection of the resulting plasmid into 293 cells with pJM17 yielded a replication-defective recombinant adenovirus (Ad5-CMV-CINC). The virus was administered to intact rats via portal vein injection; four days after infection, CINC mRNA and protein were measured in liver and plasma, along with biochemical and histologic parameters of liver injury. Results: Rats infected with Ad5-CMV-CINC exhibited a dose-dependent increase in CINC expression at 4 days. 1 x 109 pfu were sufficient to induce CINC mRNA; 5 x 109 pfu were required to stimulate CINC production. Virusinfected rats exhibited substantially higher levels of liver and plasma CINC (liver, 62.4 vs 4.2 pg,/mg protein; plasma, 624 vs 228 pg/ml) and significantly larger numbers of circulating neutropbils (5.6 vs 0.8 K/I.d) than did uninfected rats. Hepatic transaminases and bilirubin were also increased (ALT, 263 vs 60 IU/L; Bili, 1.3 vs 0.1 mg/dl). Histology revealed a severe hepatitis, with hepatocytes surrounded by nentrophils. Control rats, infected with 1 x 10TM pfu of At5 without CINC, exhibited mild neutrophilia but no biochemical or histologic liver damage. Summary: CINC/gro, when produced in sufficient quantity by rat liver cells, induces a nentrophilic hepatitis. Conclusion: The data suggest that in humans, IL-8 may cause nentrophil recruitment to the liver and contribute importantly to conditions such as alcoholic hepatitis. (Supported by AA07810 and the ABMRF).
727 IDENTIFICATION
OF A DYSFUNCTIONAL MUTATION IN THE HUMAN ILEAL BILE A C I D TRANSPORTER GENE. P Oelkers. MH Won(]. and PA Dawson. Dept. of Internal Medicine, Bowman Gray School of Medicine, Winston-Salem, NC 27157 In order to screen for mutations in the human ileal Na*dependent bile acid transporter gene that may be associated with human disease, we have employe d cDNA cloning, COS cell expression, genomic cloning; and single-stranded DNA conformation polymorphism techn!ques. In the course of these studies, a dysfunctional ileal bile acid transporter isoform was isolated from a cDNA library. The library had been constructed using ileal mRNA isolated from a patient with Crohn's disease. Sequence analysis of the isoform revealed a single C to T transition resulting in a proline to serine substitution at amino acid position 290. Proline 290 is located near the extracellular face of the seventh predicted transmembrane domain of the human ileal bile acid transporter. In transfected COS cells, the single amino acid change abolished taurocholate transport activity but did not alter the transporter's synthesis or subcellular distribution. Sequence analysis revealed that affected proband with Crohn's disease was heterozygous for ti3e mutation. The inheritance of this mutation in the proband's family was confirmed by single stranded DNA conformation polymorphism analysis and DNA sequencing. This is the first identification and characterization of a naturally occurring defect in a human Na*-dependent bile acid transporter. The identification of this mutation will facilitate understanding the molecular mechanism of ileal bile acid transport and its role in humandiseaSe states.
HEPATOLOGYOctober 1995
726 DOES ANTIBODY RESPONSE TO CARBONIC ANHYDRASE II RELATE TO CHOLANGIOPATHIES? AN APPROACH USING A RODENT MODEL. Y Ueno. M Ishii. S. Takahashi. T. Toyota. NF LaRusso*. JR Sendal Hospital, Tohoku University, Sendal, Japan, and *Mayo Clinic, Rochester, MN. Autoimmune cholangitis is a newly described syndrome with histologic features similar to primary bfliary cirrhosis but negative for antimitochondrial antibody. Recent reports suggest a close relationship between autoimmune cholangitis and an autoantibody response to carbonic anhydrase II (CA-H), a protein known to be expressed in the liver in bile duct epithelial cells (cholangiocytes) but not hepatoeytes. Recently, we developed a rodent model of immune-cholangitis via active immunization with purified cholangiocytes (Hepatology 18.'170A, 1993). The AIM of this study was to investigate the relationship between antibody response to CA-l] and cholangitis using a rodent model. Methods: Ten Balb/c mice were immunized three times with 20 lag CAII intraperitoneally on an every other week schedule. Control animals were immunized with bovine serum albumin (BSA). After fmal immunization, tissues (liver, colon, kidney and salivary gland) were evaluated under coded identification. Antibody responses were examined by immunoblotting and immunohistochemistry. Serum liver enzymes (ALT, ALP, 'yGT) were also measured. Results: 1) All the animals immunized with CA-II developed histological evidence of moderate colitis. Six of ten CA-II-immunized animals developed mild cholangitis characterized by mononuclear cell infiltration in the portal tracts. No animals showed inflammation in the salivary gland or kidney. Tissue from BSA-immunized animals was negative for inflammation in all organs. 2) All animals immunized with CA-II developed serum antibodies toward CA-II as assessed by immunoblotting. Immunohistochemistry of the liver with CA-lI-immunized mice sera showed staining of intrahepatic bile ducts, whereas control-immunized sera did not show such staining. 3) No serum enzyme changes eecurred throughout the experiment. Conclusions: These results show that immunization with CA-l] induces eholangl'tis and colitis in mice; these changes are associated with the development of antibodies to CA-l] which appears to decorate intrahepatic bile duct epithelial cells. This animal model should prove useful in studying the mechanisms of immune-mediated cholangitis.
