PROCESSING OF ENDOTOXINS BY LIVER MACROPHAGES

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Infection due to gram negative bacteria is a major clinical problem and bacterial endotoxins in the circulation cause life threatening systemic reactions. Endotoxin is also involved in the pathology of a number of liver diseases including cirrhosis. The liver is the major site of metabolism for gut derived endotoxins. The cells responsible for endotoxin clearance from the portal blood is the liver macrophage (Kupffer cell) and co-operates with hepatocytes in detoxification. Because of continuous chronic exposure to low levels of endotoxin the Kupffer cell response to endotoxin is different to other macrophages and monocytes and the molecular details of endotoxin uptake and response in Kupffer cells in unclear. To investigate this we will: 1) Characterize the structures necessary for the binding of endotoxin to the Kupffer cells. We have identified two novel endotoxin binding proteins of 31 and 34 kD on rat Kupffer cells that are internal proteins that appear to be involved in transducing the signal for the cytokine response. Experiments are designed to purify these proteins and to determine amino acid sequences. We will produce monoclonal antibodies to both the 31 and 34 kD binding proteins using multiple antigenic peptide (MAP) technology. These antibodies will be used to study the functional significance and distribution of these binding proteins. 2) We will identify and clone and sequence the genes encoding the 31 and 34 kD endotoxin binding proteins using peptide sequences to synthesize specific oligonucleotide probes. The sequence data will be used to create a potential domain model for the binding proteins and to examine their structural relationships to other proteins. The cloned genes will also be available for transfection into eukaryotic cells and provide a model to study the biological role of these novel LPS binding proteins. 3) We will determine the role of the 31 and 34 kD binding proteins in signaling events associated with LPS interaction with Kupffer cells and in the secretion of cytokines by Kupffer cells in response to LPS. To examine the protective effects of Mn/++ and other transition metals against endotoxin and their inhibition of LPS binding to the 31 and 34 kD proteins. These studies will increase our understanding of the biological effects of endotoxins and lead to novel therapies against toxic shock.