Chris J. Weston

Distinct Roles for CCR4 and CXCR3 in the Recruitment and Positioning of Regulatory T Cells in the Inflamed Human Liver

Regulatory T cells (Tregs) are found at sites of chronic inflammation where they mediate bystander and Ag-specific suppression of local immune responses. However, little is known about the molecular control of Treg recruitment into inflamed human tissues. We report that up to 18% of T cells in areas of inflammation in human liver disease are forkhead family transcriptional regulator box P3 (FoxP3)+ Tregs. We isolated CD4+CD25+CD127lowFoxP3+ Tregs from chronically inflamed human liver removed at transplantation; compared with blood-derived Tregs, liver-derived Tregs express high levels of the chemokine receptors CXCR3 and CCR4. In flow-based adhesion assays using human hepatic sinusoidal endothelium, Tregs used CXCR3 and α4β1 to bind and transmigrate, whereas CCR4 played no role. The CCR4 ligands CCL17 and CCL22 were absent from healthy liver, but they were detected in chronically inflamed liver where their expression was restricted to dendritic cells (DCs) within inflammatory infiltrates. These DCs were closely associated with CD8 T cells and CCR4+ Tregs in the parenchyma and septal areas. Ex vivo, liver-derived Tregs migrated to CCR4 ligands secreted by intrahepatic DCs. We propose that CXCR3 mediates the recruitment of Tregs via hepatic sinusoidal endothelium and that CCR4 ligands secreted by DCs recruit Tregs to sites of inflammation in patients with chronic hepatitis. Thus, different chemokine receptors play distinct roles in the recruitment and positioning of Tregs at sites of hepatitis in chronic liver disease.

Isolation of Primary Human Hepatocytes from Normal and Diseased Liver Tissue: A One Hundred Liver Experience

Successful and consistent isolation of primary human hepatocytes remains a challenge for both cell-based therapeutics/transplantation and laboratory research. Several centres around the world have extensive experience in the isolation of human hepatocytes from non-diseased livers obtained from donor liver surplus to surgical requirement or at hepatic resection for tumours. These livers are an important but limited source of cells for therapy or research. The capacity to isolate cells from diseased liver tissue removed at transplantation would substantially increase availability of cells for research. However no studies comparing the outcome of human hepatocytes isolation from diseased and non-diseased livers presently exist. Here we report our experience isolating human hepatocytes from organ donors, non-diseased resected liver and cirrhotic tissue. We report the cell yields and functional qualities of cells isolated from the different types of liver and demonstrate that a single rigorous protocol allows the routine harvest of good quality primary hepatocytes from the most commonly accessible human liver tissue samples.

Activation of vascular adhesion protein‐1 on liver endothelium results in an NF‐κB–dependent increase in lymphocyte adhesion

Vascular adhesion protein‐1 (VAP‐1) is an adhesion molecule and amine oxidase that is expressed at high levels in the human liver. It promotes leukocyte adhesion to the liver in vivo and drives lymphocyte transmigration across hepatic sinusoidal endothelial cells in vitro. We report that in addition to supporting leukocyte adhesion, provision of specific substrate to VAP‐1 results in hepatic endothelial cell activation, which can be abrogated by treatment with the enzyme inhibitor semicarbazide. VAP‐1–mediated activation was rapid; dependent upon nuclear factor‐κB, phosphatidylinositol‐3 kinase, and mitogen‐activated protein kinase pathways; and led to upregulation of the adhesion molecules E‐selectin, intercellular adhesion molecule‐1, and vascular cell adhesion molecule‐1 and secretion of the chemokine CXCL8. This response resulted in enhanced lymphocyte adhesion, was restricted to hepatic endothelial cells that expressed VAP‐1, and was not observed in human umbilical vein endothelial cells. Conclusion: We propose that as well as directly promoting adhesion via interactions with the as yet unknown ligand, binding of enzyme substrate to VAP‐1 can indirectly promote inflammatory cell recruitment via upregulation of adhesion molecules and chemokines. This response is likely to be important for the recruitment of leukocytes to the liver and suggests that VAP‐1 inhibitors have therapeutic potential for treating chronic inflammatory liver disease. (HEPATOLOGY 2007;45:465–474.)

Common lymphatic endothelial and vascular endothelial receptor-1 mediates the transmigration of regulatory T cells across human hepatic sinusoidal endothelium.

The common lymphatic endothelial and vascular endothelial receptor (CLEVER-1; also known as FEEL-1 and stabilin-1) is a recycling and intracellular trafficking receptor with multifunctional properties. In this study, we demonstrate increased endothelial expression of CLEVER-1/stabilin-1 at sites of leukocyte recruitment to the inflamed human liver including sinusoids, septal vessels, and lymphoid follicles in inflammatory liver disease and tumor-associated vessels in hepatocellular carcinoma. We used primary cultures of human hepatic sinusoidal endothelial cells (HSEC) to demonstrate that CLEVER-1/stabilin-1 expression is enhanced by hepatocyte growth factor but not by classical proinflammatory cytokines. We then showed that CLEVER-1/stabilin-1 supports T cell transendothelial migration across HSEC under conditions of flow with strong preferential activity for CD4 FoxP3+ regulatory T cells (Tregs). CLEVER-1/stabilin-1 inhibition reduced Treg transendothelial migration by 40% and when combined with blockade of ICAM-1 and vascular adhesion protein-1 (VAP-1) reduced it by >80%. Confocal microscopy demonstrated that 60% of transmigrating Tregs underwent transcellular migration through HSEC via ICAM-1– and VAP-1–rich transcellular pores in close association with CLEVER-1/stabilin-1. Thus, CLEVER-1/stabilin-1 and VAP-1 may provide an organ-specific signal for Treg recruitment to the inflamed liver and to hepatocellular carcinoma.