728 HEPATOCELLULAR THYROID HORMONE EFFLUX IS A NOVEL MECHANISM FOR REGULATION OF THYROID HORMONE ACTION. RCJ Ribeiro. RR Cavalieri. N Lomri. C Rahmaoui. JD Baxter. and BF Scharschmidt, Metabolic Research Unit and Liver Center, Univ. of CA, San Francisco 94143. TH regulate growth, differentiation and other critical functions in many cells, including hepatocytes, through nuclear TH receptors (TRs). While TH response is dependent on access of TH to TRs, prior studies of cellular TH transport have been inconclusive and there is no prior evidence that TH transport can affect TR occupancy or TH response. We have recently developed specially adapted HTC rat hepatoma cells (HTCR cells), selected for resistance to a bile acid ester; that exhibit enhanced bile acid secretion and plasma membrane over-expression of novel ATPbinding cassette (ABC)/multidrug-resistance (mdr)-related proteins (PNAS 92:5421, 1995). Because most ABC proteins are transporters, we tested the hypothesis that HTC-R cells exhibit altered responsiveness to, and enhanced transport of TH. Methods and Results, (a) Sensitivity to T3 and T4, measured following co-transfection with cDNAs encoding the 131 TR isoform and a reporter gene with a TH-responsive element upstream of CAT coding sequences, was ~10-tbld less in HTC-R ceils than in parental HTC cells; maximal TH response was equal. HTC-R and HTC cells exhibited equal sensitivity to estradiol. (b) T3 entry (to 5min) into HTC-R and HTC cells was equal, yetT3 content in HTC-R cells at 1-4 hours was only half that in HTC cells, a difference not explained by altered TH metabolism. (c) T3 efflux from HTC-R cells was ~4x faster than from HTC cells, but was reduced to the rate seen in HTC cells by 4°C incubation, excess (10-4M) T4 or T3, and verapamil, a known inhibitor of transport by mdr/P-glycoproteins. Estradiol efflux was equal from HTC and HTC-R cells. (d) Verapamil increased the cellular and nuclear content ofT3 in HTC-R cells -2-fold, indicating that their lower TH content was due to enhanced TH eftlux. (el Verapamil-sensitive TH efflux was also observed in rat hepatocytes, ventriculocytes, atriocytes, and fibroblasts in primary culture. Conclusions. Active, saturable and selective extrusion of TH, likely mediated by novel ABC/mdr-related protein(s), governs cellular levels of, and response to TH in HTC-R cells, presumably by regulating access of TH to TRs. This novel / transport mechanism, which appears operative in hepatocytes and other TH target cells, may explain tissue-specific TH responsiveness.
