Systemic Inflammatory Response Syndrome (SIRS) or septic shock from bacterial infection, trauma or burn injury can be fatal. Over one-half of septic shock cases are a result of gram-negative bacterial infection. Structure/function studies of gram-negative lipopolysaccharide (LPS) have revealed a group of compounds that act as LPS antagonists. Lipid Iva is an LPS precursor molecule that has a unique species-specific bioactivity. Lipid Iva is an LPS antagonist in human cells and exhibits full LPS agonist activity in murine cells. This species-specific difference in bioactivity is believed to be a result of the differences in structure between the murine and human LPS signaling receptor. We used the species-specific difference to isolate, by expression cloning, a molecule that may be involved in LPS signaling. Our preliminary results describe the isolation and DNA sequence analysis of a partial murine cDNA (G21) that enables human cells to respond to lipid Iva. In addition we demonstrate that a stable human cell line containing the murine cDNA responds to lipid Iva stimulation in a CD14-dependent manner. Therefore this murine cDNA may be a part of the LPS recognition complex. Our goal for the two years of this proposal is to isolate and characterize both the murine and human full-length cDNAs encoding this receptor. These cDNAs will be used to construct stable cell lines to confirm lipid Iva and LPS dependent induction of inflammatory mediators (TNFalpha,ICAM-1). In vitro mutagenesis studies will be employed to identify the areas(s) involved in ligand recognition. If this receptor is part of the innate immune response to bacteria, we will have a new target for the design of drugs to fight septic shock.
