The principal aim of this proposal is to describe the molecular interactions that enable bacterial lipopolysaccharide (LPS, endotoxin) to stimulate leukocytes in vitro and to cause endotoxic shock in animals. Our hypothesis is that binding proteins in serum first interact with LPS, then the resulting LPS-binding protein complexes interact with a specific receptor on leukocytes. In preliminary experiments, a novel human serum protein called septin was isolated. This protein binds LPS and the resulting septin-LPS complexes are recognized by human monocytes, macrophages, and PMN. The molecule on phagocytes responsible for binding septin-LPS complexes vas shown to be CD14, a well-characterized membrane protein with no previously described function. CD14 appears critical to cellular responses since blockade of CD14 with mAbs blocked TNF synthesis by monocytes in blood incubated with LPS. This proposal seeks to first characterize the structure of septin, then to characterize the binding interactions between septin and LPS, and between septin-LPS complexes and CD14. The role of septin and CD14 in the stimulation of leukocytes will be examined by assaying several activities of isolated cells including enhanced CD18-dependent adhesivity of PMN and TNF secretion by monocytes in the presence and absence of septin and in the presence or absence of anti-CD14 mAbs. Preliminary studies indicate that CD14 is lost from the cell surface during responses to LPS. The fate of the lost CD14 will be determined, and experiments will be done to test the hypothesis that loss of CD14 plays a role in low-dose desensitization to LPS. The role of septin and CD14 in signal transduction will be characterized by separately measuring the time course of binding, uptake and/or shedding, and degradation of septin, CD14, and LPS and then comparing these time courses with that of signalling. Finally, we will test the hypothesis that septin and CD14 play a role in endotoxic shock by studies of a baboon model of sepsis. Blood levels of soluble and cell bound septin, and soluble and cell-bound CD14 will be measured at time intervals during experimental sepsis. In addition, animals will be treated with anti-CD14 or anti-septin prior to challenge with bacteria to determine the role of these proteins in all of the principal manifestations of endotoxemia.
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