Binding and internalization of gold-labeled IFN-gamma by human Raji cells

Binding and internalization of gold-labeled IFN-gamma (IFN-gamma/Au) by human Raji cells was examined by scanning and transmission electron microscopy. For SEM, visualization of gold particles was enhanced by the silver enhancement technique and by backscattered electron imaging. Binding studies revealed distinct labeling of microvilli-bearing cells after incubation with at least 10 U/ml IFN-gamma/Au, whereas cells with a smooth surface showed substantially lower labeling. After application of higher IFN-gamma (greater than 200 U/ml) concentrations, labeling intensity remained constant, which is consistent with the concentration of radiolabeled IFN-gamma required for saturating receptors on Raji cells. The specificity of IFN-gamma/Au binding was demonstrated by complete displacement with unlabeled IFN-gamma and by partial inhibition of labeling with a monoclonal anti-IFN-gamma R antibody. Thus, colloidal gold represents a valuable tag for visualizing the interaction of IFN-gamma with its receptor. Internalization of IFN-gamma/Au was initiated by accumulation of gold particles in coated pits which occurred within 10 min after warming of Raji cells. Additional incubation at 37 degrees C (up to 2 h) led to the appearance of gold particles in endocytic vesicles and lysosomes. Thus, our studies indicate that IFN-gamma/Au enters the Raji cells via the typical endocytotic pathway.