Liver-infiltrating Mononuclear Cells Research Articles

GVHD targets organoid-forming bile duct stem cells in a TGF-β–dependent manner

AbstractGraft-versus-host disease (GVHD) is a major life-threatening complication that occurs after allogeneic hematopoietic cell transplantation (HCT). Although adult tissue stem cells have been identified as targets of GVHD in the skin and gut, their role in hepatic GVHD is yet to be clarified. In the current study, we explored the fate of bile duct stem cells (BDSCs), capable of generating liver organoids in vitro, during hepatic GVHD after allogeneic HCT. We observed a significant expansion of biliary epithelial cells (BECs) on injury early after allogeneic HCT. Organoid-forming efficiency from the bile duct was also significantly increased early after allogeneic HCT. Subsequently, the organoid-forming efficiency from bile ducts was markedly decreased in association with the reduction of BECs and the elevation of plasma concentrations of bilirubin, suggesting that GVHD targets BDSCs and impairs the resilience of BECs. The growth of liver organoids in the presence of liver-infiltrating mononuclear cells from allogeneic recipients, but not from syngeneic recipients, was significantly reduced in a transforming growth factor-β (TGF-β)–dependent manner. Administration of SB-431542, an inhibitor of TGF-β signaling, from day 14 to day 28, protected organoid-forming BDSCs against GVHD and mitigated biliary dysfunction after allogeneic HCT, suggesting that BDSCs are a promising therapeutic target for hepatic GVHD.

2,3,7,8-Tetrachlorodibenzo-p-dioxin Induces Gimap5 Expression to Ameliorate Concanavalin-Induced T Cell-Mediated Liver Injury

Abstract GTPase of the immune-associated protein 5 (Gimap5) has been implicated in immune cell maintenance and development. A mouse model comparable to autoimmune hepatitis in humans was used to study the alterations induced in the gene expression profile of immune cells upon administration of environmental toxin 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). In this study, we tested the hypothesis that TCDD is capable of modulating gene expression to ameliorate autoimmune hepatitis severity. Mice were injected intravenously with 12.5 mg/kg concanavalin A (ConA) and treated intraperitoneally one hour after challenge with vehicle or 10 μg/kg TCDD. TCDD-treated mice showed lower levels of liver injury enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as compared to vehicle-treated mice. Histopathological analysis revealed a reduction in liver damage in the TCDD-treated group as compared to the vehicle-treated control. Furthermore, flow cytometric analysis of liver-infiltrating mononuclear cells demonstrated that following treatment with TCDD, there was an increase in FoxP3+CD4+ Tregs as well as a decrease in proinflammatory Tbet+ Th1 and RORγt+ Th17 T helper cell subtypes after induction of the disease. Single cell RNA-sequencing was conducted on liver mononuclear cells to further assess the changes induced by TCDD. The gene expression profile showed that TCDD treatment significantly upregulated the expression of vital genes such as Gimap5, a maintenance gene for normal liver function and survival of T cells. In summary, our data suggests that TCDD is capable of inducing Gimap5 expression in autoimmune hepatitis, which in turn modulates the progression of this disease.

Anti-drug Antibodies Against a Novel Humanized Anti-CD20 Antibody Impair Its Therapeutic Effect on Primary Biliary Cholangitis in Human CD20- and FcγR-Expressing Mice.

There is considerable interest in expanding B cell-targeted therapies in human autoimmune diseases. However, clinical trials in human primary biliary cholangitis (PBC) using a chimeric antibody against human CD20 (hCD20) have showed limited efficacy. Two potential explanations for these disappointing results are the appearance of anti-drug antibodies (ADAs) and the high frequency of patients with moderate PBC or patients who had failed ursodeoxycholic acid treatment. Here, we studied a novel humanized IgG1 antibody against hCD20 and explored its efficacy in early stage PBC using a well-defined murine model. We developed a unique murine model consisting of dnTGF-βRII mice expressing hCD20 and human Fcγ receptors (hFcγRs). Beginning at 4–6 weeks of age, equivalent to stage I/II human PBC, female mice were given weekly injections of an anti-hCD20 antibody (TKM-011) or vehicle control, and monitored for liver histology as well as a broad panel of immunological readouts. After 16 weeks' treatment, we observed a significant reduction in portal inflammation, a decrease in liver-infiltrating mononuclear cells as well as a reduction in liver CD8+ T cells. Importantly, direct correlations between numbers of liver non-B cells and B cells (r = 0.7426, p = 0.0006) and between numbers of liver memory CD8+ T cells and B cells (r = 0.6423, p = 0.0054) were apparent. Accompanying these changes was a dramatic reduction in anti-mitochondrial antibodies (AMAs), interleukin (IL)-12p40 and IL-5, and elevated levels of the anti-inflammatory chemokine CXCL1/KC. In mice that developed ADAs, clinical improvements were less pronounced. Sustained treatment with B cell-targeted therapies may broadly inhibit effector pathways in PBC, but may need to be administered early in the natural history of PBC.

