title: Endocytosis of uncleaved tumor necrosis factor-alpha in macrophages.
abstract: Activated monocytes and macrophages secrete the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). TNF-alpha is produced as a 26 kd transmembrane protein that is cleaved to release a 17 kd soluble protein. TNF-alpha in both forms is biologically active. The intracellular trafficking of membrane-associated TNF-alpha in lipopolysaccharide-activated mouse macrophages was assessed after treatment with the metalloprotease inhibitor BB-3103, which prevents the cleavage of pro-TNF-alpha. Immunoprecipitation and immunofluorescence studies showed sustained expression of cell-associated TNF-alpha in the presence of the inhibitor. Cell immunoreactivity and surface biotinylation revealed that uncleaved TNF-alpha accumulated on the cell surface and was endocytosed, appearing in intracellular vesicles. Perturbation of post-Golgi traffic blocked the surface expression of 26 kd TNF-alpha. Tracking a bolus of TNF-alpha over time in cycloheximide-treated cells confirmed that uncleaved TNF-alpha is first transported to the cell surface and subsequently endocytosed. Vesicular structures immunoreactive for TNF-alpha were identified as endosomes by double labeling. The secretory and membrane-associated endocytic trafficking of TNF-alpha provides a mechanism for modulating the quantity of biologically active 26 kd TNF-alpha expressed on macrophages, allowing regulation of paracrine and autocrine responses.
authors.affiliation: The Institute for Molecular Bioscience, The University of Queensland, Australia.
authors.forename: W
authors.initials: W
authors.lastname: Shurety
authors.forename: J K
authors.initials: JK
authors.lastname: Pagan
authors.forename: J B
authors.initials: JB
authors.lastname: Prins
authors.forename: J L
authors.initials: JL
authors.lastname: Stow
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology; D004705:Endocytosis; D006056:Golgi Apparatus / Q000276:immunology; D006877:Hydroxamic Acids / Q000494:pharmacology; D007150:Immunohistochemistry; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation; D008264:Macrophages / Q000187:drug effects / Q000276:immunology*; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors; D051379:Mice; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000378:metabolism*
chemical_list: C108282:BB 3103; D006877:Hydroxamic Acids; D008070:Lipopolysaccharides; D011480:Protease Inhibitors; D014409:Tumor Necrosis Factor-alpha; D008666:Metalloendopeptidases