Endocytosis of Uncleaved Tumor Necrosis Factor-α in Macrophages

Activated monocytes and macrophages secrete the inflammatory cytokine tumor necrosis factor-α (TNF-α). TNF-α is produced as a 26 kd transmembrane protein that is cleaved to release a 17 kd soluble protein. TNF-α in both forms is biologically active. The intracellular trafficking of membrane-associated TNF-α in lipopolysaccharide-activated mouse macrophages was assessed after treatment with the metalloprotease inhibitor BB-3103, which prevents the cleavage of pro–TNF-α. Immunoprecipitation and immunofluorescence studies showed sustained expression of cell-associated TNF-α in the presence of the inhibitor. Cell immunoreactivity and surface biotinylation revealed that uncleaved TNF-α 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-α. Tracking a bolus of TNF-α over time in cycloheximide-treated cells confirmed that uncleaved TNF-α is first transported to the cell surface and subsequently endocytosed. Vesicular structures immunoreactive for TNF-α were identified as endosomes by double labeling. The secretory and membrane-associated endocytic trafficking of TNF-α provides a mechanism for modulating the quantity of biologically active 26 kd TNF-α expressed on macrophages, allowing regulation of paracrine and autocrine responses.