Loss of CD28 Expression by Liver-Infiltrating T Cells Contributes to Pathogenesis of Primary Sclerosing Cholangitis

T-cell-mediated biliary injury is a feature of primary sclerosing cholangitis (PSC). We studied the roles of CD28(-) T cells in PSC and their regulation by vitamin D. Peripheral and liver-infiltrating mononuclear cells were isolated from blood or fresh liver tissue. We analyzed numbers, phenotypes, functions, and localization patterns of CD28(-) T cells, along with their ability to activate biliary epithelial cells. We measured levels of tumor necrosis factor (TNF)α in liver tissues from patients with PSC and the effects of exposure to active vitamin D (1,25[OH]2D3) on expression of CD28. A significantly greater proportion of CD4(+) and CD8(+) T cells that infiltrated liver tissues of patients with PSC were CD28(-), compared with control liver tissue (CD4(+): 30.3% vs 2.5%, P < .0001; and CD8(+): 68.5% vs 31.9%, P < .05). The mean percentage of CD4(+)CD28(-) T cells in liver tissues from patients with PSC was significantly higher than from patients with primary biliary cirrhosis or nonalcoholic steatohepatitis (P< .05). CD28(-) T cells were activated CD69(+)CD45RA(-) C-C chemokine receptor (CCR)7(-) effector memory and perforin(+) granzyme B(+) cytotoxic cells, which express CD11a, CX3CR1, C-X3-C motif receptor 6 (CXCR6), and CCR10-consistent with their infiltration of liver and localization around bile ducts. Compared with CD28(+) T cells, activated CD28(-) T cells produced significantly higher levels of interferon γ and TNFα (P < .05), and induced up-regulation of intercellular cell adhesion molecule-1, HLA-DR, and CD40 by primary epithelial cells (3.6-fold, 1.5-fold, and 1.2-fold, respectively). Liver tissue from patients with PSC contained high levels of TNFα; TNFα down-regulated the expression of CD28 by T cells invitro (P<.01); this effect was prevented by administration of 1,25(OH)2D3 (P < .05). Inflammatory CD28(-) Tcells accumulate in livers of patients with PSC and localize around bile ducts. The TNFα-rich microenvironment of this tissue promotes inflammation; these effects are reversed by vitamin D invitro.

Baicalein Selectively Induces Apoptosis in Activated Lymphocytes and Ameliorates Concanavalin A-Induced Hepatitis in Mice

BackgroundInsufficient apoptosis in activated lymphocytes contributes to the development of autoimmune hepatitis (AIH). Baicalein (BE), a flavonoid originally isolated from the root of Scutellaria baicalensis Georgi, possesses anti-inflammatory properties. However, whether BE can selectively induce apoptosis in activated lymphocytes and exert therapeutic effect on AIH has not been studied.Methodology/Principal FindingsThe pro-apoptotic properties of BE were evaluated in vitro on different types of immune cells, and in vivo effects of BE were examined in a murine model of Concanavalin A (Con A)-induced hepatitis. In vitro treatment with BE resulted in a higher increase in the level of apoptosis in Con A-stimulated murine splenocytes, Con A-stimulated CD3+ splenocytes, lipopolysaccharide (LPS)-stimulated CD19+ splenocytes, and phorbol 12-myristate 13-acetate/ionomycin-stimulated Jurkat T cells, compared with that in unstimulated naïve ones. Murine bone marrow-derived dentritic cells, peritoneal macrophages, and RAW264.7 cells, either stimulated with LPS or unstimulated, were all insensitive to the BE-induced apoptosis. BE treatment also led to a loss of mitochondrial membrane potential, an increase of cytochrome c release from mitochondria to the cytosol, a decrease in the ratio of Bcl-2/Bax, and activation of caspase-9,-3 in Con A-stimulated CD3+ splenocytes and LPS-stimulated CD19+ splenocytes, while showing no impact on Fas/FasL expressions and caspase-8 activation. In vivo administration of BE alleviated Con A-induced liver injury, suppressed serum level of TNF-α and IFN-γ, and reduced liver infiltration of mononuclear cells (MNCs). Furthermore, BE treatment increased the incidences of apoptosis in liver-infiltrating MNCs and splenocytes, as well as in CD3+ and CD19+ splenocytes. When liver MNCs and splenocytes from BE-treated mice were cultured in vitro for 24 h, they exhibited marked increase in apoptosis compared to vehicle-treated control.Conclusions/SignificanceThe present study demonstrates the ability of BE to promote apoptosis in activated lymphocytes through mitochondrial pathway and its potential use in the treatment of AIH.