725
AASLD ABSTRACTS
ADENOVIRUS-MEDIATEDEXPRESSION OF CINC/GRO (IL-8) IN RAT LIVER INDUCES A NEUTROPHILIC HEPATITIS. 1| Maher. MK Scott. MC Burton. Liver Center Laboratory and Department of Medicine, University of Califomia, San Francisco. Interleukin-8 (IL-8)is a potent nentrophil chemoattractant produced in the liver by hepatocytes and Kupffer cells. It has recently been implicated in the pathogenesis of alcoholic hepatitis, based on a positive correlation between liver/plasma IL-8levels and hepatic injury. Despite the association between IL-8and nentrophilic liver injury, it remains uncertain whether liver-derived IL-8 actually provokes anentrophilic hepatitis in vivo. The objective of this study was to determine the pathogenic potential of liver-derived IL-8, by overexpressing its rat analogue CINC/gro in rat liver via adenovirusmediated gene delivery. Methods: A 291-bp cDNA encoding CINC/gro was cloned and inserted into an EIA-deleted Ad5 expression vector (pABL1CMV). Cotransfection of the resulting plasmid into 293 cells with pJM17 yielded a replication-defective recombinant adenovirus (Ad5-CMV-CINC). The virus was administered to intact rats via portal vein injection; four days after infection, CINC mRNA and protein were measured in liver and plasma, along with biochemical and histologic parameters of liver injury. Results: Rats infected with Ad5-CMV-CINC exhibited a dose-dependent increase in CINC expression at 4 days. 1 x 109 pfu were sufficient to induce CINC mRNA; 5 x 109 pfu were required to stimulate CINC production. Virusinfected rats exhibited substantially higher levels of liver and plasma CINC (liver, 62.4 vs 4.2 pg,/mg protein; plasma, 624 vs 228 pg/ml) and significantly larger numbers of circulating neutropbils (5.6 vs 0.8 K/I.d) than did uninfected rats. Hepatic transaminases and bilirubin were also increased (ALT, 263 vs 60 IU/L; Bili, 1.3 vs 0.1 mg/dl). Histology revealed a severe hepatitis, with hepatocytes surrounded by nentrophils. Control rats, infected with 1 x 10TM pfu of At5 without CINC, exhibited mild neutrophilia but no biochemical or histologic liver damage. Summary: CINC/gro, when produced in sufficient quantity by rat liver cells, induces a nentrophilic hepatitis. Conclusion: The data suggest that in humans, IL-8 may cause nentrophil recruitment to the liver and contribute importantly to conditions such as alcoholic hepatitis. (Supported by AA07810 and the ABMRF).
727 IDENTIFICATION
OF A DYSFUNCTIONAL MUTATION IN THE HUMAN ILEAL BILE A C I D TRANSPORTER GENE. P Oelkers. MH Won(]. and PA Dawson. Dept. of Internal Medicine, Bowman Gray School of Medicine, Winston-Salem, NC 27157 In order to screen for mutations in the human ileal Na*dependent bile acid transporter gene that may be associated with human disease, we have employe d cDNA cloning, COS cell expression, genomic cloning; and single-stranded DNA conformation polymorphism techn!ques. In the course of these studies, a dysfunctional ileal bile acid transporter isoform was isolated from a cDNA library. The library had been constructed using ileal mRNA isolated from a patient with Crohn's disease. Sequence analysis of the isoform revealed a single C to T transition resulting in a proline to serine substitution at amino acid position 290. Proline 290 is located near the extracellular face of the seventh predicted transmembrane domain of the human ileal bile acid transporter. In transfected COS cells, the single amino acid change abolished taurocholate transport activity but did not alter the transporter's synthesis or subcellular distribution. Sequence analysis revealed that affected proband with Crohn's disease was heterozygous for ti3e mutation. The inheritance of this mutation in the proband's family was confirmed by single stranded DNA conformation polymorphism analysis and DNA sequencing. This is the first identification and characterization of a naturally occurring defect in a human Na*-dependent bile acid transporter. The identification of this mutation will facilitate understanding the molecular mechanism of ileal bile acid transport and its role in humandiseaSe states.
