Localization and post-Golgi trafficking of tumor necrosis factor-alpha in macrophages

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine secre ted by activated macrophages, In this study, we examined the intracellular distribution and trafficking of TNF-alpha, Immunofluorescence and immunogol d localization demonstrated that in lipopolysaccharide (LPS)-stimulated RAW 264 macrophages, the greatest concentration of TNF-alpha is found in the pe rinuclear Golgi complex, Staining of the Golgi complex appeared 20 min afte r activation of cells and persisted for 2-12 h, and TNF-cu appeared on the cell surface only transiently during this time. The rate of disappearance o f Golgi staining correlated with the release of the cleaved, mature TNP-alp ha into the medium, Pulse chase labeling and subcellular fractionation stud ies indicated that both 26-kDa and 17-kDa forms of TNF-alpha may be present at the level of the Golgi complex, Post-Golgi trafficking of TNF-alpha was modulated by agents that disrupt the cytoskeleton, Interferon-gamma (IFN-g amma), which primes macrophages for TNF-alpha-dependent cellular cytotoxici ty, potentiated the effect of LPS by sustaining enhanced intracellular pool s of TNF-alpha and also promoted redistribution of TNF-alpha into post-Golg i vesicular compartments. We propose that the primary pool of biologically active TNF-alpha in activated macrophages is held in the Golgi complex and that the cytokine is recruited directly from this intracellular pool for re lease in response to tumor cells or pathogens.