944 PHENOTYPIC AND FUNCTIONAL PLASTICITY OF CD4posCD25highCD127pos AND CD4posCD25highCD127neg T CELLS IN AUTOIMMUNE HEPATITIS

Background and Aims: Primary sclerosing cholangitis (PSC) is a chronic liver disease characterised by progressive inflammation, fibrosis and destruction of the bile ducts. The aetiology of PSC remains enigmatic although lymphocytic infiltration in areas of portal damage suggests an autoimmune mediated disease. CD28 is an important T cell co-stimulatory molecule and CD28− T cells have been reported as being involved in several autoimmune diseases. CD28− T cells are able to produce high amounts of IFNg, resist to apoptosis and show enhanced cytolytic activity. Given this we sought to further characterise CD28− T cells in PSC. Methods: Immunohistochemistry was used to localise CD4 and CD8 subsets in human liver tissue. Liver-infiltrating mononuclear cells (LIMCs) and peripheral blood mononuclear cells (PBMCs) were isolated from normal individuals and patients with PSC. Flow cytometry was used to determine the frequency and phenotypic characteristics of CD4 and CD8 T cells in association with CD28 expression. Results: Immunohistochemical analysis revealed high CD4 and CD8 T cell infiltration in the portal areas of human PSC liver, which was much higher when compared to normal donor livers (n = 5). CD4+ and CD8+ T cells in peripheral blood of PSC patients were present at equal frequencies (ratio of CD4:CD8 1:1) whereas in PSC LIMCs an increased frequency of CD8 T cells was observed (ratio 1:1.5). CD4+CD28− T cells were present at a frequency of 5% in normal peripheral blood, 10% in PSC patients’ peripheral blood and 52% in human PSC liver. 50% of the CD8+ T cells in normal peripheral blood were CD28−. In PSC patients, in both peripheral blood and liver the frequency of CD8+CD28− T cells was 75% and 71% respectively. Conclusions: Our results show a 5-fold increase in the frequency of CD4+CD28− T cells in human PSC liver when compared to PSC patients’ peripheral blood, and an increased frequency of CD8+CD28− T cells in both peripheral blood and liver of PSC patients. Our data suggest that CD28− T cells may be involved in the pathogenesis of PSC.

945 PHENOTYPIC CHARACTERISATION OF CD4 EFFECTOR IMMUNE RESPONSES IN CHILDHOOD AUTOIMMUNE LIVER DISEASE

Background and Aims: Primary sclerosing cholangitis (PSC) is a chronic liver disease characterised by progressive inflammation, fibrosis and destruction of the bile ducts. The aetiology of PSC remains enigmatic although lymphocytic infiltration in areas of portal damage suggests an autoimmune mediated disease. CD28 is an important T cell co-stimulatory molecule and CD28− T cells have been reported as being involved in several autoimmune diseases. CD28− T cells are able to produce high amounts of IFNg, resist to apoptosis and show enhanced cytolytic activity. Given this we sought to further characterise CD28− T cells in PSC. Methods: Immunohistochemistry was used to localise CD4 and CD8 subsets in human liver tissue. Liver-infiltrating mononuclear cells (LIMCs) and peripheral blood mononuclear cells (PBMCs) were isolated from normal individuals and patients with PSC. Flow cytometry was used to determine the frequency and phenotypic characteristics of CD4 and CD8 T cells in association with CD28 expression. Results: Immunohistochemical analysis revealed high CD4 and CD8 T cell infiltration in the portal areas of human PSC liver, which was much higher when compared to normal donor livers (n = 5). CD4+ and CD8+ T cells in peripheral blood of PSC patients were present at equal frequencies (ratio of CD4:CD8 1:1) whereas in PSC LIMCs an increased frequency of CD8 T cells was observed (ratio 1:1.5). CD4+CD28− T cells were present at a frequency of 5% in normal peripheral blood, 10% in PSC patients’ peripheral blood and 52% in human PSC liver. 50% of the CD8+ T cells in normal peripheral blood were CD28−. In PSC patients, in both peripheral blood and liver the frequency of CD8+CD28− T cells was 75% and 71% respectively. Conclusions: Our results show a 5-fold increase in the frequency of CD4+CD28− T cells in human PSC liver when compared to PSC patients’ peripheral blood, and an increased frequency of CD8+CD28− T cells in both peripheral blood and liver of PSC patients. Our data suggest that CD28− T cells may be involved in the pathogenesis of PSC.

