Activation of macrophages by LPS results in production of various mediators, such as IL-1, IL-6, TNFa and GM-CSF, which are involved in regulating hematopoiesis, inflamation and immune responses. Recently it has been shown that agents that interact with microtubules, such as taxol and colchicine, can induce production of some of these mediators. In an effort to clarify the role of microtubules in the regulation of GM-CSF, we examined whether depolymerization of microtubules will affect GM-CSF production in macrophages. The murine macrophage cell line RAW264.7 was treated with colchicine (1 micromole) and LPS (50 ng/ml) and GM-CSF mRNA and protein expression examined. Colchicine did not induce GM-CSF expression in RAW 264.7 cells. However, pretreatment of the cells with colchicine resulted in a time-dependent down-regulation of LPS-induced GM-CSF steady-state mRNA expression and protein production. Maximun inhibition of LPS-induced GM-CSF expression was observed when cells were pre-treated for 4 hours with colchicine. Actinomycin D chase experiments revealed colchicine pre-treatment did not alter GM-CSF mRNA half-life.Studies using stable transfectants containing the GM-CSF promoter and the lucifersae reporter gene coding region showed that colchicine pre-treatment inhibited LPS-induced transcription of luciferase. In addition, to GM-CSF, colchicine also inhibited LPS-induced IL-6 mRNA expression but not iNOS expression. Using b-tubulin mRNA expression as an indirect measure of tubulin depolymerization, colchicine treatment of RAW264.7 cells decreased b-tubulin mRNA expression in a time dependent manner and correlated, temporally, with the decrease in GM-CSF mRNA expression. Thus, LPS and colchicine regulate GM-CSF expression at the level of transcription and the de-polymerization of microtubules can differentially affect LPS induced gene expression.
