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Structural and functional studies of human hepatocytes and biliary epithelial cells cocultured in collagen gels
HEPATOLOGY Vol. 22, N o . 4, P t . 2, 1 9 9 5
433
AASLD
RECURRENT AUTOIMMUNE HEPATITIS FOLLOWING ORTHOTOPIC LIVER TRANSPLANTATION. S Roberts*, AJ Czaia, MR Chaflton, JE Hay, RAF Krom, MK Porayko, RH Wiesner. Liver Transplantation Unit, Mayo Clinic and Foundation, Rochester, MN.
ABSTRACTS
434 CMV INFECTION OF HEPATOCYTES AND BILIARY EPITHELIAL CELLS IN-VITRO JC. Arnold, G. Otto, A, Knuth, L.Theilmann. Dept. of Gastroenterology and Surgery; University of Heidelberg, Nordwest Hospital Frankfurt, FRG
Few reports have documented recurrence of autoimmune hepatitis (AH) following liver transplantation. We therefore analyzed our experience with severe AH to determine the frequency and outcome of disease recurrence after OLT. Methods: Thirty-five patients who satisfied standard diagnostic criteria for type t autnimmune hepatitis and who had undergone OLT at our institution between March 1985 and October 1994 were assessed. All patients had been followed according to protocol after OLT and were treated with standard immunosuppressive regimens with cyclosporine or FK506. When liver biochemstries and histology suggested recurrent hepatitis, serum autoanfibndies and HAV, IgM, HAV lgM, HBsAg, anti-HBc IgM, anti-HCV, HCV RNA by PCR were evaluated. We also compared the frequency of OKT3-treated steroid-resistant rejection (SPY,) after OLT for AH to that for all other OLT indications. Results: After OLT, 4 (11%) patients developed recurrent AH during 91±13 mo of follow up. The mean AST level at recurrence was 296:t:155 U (range: 68-739). Three patients had elevated y-globulin levels (range: 1.7-2.6 g/dl). Liver biopsy showed periportal hepatitis (n=3) and portal with lobalar hepatitis (n=l); there were no histologic findings for rejection. Serum anti-SMA and/or ANA antibodies were positive in 2 patients. Each patient had negative viral markers, including serologic and/or histologic evidence for EBV and CMV. The mean interval to recurrence was 72=20 mo; 3 patients had recurrent AH at _~90 mo post OLT. In 2 patients , recurrence followed a reduction in immunosuppression. Remission was achieved in 3 patients with an increase in immunosuppressive therapy; 1 patient continues to show evidence of recurrent disease. No patient with recurrent AH has died or required retransplantation during an additional 17:i:9 mo of follow up. Two patients were mismatched at the DR3 allele. Patients with recurrent AH were clinically and biochemically similar at OLT m those without recurrence, and had a similar number of rejection episodes and frequency of OKT3 therapy. The frequency of SRR requiring OKT3 (51% v 11%) was higher (P<.0.05) in AH compared to other liver diseases. Conclusions: After OLT, recurrence of AH is not infrequent hut usually mild, and may occur with reduction in immunesuppression several years following OLT. Remission can usually be induced by an increase in immtmosuppressive therapy. The high frequency of SRR requiring OKT3 raises the possibility that recurrent AH is anderdiagonsed. Care should be taken when attempting to reduce immunosuppression during the long term follow-up of this group of patients.
435
HUMAN INTRAHEPATIC BILIARY EPITHELIAL CELLS FAIL TO STIMULATE PRIMARY ALLOGENEIC RESPONSES DUE TO LACK OF COSTIMULATION. MP Loon', JA Kirbyt, P Gibbs~, M Thiek~, JL Wilson~ MF Bassendine'. Departments of "Medicine and tSurgery, University of Newcastle upon Tyne, NE2 4HH, UK.
