MOLECULAR ANALYSIS OF MACROPHAGE ACTIVATION VIA LBP:LPS

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CD14 is a myelomonocytic differentiation antigen expressed strongly on the surface of monocytes and weakly on granulocytes. CD14 has recently been shown to be a receptor for a complex consisting of lipopolysaccharide (LPS) and an acute phase serum protein called LBP (lipopolysaccharide binding protein). Binding of LBP:LPS complex to monocytes via CD14 results in strong activation of macrophages as measured by the production of tumor necrosis factor (TNF). Activation of macrophages by this pathway is thought to be the initial step in the production of TNF found in endotoxic shock and mediating the collapse of the cardiovascular-pulmonary-renal systems. The primary goals of this proposal are to determine whether soluble forms of CD14 can inhibit macrophage activation via the LBP:LPS pathway and to further define this pathway of macrophage activation at the molecular level by mapping the LBP:LPS binding site on the CD14 molecule. In addition, we will define peptides which can reduce and/or inhibit macrophage activation via this pathway. Soluble CD14 and its peptides will be analyzed both in vitro and in vivo for the ability to inhibit LPS-induced macrophage activation and/or death due to endotoxin shock. The in vivo experiments will utilize a transgenic mouse which we have recently produced which expresses human CD14 and which is more susceptible to LPS than normal mice. The long term goal of this proposal is to use CD14 peptides to reduce and/or inhibit TNF production in gram negative septic shock with the prospect of allowing time for the patient to respond to appropriate alternative therapy.