Regulation of mucosal addressin cell adhesion molecule 1 expression in human and mice by vascular adhesion protein 1 amine oxidase activity

Primary sclerosing cholangitis (PSC) and autoimmune hepatitis are hepatic complications associated with inflammatory bowel disease (IBD). The expression of mucosal addressin cell adhesion molecule 1 (MAdCAM‐1) on mucosal endothelium is a prerequisite for the development of IBD, and it is also detected on the hepatic vessels of patients with liver diseases associated with IBD. This aberrant hepatic expression of MAdCAM‐1 results in the recruitment of effector cells initially activated in the gut to the liver, in which they drive liver injury. However, the factors responsible for the aberrant hepatic expression of MAdCAM‐1 are not known. In this study, we show that deamination of methylamine (MA) by vascular adhesion protein 1 (VAP‐1) [a semicarbazide‐sensitive amine oxidase (SSAO) expressed in the human liver] in the presence of tumor necrosis factor α induces the expression of functional MAdCAM‐1 in hepatic endothelial cells and in intact human liver tissue ex vivo. This is associated with increased adhesion of lymphocytes from patients with PSC to hepatic vessels. Feeding mice MA, a constituent of food and cigarette smoke found in portal blood, led to VAP‐1/SSAO–dependent MAdCAM‐1 expression in mucosal vessels in vivo. Conclusion: Activation of VAP‐1/SSAO enzymatic activity by MA, a constituent of food and cigarette smoke, induces the expression of MAdCAM‐1 in hepatic vessels and results in the enhanced recruitment of mucosal effector lymphocytes to the liver. This could be an important mechanism underlying the hepatic complications of IBD. (HEPATOLOGY 2011;53:661‐672)

Reactive oxygen species mediate human hepatocyte injury during hypoxia/reoxygenation

Increasing evidence shows that reactive oxygen species (ROS) may be critical mediators of liver damage during the relative hypoxia of ischemia/reperfusion injury (IRI) associated with transplant surgery or of the tissue microenvironment created as a result of chronic hepatic inflammation or infection. Much work has been focused on Kupffer cells or liver resident macrophages with respect to the generation of ROS during IRI. However, little is known about the contribution of endogenous hepatocyte ROS production or its potential impact on the parenchymal cell death associated with IRI and chronic hepatic inflammation. For the first time, we show that human hepatocytes isolated from nondiseased liver tissue and human hepatocytes isolated from diseased liver tissue exhibit marked differences in ROS production in response to hypoxia/reoxygenation (H‐R). Furthermore, several different antioxidants are able to abrogate hepatocyte ROS–induced cell death during hypoxia and H‐R. These data provide clear evidence that endogenous ROS production by mitochondria and nicotinamide adenine dinucleotide phosphate oxidase drives human hepatocyte apoptosis and necrosis during hypoxia and H‐R and may therefore play an important role in any hepatic diseases characterized by a relatively hypoxic liver microenvironment. In conclusion, these data strongly suggest that hepatocytes and hepatocyte‐derived ROS are active participants driving hepatic inflammation. These novel findings highlight important functional/metabolic differences between hepatocytes isolated from normal donor livers, hepatocytes isolated from normal resected tissue obtained during surgery for malignant neoplasms, and hepatocytes isolated from livers with end‐stage disease. Furthermore, the targeting of hepatocyte ROS generation with antioxidants may offer therapeutic potential for the adjunctive treatment of IRI and chronic inflammatory liver diseases. Liver Transpl 16:1303‐1313, 2010.

CX3CR1 and vascular adhesion protein‐1‐dependent recruitment of CD16+ monocytes across human liver sinusoidal endothelium