Phenotypic evaluation of CD28 negative T cells in primary sclerosing cholangitis

Event Abstract Back to Event Phenotypic evaluation of CD28 negative T cells in primary sclerosing cholangitis Evaggelia Liaskou1*, Louisa Jeffery2, Shankar Suresh1, Palak Trivedi1, Tony Bruns1, 3, David Adams1 and David Sansom2 1 University of Birmingham, Centre for Liver Research and NIHR Biomedical Research Unit in Liver Disease, Institute of Biomedical Research, United Kingdom 2 Medical Research Council Centre for Immune Regulation, School of Immunity & Infection, Institute of Biomedical Research,, United Kingdom 3 Friedrich Schiller University of Jena, Germany Background/Aims: Genetic, functional and epidemiologic studies implicate CD28 and vitamin D (vitD) as relevant to autoimmune diseases including primary sclerosing cholangitis (PSC). Here we studied the role of CD28-ve T cells in PSC and the effects of vitD on their phenotype. Methods: Liver-infiltrating (LIMCs) and peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors or patients with PSC. The frequency and phenotype of CD28-ve cells [expression of activation (CD69, CD25) and exhaustion (PD-1) markers, perforin and granzyme B] were studied by flow cytometry. PSC LIMCs and PBMCs were activated and the release of IFNγ was examined. CD28-ve and CD28+ve cells from PSC PBMCs were stimulated with anti-CD3, autologous CD14+monocytes (5:1 ratio) with/without vitD for 4 days. Results: CD4+CD28-ve and CD8+CD28-ve T cells were highly expanded in PSC peripheral blood (3.9- and 2.1-fold increase respectively versus normal blood) and in PSC liver (17.8- and 2.45-fold increase respectively versus normal liver). The frequency of CD4+CD28-ve T cells in PSC liver was significantly higher than PSC peripheral blood (p<0.01). Liver-infiltrating CD4+CD28-ve T cells showed an activated CD25+CD69+PD-1- and cytotoxic phenotype granzymeB+perforin+. In-vitro stimulation of PSC liver-infiltrating CD4+CD28-ve cells induced the production of IFNγ (41%), which was much higher compared to their CD28+ve counterparts (12%). Stimulation of PSC peripheral blood CD28-ve and CD28+ve cells with vitD induced CD28 expression on CD28-ve (+52%) and CD28+ve (+65%) cells and suppressed IFNγ production (-43% and 67%, respectively). Conclusion: In PSC, CD28 negative T cells are pro-inflammatory, an effect that can be reduced by vitamin D supplementation. References Krones E., et al, Liver Int. 2012; 32(3):352-269 Ludwig J., et al, Hepatology 1981 Bo X., et al., Gut 2001; 49: 131-141 Weng NP., et al, Trends Immunol 2009; 30(7):306-312 Keywords: Vitamin D, CD28null, primary sclerosing cholangitis, biliary epithelial cells, Cytotoxicity Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune-mediated disease pathogenesis Citation: Liaskou E, Jeffery L, Suresh S, Trivedi P, Bruns T, Adams D and Sansom D (2013). Phenotypic evaluation of CD28 negative T cells in primary sclerosing cholangitis. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00085 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 08 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Evaggelia Liaskou, University of Birmingham, Centre for Liver Research and NIHR Biomedical Research Unit in Liver Disease, Institute of Biomedical Research, Birmingham, United Kingdom, e.liaskou@bham.ac.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Evaggelia Liaskou Louisa Jeffery Shankar Suresh Palak Trivedi Tony Bruns David Adams David Sansom Google Evaggelia Liaskou Louisa Jeffery Shankar Suresh Palak Trivedi Tony Bruns David Adams David Sansom Google Scholar Evaggelia Liaskou Louisa Jeffery Shankar Suresh Palak Trivedi Tony Bruns David Adams David Sansom PubMed Evaggelia Liaskou Louisa Jeffery Shankar Suresh Palak Trivedi Tony Bruns David Adams David Sansom Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Activated natural killer T cells producing interferon-gamma elicit promoting activity to murine dendritic cell-based autoimmune hepatic inflammation

As natural killer (NK) T cells play an important role in the development of autoimmune diseases, they should have significant roles for the pathogenesis of autoimmune liver disease. Implication of the NK T cells in the generation of autoimmune-related hepatic inflammation was investigated using a novel mouse model. Immunization of mice with dendritic cells (DCs) loaded with hepatocyte-mimicking hepatocellular carcinoma cells (DC/Hepa1-6) induces cytotoxic T lymphocytes (CTL) capable of killing hepatocytes. Subsequent administration of interleukin (IL)-12, a potent interferon-gamma (IFN-γ) inducer, to the immunized mice generates autoimmune hepatic inflammation (AHI), as reported previously. Upon onset of the AHI response, the number of intrahepatic CD3(+) NK1 · 1(+) NK T cells increased markedly, along with a decrease in the number of splenic NK T cells, augmented expression of CXCR6 on intrahepatic NK T cells and CXCL16 in hepatic tissue, suggesting that NK T cells were recruited into the inflamed liver. The NK T cells were strongly positive for CD69 and produced IFN-γ, but not IL-4. AHI activity was attenuated markedly in CD1d(-/-) NK T cell-deficient mice, indicating that NK T cells play a pivotal role in the development of AHI. Mice treated with DC/Hepa1-6 and alpha-galactosylceramide, a potent NK T cell activator, also exhibited similar hepatic inflammation, in which activated NK T cells producing IFN-γ and CD8(+) T cells cytotoxic to hepatocytes were induced in liver-infiltrating mononuclear cells. Activated NK T cells producing IFN-γ potentiate DC-based AHI in the mouse model.

