Immunohistochemical demonstration of cytoplasmic and membrane-associated tumor necrosis factor in murine macrophages

Abstract

Using a highly specific rabbit antisera directed against murine tumor necrosis factor (TNF), immunohistochemical localization of this monokine was performed in cultured mouse peritoneal macrophages. Resident macrophages did not express TNF even after stimulus with lipopolysaccharide (LPS). In contrast, 12% of macrophages elicited with Freund's adjuvant stained positively and up to 60% were positive after LPS stimulation. Analysis of the kinetics of expression revealed that maximal staining occurred from 1-3 hours after stimulus with disappearance of staining by 12 hours. Both a membrane and cytoplasmic pattern of staining could be demonstrated. The presence of plasma membrane TNF was confirmed by scanning electron microscopy. Northern blot analysis and bioassay revealed that the kinetics of TNF mRNA synthesis corresponded to the appearance of the protein while its disappearance corresponded to the appearance of TNF in the supernate. Thus, TNF synthesis and secretion could be histochemically demonstrated. These findings support the notion that TNF production is a characteristic of activated macrophages and that such cells display membrane-associated TNF at least transiently after stimulation.