Simon C. Afford

Cytokines induced during chronic hepatitis B virus infection promote a pathway for NK cell–mediated liver damage

Hepatitis B virus (HBV) causes chronic infection in more than 350 million people worldwide. It replicates in hepatocytes but is non-cytopathic; liver damage is thought to be immune mediated. Here, we investigated the role of innate immune responses in mediating liver damage in patients with chronic HBV infection. Longitudinal analysis revealed a temporal correlation between flares of liver inflammation and fluctuations in interleukin (IL)-8, interferon (IFN)-α, and natural killer (NK) cell expression of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) directly ex vivo. A cross-sectional study confirmed these findings in patients with HBV-related liver inflammation compared with healthy carriers. Activated, TRAIL-expressing NK cells were further enriched in the liver of patients with chronic HBV infection, while their hepatocytes expressed increased levels of a TRAIL death–inducing receptor. IFN-α concentrations found in patients were capable of activating NK cells to induce TRAIL-mediated hepatocyte apoptosis in vitro. The pathogenic potential of this pathway could be further enhanced by the ability of the IFN-α/IL-8 combination to dysregulate the balance of death-inducing and regulatory TRAIL receptors expressed on hepatocytes. We conclude that NK cells may contribute to liver inflammation by TRAIL-mediated death of hepatocytes and demonstrate that this non-antigen–specific mechanism can be switched on by cytokines produced during active HBV infection.

CD40 Induces Apoptosis in Carcinoma Cells through Activation of Cytotoxic Ligands of the Tumor Necrosis Factor Superfamily

ABSTRACT CD40, a tumor necrosis factor (TNF) receptor (TNFR) family member, conveys signals regulating diverse cellular responses, ranging from proliferation and differentiation to growth suppression and cell death. The ability of CD40 to mediate apoptosis in carcinoma cells is intriguing given the fact that the CD40 cytoplasmic C terminus lacks a death domain homology with the cytotoxic members of the TNFR superfamily, such as Fas, TNFR1, and TNF-related apoptosis-inducing ligand (TRAIL) receptors. In this study, we have probed the mechanism by which CD40 transduces death signals. Using a trimeric recombinant soluble CD40 ligand to activate CD40, we have found that this phenomenon critically depends on the membrane proximal domain (amino acids 216 to 239) but not the TNFR-associated factor-interacting PXQXT motif in the CD40 cytoplasmic tail. CD40-mediated cytotoxicity is blocked by caspase inhibitors, such as zVAD-fmk and crmA, and involves activation of caspase 8 and caspase 3. Interestingly, CD40 ligation was found to induce functional Fas ligand, TRAIL (Apo-2L) and TNF in apoptosis-susceptible carcinoma cells and to up-regulate expression of Fas. These findings identify a novel proapoptotic mechanism which is induced by CD40 in carcinoma cells and depends on the endogenous production of cytotoxic cytokines and autocrine or paracrine induction of cell death.

Hepatic Stellate Cells Express the Low Affinity Nerve Growth Factor Receptor p75 and Undergo Apoptosis in Response to Nerve Growth Factor Stimulation

We have examined the expression of p75, a member of the TNF receptor superfamily in hepatic stellate cells (HSC) and pancreatic stellate cells (PSC). Activated HSC and PSC were demonstrated by Western blot analysis to express p75. p75 was immunolocalized to cells with a myofibroblast-like morphology in the fibrotic bands of six fibrotic and cirrhotic liver biopsies and three biopsies of fibrotic human pancreas. Immunostaining of parallel sections indicated that these cells were alpha-smooth muscle actin-positive, identifying them as activated HSC and PSC, respectively. HSC apoptosis in tissue culture in the presence of serum was quantified after addition of 0.1 to 100 ng/ml of nerve growth factor (NGF) a ligand for p75, by in situ counting of apoptotic bodies after addition of acridine orange. HSC demonstrated a significant increase in apoptosis in response to 100 ng/ml NGF (0.05 > P by Wilcoxons rank; n = 7) after 24 hours. NGF 100 ng/ml had no effect on HSC proliferation, but reduced total HSC DNA by 19% relative to control after 24 hours (n = 3). These data demonstrate that activated HSC express p75 and respond to NGF stimulation by undergoing apoptosis. We therefore report p75 as a novel marker of activated HSC and suggest that signaling via ligand binding to p75 may provide a mechanism for selective apoptosis of HSC.

