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Vascular adhesion protein-1 mediates adhesion and transmigration of lymphocytes on human hepatic endothelial cells
212
Poster Sessions
•7-5-•
VASCULAR ADHESION PROTEIN-1 MEDIATES ADHESION AND TRANSMIGRATION OF LYMPHOCYTES ON HUMAN HEPATIC ENDOTHELIAL CELLS
P.F. Lalor, S. Edwards, C.M. Morland, M. Salmi, S. Jalkanen, D.H. Adams. Liver Research Labs, University of Birmingham, UK Vascular Adhesion Protein-1 is a novel endothelial adhesion receptor which has restricted tissue expression. In normal conditions expression is restricted to lymph node HEV and sinusoidal and vascular endothelium in the liver suggesting it may act as a fiver addressin. In the present study we demonstrate expression of functional VAP- 1 on primary human hepatic endothelial cells in vitro and report a role for VAP- 1 in adhesion and transmigration of lymphocytes across liver endothelial cells under conditions of physiological shear stress. Human hepatic endothelial cells constitutively expressed VAP-1 which was upregulated in response to TNFb and LPS but not to TNFa or IL-1. Under static conditions adhesion of peripheral blood lymphocytes to TNF-stimulated hepatic endothelial cells was inhibited by antibodies to VAP-1 whereas VAP-1 inhibition had no effect on binding to umbilical vein or dermal endothelial cells. Under conditions of shear stress in a flow-based adhesion assay anti-VAP-1 inhibited adhesion of lymphocytes to liver endothelial cells by approx 50%. Rolling adhesion of lymphocytes was consistently reduced by treatment with VAP-1 antibody which also significantly reduced the percentage of adherent lymphocytes which transmigrated through hepatic endothelial monolayers. In conclusion we describe for the first time a primary endothelial cell line which expresses VAP-I and report the ability of VAP-1 to support both adhesion and transmigration of lymphocytes. We suggest that VAP-1 may provide a tissue-specific signal for lymphocyte recruitment to the human liver and that it plays an important role in lymphocyte transendothelial migration.
57 THE INFLUENCE OF NITRIC OXIDE INCREASE IN ETHANOL CHRONIC EXPOSURE O. Gabor, C. Ghiciuc, C. Lupusoru, I. Popovici, M. Savin. University of
Medicine and Pharmacy, lasi, Romania Aim: We studied the influence of NO increase (induced by a NO releasing substance - methyl 3-morpholinosydnonimine-N-ethylcarbamide (methyl SIN-l) in rats with chronic ethanol exposure. Material and Method: We used 10 groups, with 10 white, male rats in each. Group 1 got saline (0.05 ml/100 g/day), group 2 got methyl SIN-1 (0.55 mg/kg/day); group 3 got methyl SIN-1 (1.12 mg/kg/day); group 4 got methyl SIN-1 (4.5 mg/kg/day) and group 5 got Levamisole 10 mg/kg/day. All substances were administrated i.p., 14 days/months, 3 months. Other 5 groups (1E, 2E, 3E, 4E, 5E) got the same substances in the same doses together with ethanol 10% per os, ad libitum, 3 months. We assessed in blood sample, at the beginning and at the end of experiment, the following data: the phagocytic capacity of neutrophiles (NBT test), serum complement activity, cytolithic markers, albumin levels and oxidative enzymes. Levamisole groups were used as reference for immunological and hepatic effects. Results and Conclusions: In groups treated with minimal dose, methyl SIN-1 had hepatoprotective effects (expressed by evolution of cytolithic enzymes and albumin levels) on rats chronically expose to ethanol and also reduced the negative effects of ethanol on immunitary system and oxidative enzymes. These hepatoprotective and immunoprotective effects of methyl SIN-1 are weaker than Levamisole and are dosedepending.