Fractalkine and other chemokines in primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is characterized by the autoimmune injury of small intrahepatic bile duct. On this basis, it has been suggested that the targeted biliary epithelial cells (BEC) play an active role in the perpetuation of autoimmunity by attracting immune cells via chemokine secretion. To address this issue, we challenged BEC using multiple toll-like receptor (TLR) ligands as well as autologous liver infiltrating mononuclear cells (LMNC) with subsequent measurement of BEC phenotype and chemokine production and LMNC chemotaxis by quantifying specific chemokines, specially CX3CL1 (fractalkine). We submit the hypothesis that BEC are in fact the innocent victims of the autoimmune injury and that the adaptive immune response is critical in PBC.

Cx3cl1 (Fractalkine): A Signpost for Biliary Inflammation in Primary Biliary Cirrhosis

Improvements in the treatment of primary biliary cirrhosis (PBC) may depend upon dissection of mechanisms that determine recruitment of mononuclear cells to intralobular bile ducts, including the role of the chemokine-adhesion molecule CX3CL1 (fractalkine). We submit that there are unique interactions between intrahepatic biliary epithelial cells (BECs), endothelial cells (ECs), liver sinusoidal endothelial cells (LSECs), and liver-infiltrating mononuclear cells (LMCs), and that such interactions will in part dictate the biliary-specific inflammatory response. To address this, we studied fresh explanted livers from pretransplantation patients with PBC and with inflammatory liver disease due to viral infection (disease controls) and biopsy material from patients with a discrete liver tumor (normal controls). Using this clinical material, we isolated and stimulated BECs, ECs, LSECs, and LMCs with a panel of Toll-like receptor ligands. We also studied the interactions of these cell populations with LMCs with respect to adhesion capability and production of tumor necrosis factor alpha (TNF-alpha). Finally, we used fresh biopsy samples to evaluate mononuclear cells around intrahepatic biliary ductules using monoclonal antibodies specific to CD68 or CD154, markers for monocytes/macrophages, and activated T cells, respectively. There are common properties of ECs, LSECs, and BECs, whether derived from PBC or viral hepatitis, but there are also significant differences, particularly in the potential in PBC for LMCs to adhere to ECs and BECs and to produce TNF-alpha; such properties were associated with augmented CX3CL1 production by BEC from PBC liver. The processes defined herein suggest potential novel biotherapies for biliary specific inflammation.

Common Transcriptional Signature of Tumor-Infiltrating Mononuclear Inflammatory Cells and Peripheral Blood Mononuclear Cells in Hepatocellular Carcinoma Patients

Hepatocellular carcinoma (HCC) is frequently associated with infiltrating mononuclear inflammatory cells. We performed laser capture microdissection of HCC-infiltrating and noncancerous liver-infiltrating mononuclear inflammatory cells in patients with chronic hepatitis C (CH-C) and examined gene expression profiles. HCC-infiltrating mononuclear inflammatory cells had an expression profile distinct from noncancerous liver-infiltrating mononuclear inflammatory cells; they differed with regard to genes involved in biological processes, such as antigen presentation, ubiquitin-proteasomal proteolysis, and responses to hypoxia and oxidative stress. Immunohistochemical analysis and gene expression databases suggested that the up-regulated genes involved macrophages and Th1 and Th2 CD4 cells. We next examined the gene expression profile of peripheral blood mononuclear cells (PBMC) obtained from CH-C patients with or without HCC. The expression profiles of PBMCs from patients with HCC differed significantly from those of patients without HCC (P < 0.0005). Many of the up-regulated genes in HCC-infiltrating mononuclear inflammatory cells were also differentially expressed by PBMCs of HCC patients. Analysis of the commonly up-regulated or down-regulated genes in HCC-infiltrating mononuclear inflammatory cells and PBMCs of HCC patients showed networks of nucleophosmin, SMAD3, and proliferating cell nuclear antigen that are involved with redox status, the cell cycle, and the proteasome system, along with immunologic genes, suggesting regulation of anticancer immunity. Thus, exploring the gene expression profile of PBMCs may be a surrogate approach for the assessment of local HCC-infiltrating mononuclear inflammatory cells.