HEPATOLOGYOctober 1995
726 DOES ANTIBODY RESPONSE TO CARBONIC ANHYDRASE II RELATE TO CHOLANGIOPATHIES? AN APPROACH USING A RODENT MODEL. Y Ueno. M Ishii. S. Takahashi. T. Toyota. NF LaRusso*. JR Sendal Hospital, Tohoku University, Sendal, Japan, and *Mayo Clinic, Rochester, MN. Autoimmune cholangitis is a newly described syndrome with histologic features similar to primary bfliary cirrhosis but negative for antimitochondrial antibody. Recent reports suggest a close relationship between autoimmune cholangitis and an autoantibody response to carbonic anhydrase II (CA-H), a protein known to be expressed in the liver in bile duct epithelial cells (cholangiocytes) but not hepatoeytes. Recently, we developed a rodent model of immune-cholangitis via active immunization with purified cholangiocytes (Hepatology 18.'170A, 1993). The AIM of this study was to investigate the relationship between antibody response to CA-l] and cholangitis using a rodent model. Methods: Ten Balb/c mice were immunized three times with 20 lag CAII intraperitoneally on an every other week schedule. Control animals were immunized with bovine serum albumin (BSA). After fmal immunization, tissues (liver, colon, kidney and salivary gland) were evaluated under coded identification. Antibody responses were examined by immunoblotting and immunohistochemistry. Serum liver enzymes (ALT, ALP, 'yGT) were also measured. Results: 1) All the animals immunized with CA-II developed histological evidence of moderate colitis. Six of ten CA-II-immunized animals developed mild cholangitis characterized by mononuclear cell infiltration in the portal tracts. No animals showed inflammation in the salivary gland or kidney. Tissue from BSA-immunized animals was negative for inflammation in all organs. 2) All animals immunized with CA-II developed serum antibodies toward CA-II as assessed by immunoblotting. Immunohistochemistry of the liver with CA-lI-immunized mice sera showed staining of intrahepatic bile ducts, whereas control-immunized sera did not show such staining. 3) No serum enzyme changes eecurred throughout the experiment. Conclusions: These results show that immunization with CA-l] induces eholangl'tis and colitis in mice; these changes are associated with the development of antibodies to CA-l] which appears to decorate intrahepatic bile duct epithelial cells. This animal model should prove useful in studying the mechanisms of immune-mediated cholangitis.
728 HEPATOCELLULAR THYROID HORMONE EFFLUX IS A NOVEL MECHANISM FOR REGULATION OF THYROID HORMONE ACTION. RCJ Ribeiro. RR Cavalieri. N Lomri. C Rahmaoui. JD Baxter. and BF Scharschmidt, Metabolic Research Unit and Liver Center, Univ. of CA, San Francisco 94143. TH regulate growth, differentiation and other critical functions in many cells, including hepatocytes, through nuclear TH receptors (TRs). While TH response is dependent on access of TH to TRs, prior studies of cellular TH transport have been inconclusive and there is no prior evidence that TH transport can affect TR occupancy or TH response. We have recently developed specially adapted HTC rat hepatoma cells (HTCR cells), selected for resistance to a bile acid ester; that exhibit enhanced bile acid secretion and plasma membrane over-expression of novel ATPbinding cassette (ABC)/multidrug-resistance (mdr)-related proteins (PNAS 92:5421, 1995). Because most ABC proteins are transporters, we tested the hypothesis that HTC-R cells exhibit altered responsiveness to, and enhanced transport of TH. Methods and Results, (a) Sensitivity to T3 and T4, measured following co-transfection with cDNAs encoding the 131 TR isoform and a reporter gene with a TH-responsive element upstream of CAT coding sequences, was ~10-tbld less in HTC-R ceils than in parental HTC cells; maximal TH response was equal. HTC-R and HTC cells exhibited equal sensitivity to estradiol. (b) T3 entry (to 5min) into HTC-R and HTC cells was equal, yetT3 content in HTC-R cells at 1-4 hours was only half that in HTC cells, a difference not explained by altered TH metabolism. (c) T3 efflux from HTC-R cells was ~4x faster than from HTC cells, but was reduced to the rate seen in HTC cells by 4°C incubation, excess (10-4M) T4 or T3, and verapamil, a known inhibitor of transport by mdr/P-glycoproteins. Estradiol efflux was equal from HTC and HTC-R cells. (d) Verapamil increased the cellular and nuclear content ofT3 in HTC-R cells -2-fold, indicating that their lower TH content was due to enhanced TH eftlux. (el Verapamil-sensitive TH efflux was also observed in rat hepatocytes, ventriculocytes, atriocytes, and fibroblasts in primary culture. Conclusions. Active, saturable and selective extrusion of TH, likely mediated by novel ABC/mdr-related protein(s), governs cellular levels of, and response to TH in HTC-R cells, presumably by regulating access of TH to TRs. This novel / transport mechanism, which appears operative in hepatocytes and other TH target cells, may explain tissue-specific TH responsiveness.