Human intrahepatic biliary epithelial cells (HIBEC) are a major immunological target during liver allograft rejection and primary biliary cirrhosis (PBC). It has been shown .in vivo and in vitro that HIBEC express MHC antigens and some adhesion molecules during inflammation. This has led to the proposal that these cells might play an active role during initiation and amplification of immune responses within the liver. Purified HIBEC stimulated with pro-inflammatory eytokioes (TNF-et and IFN-'/) express similar levels of class I and II MHC antigens, ICAM-1 and LFA-3 to those expressed by an EBV-transformed B cell line. However, these HIBEC do net express the costimulatory molecules B7-1 or B7-2 (1), which are ligands of the CD28 molecule on T cells. The aim of this study was to determine if MHC class II expressing HIBEC could stimulate proliferation of allospeeific T cells, using other costimulatory molecules. Either lymphocytes or purifmd CD4+ T cells were co-cultured with irradiated allogeneie stimulator culls. Some of these cultures were supplemented with anti-CD28 antibody (clone 9.3; 12.5 p.g/ml) or a control IgG antibody to examine the effect of supplying artificial costimulatory stimuli. Allospecific T cells proliferated during co-culture with EBVtransformed B cells, but failed to do so during co-culture with cytokinestimulated HIBEC. However, when the anti-CD28 antibody was added to the cultures, cytokine stimulated HIBEC were able to stimulate primary proliferati0Wof allogeneic CiY4+ T cells (p<0.05). These experiments show that class II MHC molecule-expressing HIBEC are unable to initiate a primary alloimmune-response due to their inability to deliver costimulatory signals via CD28. (1) J Hepatology 1995, 22: 591-595.
215A
After liver transplantation cytomegalovirus (CMV) causes hepatocellular damage and may be associated with the development of a certain type of chronic aliograft rejection, the vanishing bile duct syndrome. In an in-vitro model we wanted to investigate whether the time course and intensity of CMV infection is different in hepatocytes and biliary epithelial cells. Cultured hepatocytes (both primary human hepatocytes and the HepG2 cell line, respectively) and a bile duct carcinoma cell line (J. Hepatol 1985; 1: 579-596) were infected with the CMV strain AD 169, multiplicity of infection 5 #u/cell, for 1 hour. CMV antigen (immediate early, early and late) expression was investigated 4-, 8-, 12-, 24-, 48 hours and 4-, 7-, 12 days post infection (p.i.) using monoclonal antibodies. To detect replicated virus shedded in the culture-medium, CMV DNA was detected by PCR CMV antigens were detectable as early as 4 hours p.i. in nuclei of hepatocytes and the bile duct carcinoma cell line. Maximum number of hepatocytes expressing CMV antigens was 10%evident at 48 hours p.i.. In contrast up to 60% of bite duct carcinoma cells expressed CMV antigens after in-vitro infection. Evidence of shedded virus was present only in the supernatent of primary human hepatocytes at 12 days p.i_ These data indicate that biliary epithelial cells are more susceptible for CMV infection, by expressing a much a higher number of CMV antigens as compared to hepatocytes. Viral replication of CMV however was shown only in primary human hepatocytes following a similar course as in cultured fibroblasts.
436
S T R U C T U R A L AND F U N C T I O N A L STUDIES O F H U M A N H E P A T O C Y T E S AND B I L I A R Y E P I T H E L I A L CELLS C O C U L T U R E D IN C O L L A G E N GELS. M K H Auth, R Joplin, L Fabris, A Keogh, RE Gailacher, JM Neuberger, P McMaster, AJ Strain. Liver Research Laboratories, Queen Elizabeth Hospital, Birmingham, UK.
Previous studies with h u m a n hepatocytes (HC) and biliary epithelial cells (BEC) have been restricted to conventional monolayers, where cells lose their membrane polarity and functional characteristics. Here, we have investigated the effect o f co-culturing ceils in collagen gels in order to improve these features. Autologous HC and BEC were purified from human liver by collagenase perfusion and immune-magnetic separation respectively. Cells were maintained on rat-tail collagen as a single gel (SG) or double gel (DG) in Williams E medium with or without 10 ng/ml EGF or HGF or conditioned medium from previous co-cultures (CM). Cultures were examined by high resolution microscopy, growth assessed by thymidine incorporation and albumin expression measured by immunoassay. In serum- and growth factor-free medium, co-cultures could be maintained for >7 weeks and cells could be harvested and replated. On SG, BEC proliferated to form confluent monolayers between HC while in DG, within 5-10 days they established three-dimensional cyst-like structures which stained positively for the biliary markers H E A l 2 5 and CK-19. Cyst formation in WE (range 14-64 per well) was increased by EGF (30-125/we11) and not HGF (25-46/we11), but was substantially enhanced by EGF+HGF (94-199/we11) or CM (82-167). From these cysts after a further 7-10 days, ductular structures with a distinct lumen developed. BEC within these ducts demonstrated features o f polarised ceils including apical micro-villi, nuclei located toward the basal pole and inter-cellular junctional complexes. Albumin expression by HC was similarly improved by co-culturing with BEC (36 vs 56 gg/ml/24h) or by addition of HGF (+30%), EGF (+150%), HGF+EGF or CM (+100%). These observations indicate that the use o f collagen gels improves the structural and functional integrity o f both human HC and BEC and this is further enhanced by the cellular interactions which occur in co-cultures. The efficacy of growth factors and CM is under investigation. (Supported by DFG grant Au 117/1 to MKHA).