The effect of recombinant interferon-alpha on lymphocyte subpopulations and HLA-DR expression on liver tissue of HBV-positive individuals

Interferon-alpha (IFN-alpha) has been reported to be beneficial in the treatment of chronic active hepatitis occurring as a result of hepatitis B virus (HBV) infection. Treatment with IFN-alpha has been proposed as a means of reducing the high rate of allograft infection in clinical liver transplantation in patients transplanted for HBV-related chronic active hepatitis and cirrhosis who are positive for hepatitis B surface antigen (HBsAg). We obtained resected whole livers from two groups of patients who received liver transplants. Group A consisted of 11 patients who were HBsAg+ but were not treated with IFN-alpha, and group B consisted of 10 patients who were also HBsAg+ but received IFN-alpha therapy for 29.4 +/- 5.6 days prior to orthotopic liver transplantation. No differences between the two groups existed in terms of a variety of demographic and clinical characteristics. The liver tissue was stained with monoclonal antibodies to cell surface antigens unique to different mononuclear cell populations by the avidin-biotin-immunoperoxidase technique to determine the effect of IFN-alpha on the lymphocyte subsets as well as HLA antigen expression on liver-infiltrating mononuclear cells. The number of HLA-DR+ lymphocytes in the liver was significantly increased (P less than 0.005) within the portal areas in group B compared with that found in group A (84 +/- 14 versus 33 +/- 5 per one high-power field). Moreover, the intensity of the HLA-DR antigen expression on lymphocytes in the portal areas (P less than 0.02) and in the hepatic lobule (P less than 0.05) was greater in group B than in group A. The number of natural killer (NK) cells was increased in the portal areas (P less than 0.05) of group B compared with group A. These alterations in the lymphocyte and NK cell populations present in the liver in response to IFN-alpha therapy presumably reflect an IFN-alpha-induced enhancement of the immune response to virus-infected cells.

Biliary epithelial cells and primary biliary cirrhosis: The role of liver-infiltrating mononuclear cells

Primary biliary cirrhosis (PBC) is characterized by the highly selective autoimmune injury of small intrahepatic bile ducts, despite widespread distribution of mitochondrial autoantigens. On this basis, it has been suggested that the targeted biliary epithelial cells (BECs) play an active role in the perpetuation of autoimmunity by attracting immune cells via chemokine secretion. To address this issue, we challenged BECs from patients with PBC and controls using multiple Toll-like receptor (TLR) ligands as well as autologous liver-infiltrating mononuclear cells (LMNCs) with subsequent measurement of BEC phenotype and chemokine production and LMNC chemotaxis by quantifying specific chemokines. Our data reflect that BECs from PBC patients and controls express similar levels of TLR subtypes, CD40, and human leukocyte antigen DRalpha (HLA-DRalpha) and produce equivalent amounts of chemokines in our experimental conditions. Interestingly, however, BEC-expressed chemokines elicit enhanced transmigration of PBC LMNCs compared with controls. Furthermore, the addition of autologous LMNCs to PBC BECs led to the production of higher levels of chemokines and enhanced the expression of CD40 and HLA-DRalpha. We submit that the proinflammatory activity of BECs in PBC is secondary to the intervention of LMNCs and is not determined per se. These data support the hypothesis that BECs are in fact "innocent victims" of autoimmune injury and that the adaptive immune response is critical in PBC.

Analysis of CD28 and bcl-2 Expression on Peripheral Blood and Liver-Infiltrating Mononuclear Cells in Patients with Autoimmune Hepatitis

Because the underlying mechanism of hepatocellular damages in autoimmune hepatitis (AIH) still remains unclear, analysis of CD28 and bcl-2 molecules, which are critical for T cell activation and survival, was performed in patients with AIH. The number of CD28(+)CD4(+) peripheral blood mononuclear cells (PBMC) in corticosteroid (CS)-treated patients was comparable to normal control individuals but decreased in untreated AIH patients. In contrast, the number of CD28(+)CD8(+) PBMC was decreased in both CS-treated and untreated AIH patients. Analysis of liver-infiltrating mononuclear cells (LIMC) showed that the number of CD28(+)CD4(+) and CD28(-)CD8(+) LIMC were positively correlated with the histology activity index score. Bcl-2(+)CD4(+) LIMC were observed in the portal area of the liver and the numbers fluctuated with disease activity during the time course after CS administration. By contrast, CD8(+) LIMC were shown not to express bcl-2. Taken collectively, these results suggest that bcl-2(+)CD28(+)CD4(+) and bcl-2(-)CD28(-)CD8(+) cells may play critical and distinct roles in hepatocellular damage in AIH.

