The interaction of the outer membrane components of bacteria with host cells in one form or another has been investigated extensively over the past decades. The lipopolysaccharide endotoxin (LPS) of Gram-negative bacteria is an example of an essential element of the outer membrane of these organisms that as amphipathic molecule has the property of binding to and stimulating various types of mammalian cells. The results of this interaction are multiple pathophysiological, pharmacological and immunological responses by the host. Over the preceding years, our long term objective has been to gain a better understanding of how LPS endotoxin affects cells of the host. The knowledge that host responses are under genetic control is key to this understanding. Consequently, we have pursued the goal of isolating the LPS gene by the use of the C3H/HeJ mouse strain whose cells possess a specific defect for LPS. By employing a differential functional screening approach, we have isolated a cDNA from splenic B cells of C3H/OuJ responder mice, and its expression in splenic B cells of C3H/OuJ responder mice, and its expression in splenic B cells of C3H/HeJ non-responder mice resulted in polyclonal B cell activation in response to LPS stimulation. In this proposal, our specific aims, which are a logical consequence of this finding, are as follows: 1) to complete the confirmatory analysis that expression of the cDNA we isolated can functionally reconstitute a macrophage cell line derived from a C3H/HeH mouse, 2) to refine the mapping analysis indicating that the gene is located on mouse chromosome four, 3) to define the nature of the Lpsd gene we isolated from C3H/HeH mice, 4) to investigate its genomic organization and to verify two single-base substitutions found in C3H/HeJ cDNA, 5) to introduce the Lpsn gene into various primary cells of the C3H/HeJ non-responder mouse and measure their responsiveness after exposure to LPS, 6) to introduce the Lpsn gene into C3H/HeJ non-responder mice using a retrovirus vector and measure the key parameters of endotoxin responsiveness in these mice including B cell mitogenesis, macrophage activation and cytokine elaboration, and resistance to endotoxemia or lethal shock, and 7) to assess the role of Lpsn in resistance to Gram negative bacterial infection.
