The role of macrophage CD154 in promoting chronic liver inflammation and apoptosis using a primary cell coculture model
Lead Research Organisation:
University of Birmingham
Department Name: Sch of Geography, Earth & Env Sciences
Abstract
The vital functions of the liver are removal of blood toxins, protein production, bile acid synthesis, iron storage and gluconeogenesis. As a result of toxic injury the liver may undergo episodes of inflammation which usually resolve. The persistence of inflammation causes permanent tissue damage leading to cirrhosis, liver failure and occasionally malignant disease. Currently the only treatment for end stage liver disease is transplantation. This has significant impact on patients‘ quality of life and constitutes a major economic burden to the NHS. Some of these end stage liver diseases are characterised by destruction of hepatocytes and the bile ducts. The liver contains a variety of inflammatory cells which are either resident or derived from the circulation. Lymphocytes and macrophages or Kuppfer cells constitute the largest populations of cells within the liver and their numbers increase during inflammation. Macrophages clear unwanted cellular debris resulting from tissue damage. They may also theoretically promote inflammation if inappropriately activated. My preliminary work has demonstrated that macrophages can destroy biliary epithelial cells that form the bile ducts that transport bile within the liver but the mechanisms responsible remain unclear. It is of crucial importance to dissect the mechanism of cell destruction in order to design more effective future therapies for treatment of chronic inflammatory liver disease.
Technical Summary
Background: We have previously reported increased expression of CD40 in the vanishing bile duct syndromes namely chronic allograft rejection and primary biliary cirrhosis. Although T cells are present, CD68 positive macrophages expressing high levels of CD154 (CD40 ligand) are the predominant inflammatory cell in zones of hepatocyte and bile duct loss (Afford et al JEM 1999; Ahmed Choudhury et al FASEB J 2001; Ahmed Choudhury et al Mol Biol Cell 2003) suggesting that macrophages are the main source of CD154 to activate biliary epithelial cell (BEC) CD40. Recent evidence that macrophages are key regulatory cells in the development and resolution of hepatic fibrosis (Iredale JP et al JCI 2005) suggest a critical role for hepatic macrophages in chronic inflammatory liver disease. WE PROPOSE THAT THE ACTIVATION OF BILIARY EPITHELIAL CELL (BEC) CD40 BY LIVER MACROPHAGE CD154 PROMOTES BEC LOSS AND CHRONIC INFLAMMATION IN LIVER DUCTOPENIAS.
Hypothesis: Bi-directional signals between macrophages and BEC mediated by the CD40-CD154 pathway promote chronic inflammation and BEC apoptosis in chronic liver disease.
Aims: To define the functional consequences of paracrine interactions between hepatic macrophages and BEC mediated by the CD40-CD154 pathway.
Experimental design: I have established coculture systems to study interactions between human liver-derived macrophages (LDM) or peripheral blood-derived macrophages (BDM) and biliary epithelial cells (BEC) and will use this system to determine how such interactions may drive local cytokine/ chemokine secretion and mediate apoptosis of BEC. The use of matched samples of LDM and BDM will allow for comparison of the actions of the hepatic and circulating macrophages.
Study 1: As a consequence of coculture of CD40 expressing primary BEC with LDM or BDM, the cytokines secreted will be determined using multiplex cytokine arrays in conjunction with conventional ELISA. These experiments will determine how paracrine interactions between these cells contribute to the local inflammatory cytokine/ chemokine milieu.
Study 2: The apoptosis of BEC as a consequence of coculture with CD154 expressing macrophages will be measured and the contribution of CD154, CD40 and Fas will be determined using specific chemical inhibitors by silencing of macrophage-expressed CD154 using transient transfection with siRNA, dominant negative mutant CD154, and similarly by silencing of BEC-expressed CD40 and CD95 using siRNA and constructs.
Study 3: CD154 mRNA expression in macrophages isolated (with or without subsequent stimulation) from a variety of ductopenic livers will be measured using real time PCR. This will similarly be done on macrophages isolated specifically from regions of BEC apoptosis by using laser capture microdissection on frozen tissue sections from a variety of ductopenic livers. This will allow for relative quantification of CD154 mRNA in different ductopenias and in different spatial locations where macrophages reside in tissue. Laser capture microdissection is established in our laboratories. Furthermore, the expression of genes and markers of cellular activation as a consequence of CD154-CD40 interaction will be determined using limited cDNA microarray to profile the expression of key cytokine and chemokine genes involved in the inflammatory response. This will be done in collaboration with Professors Young and Wakelam (CRUK Institute of Cancer Studies). For this study, in addition to ligation by cell-bound CD40, CD154 will also be engaged with cross-linking antibody and recombinant CD40.
Justification: The molecular pathogenesis of chronic inflammation is a major focus for Professor Adams‘ and Dr Afford‘s laboratories. Understanding the role of macrophage CD154 in a physiologically relevant model of chronic inflammation may identify novel targets for modulation of macrophage proinflammatory function, treatment of irreversible bile duct damage, and progression of cirrhosis.
Hypothesis: Bi-directional signals between macrophages and BEC mediated by the CD40-CD154 pathway promote chronic inflammation and BEC apoptosis in chronic liver disease.
Aims: To define the functional consequences of paracrine interactions between hepatic macrophages and BEC mediated by the CD40-CD154 pathway.
Experimental design: I have established coculture systems to study interactions between human liver-derived macrophages (LDM) or peripheral blood-derived macrophages (BDM) and biliary epithelial cells (BEC) and will use this system to determine how such interactions may drive local cytokine/ chemokine secretion and mediate apoptosis of BEC. The use of matched samples of LDM and BDM will allow for comparison of the actions of the hepatic and circulating macrophages.
Study 1: As a consequence of coculture of CD40 expressing primary BEC with LDM or BDM, the cytokines secreted will be determined using multiplex cytokine arrays in conjunction with conventional ELISA. These experiments will determine how paracrine interactions between these cells contribute to the local inflammatory cytokine/ chemokine milieu.
Study 2: The apoptosis of BEC as a consequence of coculture with CD154 expressing macrophages will be measured and the contribution of CD154, CD40 and Fas will be determined using specific chemical inhibitors by silencing of macrophage-expressed CD154 using transient transfection with siRNA, dominant negative mutant CD154, and similarly by silencing of BEC-expressed CD40 and CD95 using siRNA and constructs.
Study 3: CD154 mRNA expression in macrophages isolated (with or without subsequent stimulation) from a variety of ductopenic livers will be measured using real time PCR. This will similarly be done on macrophages isolated specifically from regions of BEC apoptosis by using laser capture microdissection on frozen tissue sections from a variety of ductopenic livers. This will allow for relative quantification of CD154 mRNA in different ductopenias and in different spatial locations where macrophages reside in tissue. Laser capture microdissection is established in our laboratories. Furthermore, the expression of genes and markers of cellular activation as a consequence of CD154-CD40 interaction will be determined using limited cDNA microarray to profile the expression of key cytokine and chemokine genes involved in the inflammatory response. This will be done in collaboration with Professors Young and Wakelam (CRUK Institute of Cancer Studies). For this study, in addition to ligation by cell-bound CD40, CD154 will also be engaged with cross-linking antibody and recombinant CD40.
Justification: The molecular pathogenesis of chronic inflammation is a major focus for Professor Adams‘ and Dr Afford‘s laboratories. Understanding the role of macrophage CD154 in a physiologically relevant model of chronic inflammation may identify novel targets for modulation of macrophage proinflammatory function, treatment of irreversible bile duct damage, and progression of cirrhosis.