Usefulness of liver infiltrating CD86-positive mononuclear cells for diagnosis of autoimmune hepatitis

Although the pathogenic mechanism underlying autoimmune hepatitis (AIH) remains unclear, the immune system is thought to be critical for the progression of the disease. Cellular immune responses may be linked to the hepatocellular damage in AIH. Recently, much attention has been focused on the critical functions of costimulatory molecules expressed on mononuclear cells in the generation of effective T cell-mediated immune responses. Analysis of costimulatory molecule expressed on mononuclear cells from the patients with AIH may give us insight into the pathogenic mechanism of hepatocellular damage in AIH. Peripheral blood mononuclear cells (PBMC) were taken from the patients with AIH (34 cases) and healthy controls (25 cases). Liver infiltrating mononuclear cells (LIMCs) were taken from the patients with AIH (18 cases), the patient with chronic hepatitis C (CH-C) (13 cases) and the patients with fatty liver (2 cases). Using flow cytometry, the cells were analyzed for the expression of costimulatory molecules, such as CD80, CD86, and CD152 (CTLA-4). The results were compared with clinical data such as the level of gammaglobulin, histological grade, presence or absence of corticosteroids administration and the response to corticosteroids. The levels of CD80+, CD86+ and CD152+ PBMC were significantly reduced in the patients with AIH as compared with healthy controls. By contrast, those cells were significantly higher in LIMC than in PBMC of the patients with AIH. Especially, the level of CD86+ LIMC showed a marked increase irrespective of the degree of disease activity in the patients with AIH, although CD86+ cells were rarely present in PBMC. The levels of CD86+ cells were present in significantly higher frequency in patients with AIH than in the patients with CH-C. Furthermore, the patients with AIH with high levels of CD86+ LIMC showed good responses to corticosteroids, whereas 2 cases of AIH with low levels of CD86+ LIMC did not respond well. These results suggest that LIMC over-expressing costimulatory molecules such as CD80 and CD86 appears to play a role in the pathogenesis of AIH. Especially, CD86 molecule expressed on the LIMC may be useful for the diagnosis of AIH and for the prediction of the therapeutic effects of corticosteroids on AIH.

CD28-negative CD8-positive cytotoxic T lymphocytes mediate hepatocellular damage in hepatitis C virus infection.

The pathogenic mechanism for hepatocellular damage in hepatitis C virus (HCV) infection has not been clearly understood. Analysis of costimulatory molecules on lymphocytes may give us insight into the pathogenic mechanism of hepatocellular damage in HCV infection. Peripheral blood mononuclear cells (PBMCs) and liver infiltrating mononuclear cells (LIMCs) isolated from the HCV-infected patients were analyzed with antibodies directed against a variety of costimulatory molecules by flow cytometry. Blocking experiment against HLA-A24-restricted HCV-specific CTLs and immunohistochemical analysis were also performed. PBMCs expressing CD8, CD28, CD80, or CD154 were significantly reduced in HCV-infected patients compared with the healthy controls. CD28(+)CD8(+) PBMCs in the patients inversely correlated with ALT levels. Conversely, levels of CD28(-)CD8(+) LIMCs correlated with ALT levels. HCV-specific CTL activity was blocked by the treatment with anti-CD8 antibody, but not with anti-CD4 or anti-CD28 antibody. Immunohistochemical analysis revealed the accumulation of CD28(+) cells around the portal area in the liver of a patient with chronic active hepatitis C. These results suggest that CD28(+)CD8(+) T cells leave the circulation, move to the livers, and are activated in the portal area in proportion to the extent of liver diseases. CD28(-)CD8(+) T cells may finally function as effector T cells causing the hepatocellular damage in HCV infection.

Post-transplant total lymphoid irradiation prolongs the survival of rat liver allografts

Background: Post-transplant total lymphoid irradiation (TLI) and anti-thymocyte globulin (ATG) followed by infusion of donor bone marrow (hematopoietic) cells have resulted in chimerism and long-term graft acceptance in rodent heart transplant models. Using this regimen, we have recently shown that macrochimerism, donor-specific tolerance and drug withdrawal were achieved in some recipients after clinical kidney transplantation. Because the liver is a rich source of leukocytes and hematopoietic progenitor cells, we tested whether a regimen of post-transplant TLI (± ATG), in the absence of donor bone marrow infusion, results in the development of chimerism and in prolonged liver allograft survival in rats. Methods: Lewis recipients were transplanted with fully allogeneic donor ACI rat livers. Post-transplant TLI was administrated to allografted recipients starting on day 1 after orthotopic liver transplantation, 200 cGy daily for 8 to 10 doses over a 14-day period. To test for the presence of donor blood cells in the recipient (chimerism), flow cytometry was performed on isolated peripheral blood lymphocytes (PBL), splenocytes, and liver infiltrating mononuclear cells (LIMC). Results: Untreated recipients rejected their allografts within 14 days. Post-transplant TLI significantly prolonged liver allograft survival to more than 50 days (n = 5). Donor-derived lymphocytes were not detected in recipient PBL in the early period (<50 days) post-transplant, but a significant population of donor-derived lymphocytes was seen in PBL (25%), splenocytes (20%), and LIMC (25%) isolated from a TLI-treated recipient who survived for more than 80 days. Conclusion: Post-transplant TLI alone is sufficient to allow for the development of macrochimerism and long-term liver allograft survival in the rat.