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ANTI-SLA ANTIBODY IS A MARKER OF SEVERITY OF LIVER DAMAGE IN PATIENTSWITH AUTOIMMUNE LIVER DISEASE
Y. Ma 1, D.P. Bogdanos 1, R. Willams 1, G. Mieli-Vergani 2, D. Vergani I.
1Institute of Hepatology, University College Medical School," 2Department of Child Health, King's College Hospital, London, UK Soluble liver antigen (SLA)FUGA suppressor tRNA-associated antigenic protein (tRNA-AP) was originally described to be specific for type 1 and 3 autoimmune hepatitis (AIH). We have established a radiofigand assay using eukaryotic SLA/tRNA-AP and reported the presence of its antibody in AIH-1, AIH-2 and autoimmune sclerosing cholangitis (ASC). To investigate the clinical value of this new assay, 335 sera from 82 patients (34 A/H-l, 31 AIH-2 and 17 ASC) were tested, 44 being investigated prospectively from the time of diagnosis (median follow-up time 22.5 mo, 21 days-123 rot). Anti-SLA/tRNA-AP antibodies were detected in 58%, 59% and 47% patients with AIH-1, AIH-2 and ASC. Amongst 44 patients investigated prospectively, 22 (46%) were seropositive at diagnosis. Antibody levels (cpm) correlated with biochemically assessed liver disease activity (p = 0.02 and p -- 0.05 for cpm vs AST and vs bilirubin). Seropotitive was also associated with severity of liver damage assessed with the histological activity index (HAI). Of 37 patients with paired serum/liver biopsy (16 taken at diagnosis and 21 during follow-up), those seropositive had more severe liver damage (median HAI 9, range 3-12) than those seronegative (median HAl 7, range 1-11, p < 0.05). Seropositive patients also needed a longer time to achieve remission than those seronegative (median 16 mo, 3-66 mo vs median 8 mo, 23 days-23 rot, p < 0.01). Conclusion: Anti-SLA/tRNA-AP antibodies against eukaryotic protein may serve as a marker of disease activity and be used as an additional factor to predict the prognosis of autoimmune liver disease.
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CELLULAR EPITOPE MAPPING OF CYTOCHROME P4502D6 IN PATIENTSWITH AUTOIMMUNE HEPATITIS
Y. Ma l, D.P. Bogdanos l, R. Williams 1, G. Mieli-Vergani 2, D. Vergani 1.1 Institute of Hepatology, University College Medical
School; 2Department of Child Health, King's College Hospital London, UK While the B cell epitope mapping of cytochrome P4502D6 (CYP2D6), the target antigen of liver kidney microsomal antibody type 1 (LKM1) in type 2 autoimmune hepatitis (AIH-2), has been thoroughly investigated, T cell epitope studies have focused on a 31 aa region representing one of the humoral immunodominant epitopes of CYP2D6. To define the CYP2D6 T cell epitope map, 61 overlapping icosameric peptides, spanning the 497 aa of the full-length CYP2D6 were constructed (Chiton Technologies). T cell reactivity was assessed by proliferation in 14 children with AIH-2 and 59 controls: 36 had autoimmune liver disease (26 A/H-l, 10 sclerosing cholangitis) and 23 non autoimmune liver disease (3 HBV infection, 6 alphal-antitrypsin deficiency, 6 Wilson disease, 5 biliary atresia, 2 Alagille's and 1 Mauriac syndromes). Peripheral blood mononuclear cells (PBMCs) were cultured in the presence of 10 mMol of individual peptides for 7 days, a stimulation index (SI) > or = 3 being considered positive. All patients with A/H-2 recognised at least one CYP2D6 peptide (median 5, range 1-38) with SI up to 29. Six regions spanning CYP2D6 65-100, 169-220, 225-260, 257-284, 329-364 and 361-436 were identified, 4 of which overlap with known B cell epitopes. Reactivity to one of these six regions was observed in one patient with A/H-1 and in none of the other controls. Summary Six regions of CYP2D6 have been identified T cell epitopes in patients with AIH-2. Their partial overlap with B cell epitopes indicates a degree of co-localisation between T and B cells epitopes on CYP2D6.