433
AASLD
RECURRENT AUTOIMMUNE HEPATITIS FOLLOWING ORTHOTOPIC LIVER TRANSPLANTATION. S Roberts*, AJ Czaia, MR Chaflton, JE Hay, RAF Krom, MK Porayko, RH Wiesner. Liver Transplantation Unit, Mayo Clinic and Foundation, Rochester, MN.
ABSTRACTS
434 CMV INFECTION OF HEPATOCYTES AND BILIARY EPITHELIAL CELLS IN-VITRO JC. Arnold, G. Otto, A, Knuth, L.Theilmann. Dept. of Gastroenterology and Surgery; University of Heidelberg, Nordwest Hospital Frankfurt, FRG
Few reports have documented recurrence of autoimmune hepatitis (AH) following liver transplantation. We therefore analyzed our experience with severe AH to determine the frequency and outcome of disease recurrence after OLT. Methods: Thirty-five patients who satisfied standard diagnostic criteria for type t autnimmune hepatitis and who had undergone OLT at our institution between March 1985 and October 1994 were assessed. All patients had been followed according to protocol after OLT and were treated with standard immunosuppressive regimens with cyclosporine or FK506. When liver biochemstries and histology suggested recurrent hepatitis, serum autoanfibndies and HAV, IgM, HAV lgM, HBsAg, anti-HBc IgM, anti-HCV, HCV RNA by PCR were evaluated. We also compared the frequency of OKT3-treated steroid-resistant rejection (SPY,) after OLT for AH to that for all other OLT indications. Results: After OLT, 4 (11%) patients developed recurrent AH during 91±13 mo of follow up. The mean AST level at recurrence was 296:t:155 U (range: 68-739). Three patients had elevated y-globulin levels (range: 1.7-2.6 g/dl). Liver biopsy showed periportal hepatitis (n=3) and portal with lobalar hepatitis (n=l); there were no histologic findings for rejection. Serum anti-SMA and/or ANA antibodies were positive in 2 patients. Each patient had negative viral markers, including serologic and/or histologic evidence for EBV and CMV. The mean interval to recurrence was 72=20 mo; 3 patients had recurrent AH at _~90 mo post OLT. In 2 patients , recurrence followed a reduction in immunosuppression. Remission was achieved in 3 patients with an increase in immunosuppressive therapy; 1 patient continues to show evidence of recurrent disease. No patient with recurrent AH has died or required retransplantation during an additional 17:i:9 mo of follow up. Two patients were mismatched at the DR3 allele. Patients with recurrent AH were clinically and biochemically similar at OLT m those without recurrence, and had a similar number of rejection episodes and frequency of OKT3 therapy. The frequency of SRR requiring OKT3 (51% v 11%) was higher (P<.0.05) in AH compared to other liver diseases. Conclusions: After OLT, recurrence of AH is not infrequent hut usually mild, and may occur with reduction in immunesuppression several years following OLT. Remission can usually be induced by an increase in immtmosuppressive therapy. The high frequency of SRR requiring OKT3 raises the possibility that recurrent AH is anderdiagonsed. Care should be taken when attempting to reduce immunosuppression during the long term follow-up of this group of patients.
435
HUMAN INTRAHEPATIC BILIARY EPITHELIAL CELLS FAIL TO STIMULATE PRIMARY ALLOGENEIC RESPONSES DUE TO LACK OF COSTIMULATION. MP Loon', JA Kirbyt, P Gibbs~, M Thiek~, JL Wilson~ MF Bassendine'. Departments of "Medicine and tSurgery, University of Newcastle upon Tyne, NE2 4HH, UK.
Human intrahepatic biliary epithelial cells (HIBEC) are a major immunological target during liver allograft rejection and primary biliary cirrhosis (PBC). It has been shown .in vivo and in vitro that HIBEC express MHC antigens and some adhesion molecules during inflammation. This has led to the proposal that these cells might play an active role during initiation and amplification of immune responses within the liver. Purified HIBEC stimulated with pro-inflammatory eytokioes (TNF-et and IFN-'/) express similar levels of class I and II MHC antigens, ICAM-1 and LFA-3 to those expressed by an EBV-transformed B cell line. However, these HIBEC do net express the costimulatory molecules B7-1 or B7-2 (1), which are ligands of the CD28 molecule on T cells. The aim of this study was to determine if MHC class II expressing HIBEC could stimulate proliferation of allospeeific T cells, using other costimulatory molecules. Either lymphocytes or purifmd CD4+ T cells were co-cultured with irradiated allogeneie stimulator culls. Some of these cultures were supplemented with anti-CD28 antibody (clone 9.3; 12.5 p.g/ml) or a control IgG antibody to examine the effect of supplying artificial costimulatory stimuli. Allospecific T cells proliferated during co-culture with EBVtransformed B cells, but failed to do so during co-culture with cytokinestimulated HIBEC. However, when the anti-CD28 antibody was added to the cultures, cytokine stimulated HIBEC were able to stimulate primary proliferati0Wof allogeneic CiY4+ T cells (p<0.05). These experiments show that class II MHC molecule-expressing HIBEC are unable to initiate a primary alloimmune-response due to their inability to deliver costimulatory signals via CD28. (1) J Hepatology 1995, 22: 591-595.