Serum levels of soluble Fas antigen in chronic hepatitis C patients

BAckground/Aims: In chronic hepatitis C, the expression of Fas antigen on hepatocytes is upregulated and Fas ligand expression is detected on liver-infiltrating mononuclear cells. Thus Fas antigen/Fas ligand-mediated apoptosis is thought be involved in hepatic injury in chronic hepatitis C. The soluble form of Fas antigen has been detected in serum and shown to inhibit Fas-mediated apoptosis. The present study was done to evaluate the relationship of serum soluble Fas antigen levels with disease activity. Methods: Serum soluble Fas antigen levels were measured by enzyme-linked immunosorbent assay for 68 chronic hepatitis C patients and compared with those in normal volunteers, chronic hepatitis B patients and autoimmune hepatitis patients. These levels were compared with histological activity, ALT leves, HCV-RNA titer and Fas expression on hepatocytes. Results: Serum soluble Fas antigen levels in chronic hepatitis C patients (3.24±1.55 ng/ml) were significantly higher than those in normal volunteers (1.70±1.01 ng/ml) ( p<0.01). They showed no difference from those in chronic hepatitis B or autoimmune hepatitis patients. Histologically, soluble Fas antigen levels showed correlation with the levels of liver inflammation ( p<0.01). However, no relationship was observed between serum soluble Fas antigen and serum ALT levels or HCV-RNA titer. Serum soluble Fas antigen levels showed correlation with the levels of Fas antigen expression in liver tissue ( p<0.05). Conclusions: These findings suggest that serum soluble Fas antigen may reflect the expression levels of Fas antigen on hepatocytes and the severity of liver inflammation in chronic hepatitis C.

CD30 EXPRESSION IDENTIFIES A NOVEL ALLOREACTIVE T CELL SUBSET THAT IS FOUND WITHIN REJECTING ALLOGRAFTS

100 Alloantigenic stimulation leads to the development of T cell subsets with immunoregulatory and effector function. The purpose of this study was to identify and characterize novel T cell subsets that participate in alloreactivity through the production of cytokines. On day 3 of human MLR we noted the development of a population of CD45R0+lymphocytes that express the CD30 antigen. Immunofluorescence and flow cytometry indicated that a significant proportion of CD4 (16.2±2.3%) and CD8 T cells(19.3±3.0%) express CD30 in response to alloantigen. Within the CD4 subset, CD30 cell reside in the CD4hi fraction. CD30 cells also express CD25 and CD28. However, CD30 is not a general marker of alloactivation since CD25+CD30- cells also arise in MLR. Fluorescence activated cell sorting was used to determine the functional properties of CD30 T cells. Purified alloactivated CD30+, CD30-, and unsorted alloactivated cells were restimulated with alloantigen or anti-CD3 mAbs for 48 h and cytokine production determined by ELISA. CD30+ cells produced significantly more IFN-γ (6.2 ng/ml) and IL-5 (5.5 ng/ml) than did the CD30- population (0.6 ng/ml and 2.1 ng/ml, respectively). however, CD30+ and CD30- cells produced comparable amounts of IL-10 (1.1 ng/ml and 1.9 ng/ml, respectively) when restimulated with anti-CD3 mAbs. Similar patterns of cytokine production were noted when cells were restimulated with alloantigen. An agonist anti-CD30 mAb (M44) synergized with anti-CD3 mAbs and led to enhanced production of IL-5 and IFN-γ but not IL-2. To examine the role of CD30 cells in allograft rejection we utilized the DA to Lewis orthotopic liver transplant model in which rejection typically occurs on day 12-14. Total RNA obtained from allograft tissue on days 2, 4, and 8 post-transplant were analyzed by RT-PCR for CD30 transcripts. CD30 mRNA could be detected as early as day 2 post-transplant and increased in intensity through day 8. Lewis to Lewis isografts did not contain CD30 mRNA. To determine if CD30 cells were found within the inflammatory infiltrate, liver infiltrating mononuclear cells (LIMC) were isolated from day 4 allografts and RT-PCR was performed. A prominent band corresponding to CD30 transcripts was evident in LIMC. To confirm the amplified sequence was derived from rat CD30 the PCR product was cloned into the pT-Adv vector and sequenced. Sequence analysis indicated that the molecule was 97.6% homologous with the previously published rat CD30 sequence. Taken together, these data indicate that CD30 acts as a signaling molecule in alloactivated T cells. Furthermore, CD30+ T lymphocytes may constitute a novel immunoregulatory population that participates in allograft rejection through the production of cytokines.