Astrocytes can function as antigen-presenting cells (APC) upon expression of class II antigens, which are induced by interferon-gamma (IFN-gamma). Tumor necrosis factor-alpha (TNF-alpha) can act synergistically with IFN-gamma with respect to class II expression on a variety of cells. As brain cells themselves can secrete TNF-like factors upon stimulation, we examined the effect of TNF-alpha on IFN-gamma-mediated class II induction on astrocytes. TNF-alpha alone had no effect on class II expression, but did synergize with IFN-gamma for enhanced expression of class II antigens. The specificity of TNF-alpha activity was demonstrated by blocking the amplifying effect of TNF-alpha with a polyclonal anti-TNF-alpha antibody. Kinetic analysis of the synergistic effect indicated that optimal TNF-alpha enhancement of class II expression was observed when astrocytes were pretreated with IFN-gamma 12-24 h prior to TNF-alpha addition. A possible mechanism for the synergistic action between IFN-gamma and TNF-alpha may be increased TNF-alpha receptor expression by IFN-gamma. Astrocytes treated with IFN-gamma for 24 h express more TNF-alpha receptors (3900/cell) than do untreated astrocytes (2483/cell), with no significant change in the binding affinity (Kd). These results suggest that the synergistic activity of TNF-alpha requires an inductive signal from IFN-gamma, which in part may be increased TNF-alpha receptor expression. Altogether, our observations indicate that TNF-alpha enhances ongoing class II major histocompatibility complex gene expression in rat astrocytes, which in this system is initially induced by IFN-gamma. TNF-alpha exerts its effect by binding to high affinity TNF-alpha receptors on astrocytes, whose expression is also enhanced by IFN-gamma. These two cytokines work in concert to elevate class II expression on astrocytes, an event which can contribute to initiation and/or perpetuation of intracerebral immune responses.