Poster Sessions
•7-5-•
VASCULAR ADHESION PROTEIN-1 MEDIATES ADHESION AND TRANSMIGRATION OF LYMPHOCYTES ON HUMAN HEPATIC ENDOTHELIAL CELLS
P.F. Lalor, S. Edwards, C.M. Morland, M. Salmi, S. Jalkanen, D.H. Adams. Liver Research Labs, University of Birmingham, UK Vascular Adhesion Protein-1 is a novel endothelial adhesion receptor which has restricted tissue expression. In normal conditions expression is restricted to lymph node HEV and sinusoidal and vascular endothelium in the liver suggesting it may act as a fiver addressin. In the present study we demonstrate expression of functional VAP- 1 on primary human hepatic endothelial cells in vitro and report a role for VAP- 1 in adhesion and transmigration of lymphocytes across liver endothelial cells under conditions of physiological shear stress. Human hepatic endothelial cells constitutively expressed VAP-1 which was upregulated in response to TNFb and LPS but not to TNFa or IL-1. Under static conditions adhesion of peripheral blood lymphocytes to TNF-stimulated hepatic endothelial cells was inhibited by antibodies to VAP-1 whereas VAP-1 inhibition had no effect on binding to umbilical vein or dermal endothelial cells. Under conditions of shear stress in a flow-based adhesion assay anti-VAP-1 inhibited adhesion of lymphocytes to liver endothelial cells by approx 50%. Rolling adhesion of lymphocytes was consistently reduced by treatment with VAP-1 antibody which also significantly reduced the percentage of adherent lymphocytes which transmigrated through hepatic endothelial monolayers. In conclusion we describe for the first time a primary endothelial cell line which expresses VAP-I and report the ability of VAP-1 to support both adhesion and transmigration of lymphocytes. We suggest that VAP-1 may provide a tissue-specific signal for lymphocyte recruitment to the human liver and that it plays an important role in lymphocyte transendothelial migration.
57 THE INFLUENCE OF NITRIC OXIDE INCREASE IN ETHANOL CHRONIC EXPOSURE O. Gabor, C. Ghiciuc, C. Lupusoru, I. Popovici, M. Savin. University of
Medicine and Pharmacy, lasi, Romania Aim: We studied the influence of NO increase (induced by a NO releasing substance - methyl 3-morpholinosydnonimine-N-ethylcarbamide (methyl SIN-l) in rats with chronic ethanol exposure. Material and Method: We used 10 groups, with 10 white, male rats in each. Group 1 got saline (0.05 ml/100 g/day), group 2 got methyl SIN-1 (0.55 mg/kg/day); group 3 got methyl SIN-1 (1.12 mg/kg/day); group 4 got methyl SIN-1 (4.5 mg/kg/day) and group 5 got Levamisole 10 mg/kg/day. All substances were administrated i.p., 14 days/months, 3 months. Other 5 groups (1E, 2E, 3E, 4E, 5E) got the same substances in the same doses together with ethanol 10% per os, ad libitum, 3 months. We assessed in blood sample, at the beginning and at the end of experiment, the following data: the phagocytic capacity of neutrophiles (NBT test), serum complement activity, cytolithic markers, albumin levels and oxidative enzymes. Levamisole groups were used as reference for immunological and hepatic effects. Results and Conclusions: In groups treated with minimal dose, methyl SIN-1 had hepatoprotective effects (expressed by evolution of cytolithic enzymes and albumin levels) on rats chronically expose to ethanol and also reduced the negative effects of ethanol on immunitary system and oxidative enzymes. These hepatoprotective and immunoprotective effects of methyl SIN-1 are weaker than Levamisole and are dosedepending.
