Bacterial LPS is a major factor in the toxic manifestations of systemic inflammatory response syndrome, a major cause of death in the US. The inflammatory cytokines TNF-alpha, IL-1 and IL-6 are produced by macrophages are major mediators of the toxic effects of LPS. The goal of the proposed studies is to understand the mechanisms of LPS-induced responses in leukocytes, in particular the gene products participating in the early response of macrophages to LPS. Two tools will be used for these studies, 1) the congenic mouse strains, C3H/HeN and C3H/HeJ, which respond differently to LPS due to a mutation in a single gene, Lps. 2) Taxol (a microtubule-binding agent) mimics actions of LPS in macrophages. It is proposed to determine if there are shared cellular targets of taxol and LPS, and to define these targets and their roles in LPS-induced signaling. It is also proposed to determine if there is a constitutive gene differentially expressed between HeN and HeJ mice, and to determine the role of this putative gene and its relationship to the LPS gene product. To accomplish these goals, the investigator will: 1) Characterize early responses of macrophages to LPS in a cell free system in order to test the role of candidate proteins that might participate in early LPS signaling. 2) Identify and clone cellular targets shared by taxol and LPS by screening a macrophage cDNA expression library with labeled taxol and anti-taxol antibody. 3) Identify genes differentially expressed in Lpsn (LPS normoresponsive) and Lpsd (LPS-hyporesponsive) cells using differential display analysis, to clone these genes and study their roles in LPS-signaling.
