Tumor necrosis factor-α analyzed within individual macrophages by combined immunocytochemistry and computer-aided image analysis

Abstract

Immunocytochemical staining procedures were combined with computer-aided image analysis to quantitate the relative intracellular production of tumor necrosis factor-α (TNF) within individual macrophages. Optimal conditions for time and methods for the activation of TNF production, fixation of cells for optimal immunocytochemical staining, and image analysis methods were determined. Thioglycolate elicited peritoneal macrophages were readily activated to significantly increased levels of intracellular TNF, as early as 1 hr after activation with lypopolysaccharide (LPS) + interferon-γ; maximum intracellular TNF was evident after 2–3 hr. Both LPS and interferon-γ was necessary to increase intracellular TNF. Normal alveolar macrophages also readily produced increased intracellular TNF, but normal peritoneal and splenic macrophages were poorly activated to TNF production. Acid stripping of receptor bound TNF allowed discrimination between intracellular TNF/integral membrane TNF, and TNF-receptor-bound TNF. Results stress the importance of studying these TNF forms early after activation. Applications for TNF quantitation by these means are discussed.