~-~
ANTI-SLA ANTIBODY IS A MARKER OF SEVERITY OF LIVER DAMAGE IN PATIENTSWITH AUTOIMMUNE LIVER DISEASE
Y. Ma 1, D.P. Bogdanos 1, R. Willams 1, G. Mieli-Vergani 2, D. Vergani I.
1Institute of Hepatology, University College Medical School," 2Department of Child Health, King's College Hospital, London, UK Soluble liver antigen (SLA)FUGA suppressor tRNA-associated antigenic protein (tRNA-AP) was originally described to be specific for type 1 and 3 autoimmune hepatitis (AIH). We have established a radiofigand assay using eukaryotic SLA/tRNA-AP and reported the presence of its antibody in AIH-1, AIH-2 and autoimmune sclerosing cholangitis (ASC). To investigate the clinical value of this new assay, 335 sera from 82 patients (34 A/H-l, 31 AIH-2 and 17 ASC) were tested, 44 being investigated prospectively from the time of diagnosis (median follow-up time 22.5 mo, 21 days-123 rot). Anti-SLA/tRNA-AP antibodies were detected in 58%, 59% and 47% patients with AIH-1, AIH-2 and ASC. Amongst 44 patients investigated prospectively, 22 (46%) were seropositive at diagnosis. Antibody levels (cpm) correlated with biochemically assessed liver disease activity (p = 0.02 and p -- 0.05 for cpm vs AST and vs bilirubin). Seropotitive was also associated with severity of liver damage assessed with the histological activity index (HAI). Of 37 patients with paired serum/liver biopsy (16 taken at diagnosis and 21 during follow-up), those seropositive had more severe liver damage (median HAI 9, range 3-12) than those seronegative (median HAl 7, range 1-11, p < 0.05). Seropositive patients also needed a longer time to achieve remission than those seronegative (median 16 mo, 3-66 mo vs median 8 mo, 23 days-23 rot, p < 0.01). Conclusion: Anti-SLA/tRNA-AP antibodies against eukaryotic protein may serve as a marker of disease activity and be used as an additional factor to predict the prognosis of autoimmune liver disease.
~
CELLULAR EPITOPE MAPPING OF CYTOCHROME P4502D6 IN PATIENTSWITH AUTOIMMUNE HEPATITIS
Y. Ma l, D.P. Bogdanos l, R. Williams 1, G. Mieli-Vergani 2, D. Vergani 1.1 Institute of Hepatology, University College Medical
School; 2Department of Child Health, King's College Hospital London, UK While the B cell epitope mapping of cytochrome P4502D6 (CYP2D6), the target antigen of liver kidney microsomal antibody type 1 (LKM1) in type 2 autoimmune hepatitis (AIH-2), has been thoroughly investigated, T cell epitope studies have focused on a 31 aa region representing one of the humoral immunodominant epitopes of CYP2D6. To define the CYP2D6 T cell epitope map, 61 overlapping icosameric peptides, spanning the 497 aa of the full-length CYP2D6 were constructed (Chiton Technologies). T cell reactivity was assessed by proliferation in 14 children with AIH-2 and 59 controls: 36 had autoimmune liver disease (26 A/H-l, 10 sclerosing cholangitis) and 23 non autoimmune liver disease (3 HBV infection, 6 alphal-antitrypsin deficiency, 6 Wilson disease, 5 biliary atresia, 2 Alagille's and 1 Mauriac syndromes). Peripheral blood mononuclear cells (PBMCs) were cultured in the presence of 10 mMol of individual peptides for 7 days, a stimulation index (SI) > or = 3 being considered positive. All patients with A/H-2 recognised at least one CYP2D6 peptide (median 5, range 1-38) with SI up to 29. Six regions spanning CYP2D6 65-100, 169-220, 225-260, 257-284, 329-364 and 361-436 were identified, 4 of which overlap with known B cell epitopes. Reactivity to one of these six regions was observed in one patient with A/H-1 and in none of the other controls. Summary Six regions of CYP2D6 have been identified T cell epitopes in patients with AIH-2. Their partial overlap with B cell epitopes indicates a degree of co-localisation between T and B cells epitopes on CYP2D6.