The liver contains macrophages and myeloid dendritic cells (mDCs) that are critical for the regulation of hepatic inflammation. Most hepatic macrophages and mDCs are derived from monocytes recruited from the blood through poorly understood interactions with hepatic sinusoidal endothelial cells (HSECs). Human CD16+ monocytes are thought to contain the precursor populations for tissue macrophages and mDCs. We report that CD16+ cells localize to areas of active inflammation and fibrosis in chronic inflammatory liver disease and that a unique combination of cell surface receptors promotes the transendothelial migration of CD16+ monocytes through human HSECs under physiological flow. CX3CR1 activation was the dominant pertussis‐sensitive mechanism controlling transendothelial migration under flow, and expression of the CX3CR1 ligand CX3CL1 is increased on hepatic sinusoids in chronic inflammatory liver disease. Exposure of CD16+ monocytes to immobilized purified CX3CL1 triggered β1‐integrin‐mediated adhesion to vascular cell adhesion molecule‐1 and induced the development of a migratory phenotype. Following transmigration or exposure to soluble CX3CL1, CD16+ monocytes rapidly but transiently lost expression of CX3CR1. Adhesion and transmigration across HSECs under flow was also dependent on vascular adhesion protein‐1 (VAP‐1) on the HSECs. Conclusion: Our data suggest that CD16+ monocytes are recruited by a combination of adhesive signals involving VAP‐1 and CX3CR1 mediated integrin‐activation. Thus a novel combination of surface molecules, including VAP‐1 and CX3CL1 promotes the recruitment of CD16+ monocytes to the liver, allowing them to localize at sites of chronic inflammation and fibrosis. (Hepatology 2010)

Autophagy A cyto-protective mechanism which prevents primary human hepatocyte apoptosis during oxidative stress

The role of autophagy in the response of human hepatocytes to oxidative stress remains unknown. Understanding this process may have important implications for the understanding of basic liver epithelial cell biology and the responses of hepatocytes during liver disease. To address this we isolated primary hepatocytes from human liver tissue and exposed them ex vivo to hypoxia and hypoxia-reoxygenation (H-R). We showed that oxidative stress increased hepatocyte autophagy in a reactive oxygen species (ROS) and class III PtdIns3K-dependent manner. Specifically, mitochondrial ROS and NADPH oxidase were found to be key regulators of autophagy. Autophagy involved the upregulation of BECN1, LC3A, Atg7, Atg5 and Atg 12 during hypoxia and H-R. Autophagy was seen to occur within the mitochondria of the hepatocyte and inhibition of autophagy resulted in the lowering a mitochondrial membrane potential and onset of cell death. Autophagic responses were primarily observed in the large peri-venular (PV) hepatocyte subpopulation. Inhibition of autophagy, using 3-methyladenine, increased apoptosis during H-R. Specifically, PV human hepatocytes were more susceptible to apoptosis after inhibition of autophagy. These findings show for the first time that during oxidative stress autophagy serves as a cell survival mechanism for primary human hepatocytes.

Vascular adhesion protein-1 promotes liver inflammation and drives hepatic fibrosis

Nonalcoholic fatty liver disease (NAFLD) encompasses a range of manifestations, including steatosis and cirrhosis. Progressive disease is characterized by hepatic leukocyte accumulation in the form of steatohepatitis. The adhesion molecule vascular adhesion protein-1 (VAP-1) is a membrane-bound amine oxidase that promotes leukocyte recruitment to the liver, and the soluble form (sVAP-1) accounts for most circulating monoamine oxidase activity, has insulin-like effects, and can initiate oxidative stress. Here, we determined that hepatic VAP-1 expression is increased in patients with chronic liver disease and that serum sVAP-1 levels are elevated in patients with NAFLD compared with those in control individuals. In 4 murine hepatic injury models, an absence or blockade of functional VAP-1 reduced inflammatory cell recruitment to the liver and attenuated fibrosis. Moreover, disease was reduced in animals expressing a catalytically inactive form of VAP-1, implicating enzyme activity in the disease pathogenesis. Within the liver, hepatic stromal cells expressed functional VAP-1, and evaluation of cultured cells revealed that sVAP-1 promotes leukocyte migration through catalytic generation of ROS, which depended on VAP-1 enzyme activity. VAP-1 enhanced stromal cell spreading and wound closure and modulated expression of profibrotic genes. Together, these results link the amine oxidase activity of VAP-1 with hepatic inflammation and fibrosis and suggest that targeting VAP-1 has therapeutic potential for NAFLD and other chronic fibrotic liver diseases.

A stable miniature protein with oxaloacetate decarboxylase activity

An 18‐residue miniature enzyme, Apoxaldie‐1, has been designed, based on the known structure of the neurotoxic peptide apamin. Three lysine residues were introduced on the solvent‐exposed face of the apamin α‐helix to serve as an active site for decarboxylation of oxaloacetate. The oxidised form of Apoxaldie‐1, in which two disulfide bonds stabilise the α‐helix, formed spontaneously. CD spectroscopy measurements revealed that, in its oxidised form, Apoxaldie‐1 adopted a stably folded structure, which was lost upon reduction of the disulfide bonds. Despite its small size and the absence of a designed binding pocket, Apoxaldie‐1 displayed saturation kinetics in its oxidised form and catalysed the decarboxylation of oxaloacetate at a rate that was almost four orders of magnitude faster than that observed with n‐butylamine. This rivals the performance of the best synthetic oxaloacetate decarboxylases reported to date. Unlike those, however, Apoxaldie‐1 displayed significant stability. It maintained its secondary structure at temperatures in excess of 75 °C, in the presence of high concentrations of guanidinium chloride and at pH values as low as 2.2. Apamin‐based catalysts have potential for the generation of miniature peptides that display activity under nonphysiological conditions.