215A
After liver transplantation cytomegalovirus (CMV) causes hepatocellular damage and may be associated with the development of a certain type of chronic aliograft rejection, the vanishing bile duct syndrome. In an in-vitro model we wanted to investigate whether the time course and intensity of CMV infection is different in hepatocytes and biliary epithelial cells. Cultured hepatocytes (both primary human hepatocytes and the HepG2 cell line, respectively) and a bile duct carcinoma cell line (J. Hepatol 1985; 1: 579-596) were infected with the CMV strain AD 169, multiplicity of infection 5 #u/cell, for 1 hour. CMV antigen (immediate early, early and late) expression was investigated 4-, 8-, 12-, 24-, 48 hours and 4-, 7-, 12 days post infection (p.i.) using monoclonal antibodies. To detect replicated virus shedded in the culture-medium, CMV DNA was detected by PCR CMV antigens were detectable as early as 4 hours p.i. in nuclei of hepatocytes and the bile duct carcinoma cell line. Maximum number of hepatocytes expressing CMV antigens was 10%evident at 48 hours p.i.. In contrast up to 60% of bite duct carcinoma cells expressed CMV antigens after in-vitro infection. Evidence of shedded virus was present only in the supernatent of primary human hepatocytes at 12 days p.i_ These data indicate that biliary epithelial cells are more susceptible for CMV infection, by expressing a much a higher number of CMV antigens as compared to hepatocytes. Viral replication of CMV however was shown only in primary human hepatocytes following a similar course as in cultured fibroblasts.
436
S T R U C T U R A L AND F U N C T I O N A L STUDIES O F H U M A N H E P A T O C Y T E S AND B I L I A R Y E P I T H E L I A L CELLS C O C U L T U R E D IN C O L L A G E N GELS. M K H Auth, R Joplin, L Fabris, A Keogh, RE Gailacher, JM Neuberger, P McMaster, AJ Strain. Liver Research Laboratories, Queen Elizabeth Hospital, Birmingham, UK.
Previous studies with h u m a n hepatocytes (HC) and biliary epithelial cells (BEC) have been restricted to conventional monolayers, where cells lose their membrane polarity and functional characteristics. Here, we have investigated the effect o f co-culturing ceils in collagen gels in order to improve these features. Autologous HC and BEC were purified from human liver by collagenase perfusion and immune-magnetic separation respectively. Cells were maintained on rat-tail collagen as a single gel (SG) or double gel (DG) in Williams E medium with or without 10 ng/ml EGF or HGF or conditioned medium from previous co-cultures (CM). Cultures were examined by high resolution microscopy, growth assessed by thymidine incorporation and albumin expression measured by immunoassay. In serum- and growth factor-free medium, co-cultures could be maintained for >7 weeks and cells could be harvested and replated. On SG, BEC proliferated to form confluent monolayers between HC while in DG, within 5-10 days they established three-dimensional cyst-like structures which stained positively for the biliary markers H E A l 2 5 and CK-19. Cyst formation in WE (range 14-64 per well) was increased by EGF (30-125/we11) and not HGF (25-46/we11), but was substantially enhanced by EGF+HGF (94-199/we11) or CM (82-167). From these cysts after a further 7-10 days, ductular structures with a distinct lumen developed. BEC within these ducts demonstrated features o f polarised ceils including apical micro-villi, nuclei located toward the basal pole and inter-cellular junctional complexes. Albumin expression by HC was similarly improved by co-culturing with BEC (36 vs 56 gg/ml/24h) or by addition of HGF (+30%), EGF (+150%), HGF+EGF or CM (+100%). These observations indicate that the use o f collagen gels improves the structural and functional integrity o f both human HC and BEC and this is further enhanced by the cellular interactions which occur in co-cultures. The efficacy of growth factors and CM is under investigation. (Supported by DFG grant Au 117/1 to MKHA).