Localization and post-Golgi trafficking of tumor necrosis factor-alpha in macrophages.
Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine secreted by activated macrophages. In this study, we examined the intracellular distribution and trafficking of TNF-alpha. Immunofluorescence and immunogold localization demonstrated that in lipopolysaccharide (LPS)-stimulated RAW264 macrophages, the greatest concentration of TNF-alpha is found in the perinuclear Golgi complex. Staining of the Golgi complex appeared 20 min after activation of cells and persisted for 2-12 h, and TNF-alpha appeared on the cell surface only transiently during this time. The rate of disappearance of Golgi staining correlated with the release of the cleaved, mature TNF-alpha into the medium. Pulse chase labeling and subcellular fractionation studies 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-gamma), which primes macrophages for TNF-alpha-dependent cellular cytotoxicity, potentiated the effect of LPS by sustaining enhanced intracellular pools of TNF-alpha and also promoted redistribution of TNF-alpha into post-Golgi 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 release in response to tumor cells or pathogens.
Shurety
W
W
The Centre for Molecular and Cellular Biology, The University of Queensland, Brisbane, Australia.
Merino-Trigo
A
A
Brown
D
D
Hume
D A
DA
Stow
J L
JL
D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D003599:Cytoskeleton / Q000502:physiology; D006056:Golgi Apparatus / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism*; D007371:Interferon-gamma / Q000378:metabolism / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism*; D051379:Mice; D011499:Protein Processing, Post-Translational / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology* / Q000378:metabolism*
D014409:Tumor Necrosis Factor-alpha; D020126:Brefeldin A; D007371:Interferon-gamma; D003513:Cycloheximide