The goal of this proposal is to further understand the mechanisms involved in LPS-mediated actions in leukocytes, focusing on the interaction between LPS and the microtubule (MT) network of murine macrophages (Mphi). LPS is known to have profound, multiple effects on Mphis. Preliminary findings that drugs (e.g. colchicine and taxol) which are known to affect microtubule organization can mimic LPS in inducing (a) down-regulation of Mphi TNFalpha receptors, (b) release of TNFalpha, and (c) secretion of nitrite, and that LPS-hyporesponsive C3H/HeJ mice lack these responses to taxol, raise the possibilities that MT may play an important role in LPS- mediated actions.
The specific aims of this study are to answer how LPS's actions are related to MT organization, and whether there is any structural or functional association between MT and LPS-binding protein(s). The proposed experiments include: (1) To determine the functional interaction between LPS and MT by examining the effect of LPS on MT modification (tyrosinolation and phosphorylation), and the effect of MT-active agents (i.e., colchicine, nocodazole, taxol) on lPS-mediated activation of Mphis (release of IL-1, TNFalpha, reactive oxygen or nitrogen intermediates; changes in intracellular calcium, Ia expression; and translocation of protein kinase c). (2) To examine whether any of the MY components can serve as an LPS-receptor or associate with an LPS receptor, by colocalizing MT and LPS binding sites with double immunofluorescent staining, by measuring the binding of labeled LPS to isolated MT, and by co- precipitating an LPS receptor with MT in the presence of taxol. (3) To determine the genetic linkage between LPS- and taxol-responsiveness in the mouse by examining the closeness of these two responses in Mphis from the F2 generation of a cross between C5/BL/6J and C3H/HeJ, possibly leading to the identification of the defective protein(s) in C3H/HeJ mice.
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