Distinct patterns of chemokine expression are associated with leukocyte recruitment in alcoholic hepatitis and alcoholic cirrhosis

Alcoholic liver disease is associated with three histologically distinct processes: steatosis (parenchymal fat accumulation), alcoholic hapatitis (characterized by parenchymal infiltration by neutrophil polymorphs), and alcoholic cirrhosis (in which chronic inflammation and fibrosis dominate). Chemokines are cytokines that promote subset‐specific leukoycte recruitment to tissues and could therefore play a crucial role in determining which leukocyte subsets are recruited to the liver in alcoholic liver disease. This paper reports that chemokine expression is increased in the liver of patients with alcoholic liver disease and, moreover, that distinct patterns of chemokine expression are associated with the different inflammatory responses to alcohol. Interleukin‐8 (IL‐8), monocyte chemoattractant protein‐1 (MCP‐1), macrophage inflammatory protein‐1α (MIP‐1α), and MIP‐1β were all detected in the parenchyma at sites of inflammation in alcoholic hepatitis, whereas in alcoholic cirrhosis, chemokines were restricted to inflammatory cells and endothelium in the fibrous septa and portal tracts. In alcoholic hepatitis, chemokine transcription was localized to sinusoidal cells, leukocytes, and fibroblasts in areas of parenchymal inflammation, but hepatocytes, despite staining strongly for chemokine protein, were negative. In alcoholic cirrhosis, chemokine mRNA was detected in portal tract endothelium, leukocytes, and fibroblasts. Thus, alcoholic hepatitis and alcoholic cirrhosis are associated with distinct patterns of chemokine expression that are likely to be important factors in determining whether a patient develops acute parenchymal inflammation and alcoholic hepatitis, or chronic septal inflammation and alcoholic cirrhosis.

CD40 activation-induced, Fas-dependent apoptosis and NF-κB/AP-1 signaling in human intrahepatic biliary epithelial cells

Fas‐mediated mechanisms of apoptosis are thought to be involved in the bile duct loss that characterizes diseases such as primary biliary cirrhosis (PBC). We have previously shown that activation of CD40 on hepatocytes can amplify Fas‐mediated apoptosis; in the present study, we investigated interactions between CD40 and Fas in biliary epithelial cells (BEC). We report that the bile ducts in PBC liver tissue frequently express increased levels of Fas, Fas ligand (FasL), and CD40 associated with apoptotic BEC. The portal mononuclear infiltrate contains CD40L+ve T cells and macrophages, thereby demonstrating a potential mechanism for CD40 engagement in vivo. Primary cultures of human BEC also expressed Fas, FasL, and CD40 but not CD40L protein or mRNA. Activation of CD40 on BEC using recombinant CD40L increased transcriptional expression of FasL and induced apoptosis, which was inhibited by neutralizing antibodies to either Fas or FasL. Thus, CD40‐induced apoptosis of BEC is mediated through Fas/FasL. We then investigated the intracellular signals and transcription factors activated in BEC and found that NF‐κB and AP‐1 were both activated after CD40 ligation. Increased functional NF‐κB was seen early after CD40 ligation, but returned to baseline levels after 4 h. In contrast, the rapid up‐regulation of AP‐1 was sustained over 24 h. This study provides further functional evidence of the ability of CD40 to induce Fas/FasL‐dependent apoptosis of liver epithelial cells supporting the importance of cross‐talk between tumor necrosis factor (TNF) receptor family members as an amplification step in apoptosis induction. Sustained activation of AP‐1 in the absence of NF‐κB signaling may be a critical factor in determining the outcome of CD40 engagement.—Afford, S. C., Ahmed‐Choudhury, J., Randhawa, S., Russell, C., Youster, J., Crosby, H. A., Eliopoulos, A., Hubscher, S. G., Young, L. S., Adams, D. H. CD40 activation‐induced, Fas‐dependent apoptosis and NFkB/AP‐1 signaling in human intrahepatic biliary epithelial cells. FASEB J. 15, 2345‐2354 (2001)

Fas/Fas Ligand Interaction in Human Colorectal Hepatic Metastases A Mechanism of Hepatocyte Destruction to Facilitate Local Tumor Invasion

This study demonstrates a novel role for the Fas pathway in the promotion of local tumor growth by inducing apoptotic cell death in normal hepatocytes at the tumor margin in colorectal hepatic metastases. Our results show that >85% of lymphocytes infiltrating colorectal liver cancer express high levels of Fas-ligand (Fas-L) by flow cytometry. Using immunohistochemistry of tumor tissue we showed strong Fas expression in noninvolved hepatocytes, whereas Fas-L expression was restricted to tumor cells and infiltrating lymphocytes at the tumor margin. Apoptosis was observed in 45 +/- 13% of the Fas(high) hepatocytes at the tumor margin whereas only 7 +/- 3% tumor cells were apoptotic (n = 10). In vitro, primary human hepatocytes expressed Fas receptor and crosslinking with anti-Fas antibody induced apoptosis in 44 +/- 5% of the cells compared with 4. 6 +/- 1.0% in untreated controls (P = 0.004). Both tumor-infiltrating lymphocytes (TIL) and human metastatic colon cancer cells cells are able to induce Fas-mediated apoptosis of primary human hepatocytes in coculture cytotoxic assays. TIL induced apoptosis in 47 +/- 9% hepatocytes compared with control 4.3 +/- 1. 0% (P = 0.009) and this effect was reduced by anti-human Fas-L mAb (18.7 +/- 1.3%, P = 0.009). SW620 cells induced apoptosis in 26 +/- 2% hepatocytes compared with control 5.6 +/- 1.7% (P = 0.004) and this was reduced to 11.2 +/- 1.8% (P = 0.004) in the presence of anti-human Fas-L mAb. These data suggest that the inflammatory response at the margin of colorectal liver metastases induces Fas expression in surrounding hepatocytes, allowing them to be killed by Fas-L-bearing TIL or tumor cells and facilitating the invasion of the tumor into surrounding liver tissue.

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.

Hepatic expression of macrophage inflammatory protein-1α and macrophage inflammatory protein-1β after liver transplantation

Two local events that are crucial for T cell emigration into tissue are (1) activation of T cell integrins to permit binding to endothelial counter-receptors and (2) directed migration through the endothelium and into tissue in response to chemotactic factors. Because the chemokines macrophage inflammatory protein-1 alpha (MIP-1 alpha) and MIP-1 beta can activate adhesion and induce migration of T cells in vitro, we investigated their expression in human liver allografts to determine whether they might be involved in regulating the recruitment of T cells to allografts in vivo. Both chemokines were expressed strongly by infiltrating leukocytes during rejection and could be detected immunohistochemically on biliary epithelium, an important target for T cell mediated graft damage. Both chemokines, but particularly MIP-1 beta, were detected on the vascular and sinusoidal endothelium of rejecting liver allografts, where they were coexpressed with the T cell beta 1-integrin receptor vascular cell adhesion molecule-1. In situ hybridization with complementary ribonucleic acid probes showed no MIP-1 alpha or MIP-1 beta mRNA in normal liver but dramatic expression of both chemokines in infiltrating leukocytes and graft endothelium during rejection. Expression was reduced after successful corticosteroid treatment of rejection but persisted in patients progressing to chronic rejection. Increased MIP-1 alpha and MIP-1 beta mRNA expression was already found in biopsies taken at the end of the transplant operation, suggesting that early induction of chemokines, possibly in response to graft reperfusion, might promote the subsequent development of graft rejection. These data demonstrate for the first time that MIP-1 alpha and MIP-1 beta are (1) expressed in human liver allografts, (2) produced by endothelial cells in vivo, and (3) induced early after transplantation. They suggest that MIP-1 alpha and MIP-1 beta produced by graft infiltrating leukocytes and graft endothelium might play a crucial role in regulating T cell recruitment to liver allografts in vivo.

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.

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.