IFN-gamma and IL-2 cooperatively activate NF kappa B in murine peritoneal macrophages.

Abstract

The combination of IFN-gamma and IL-2 has been demonstrated to induce or promote transcription from selected inflammatory cytokine genes in both monocytes and macrophages. In the present report, we have evaluated the ability of these agents to activate nuclear proteins, in murine peritoneal macrophages, that are capable of specific binding to the kappa B nucleotide consensus sequence (e.g., nuclear factor binding the kappa B consensus sequence (NF kappa B)), which is present in the region 5' of the transcription start site of many cytokine genes. Both IFN-gamma and IL-2 treatments alone caused some activation of NF kappa B, as evidenced by the appearance of a characteristic DNA-protein complex in electrophoretic mobility shift assays; in combination, the two agents cooperated to increase binding activity markedly, to a level equivalent to that seen in macrophages stimulated with LPS, another potent stimulus of macrophage cytokine gene expression. The binding activity was directed toward the kappa B sequence, based upon competition studies with several different oligonucleotide probes containing the kappa B motif in different contexts. The complex formed in cells stimulated with either IFN-gamma/IL-2 or LPS contained two proteins, of approximately 50 kDa and 65 kDa, based upon UV cross-linking experiments with a bromodeoxyuridine-substituted oligonucleotide probe. Although the effects of LPS and IFN-gamma/IL-2 on NF kappa B activity were both transient, response to LPS was detected earlier and declined more rapidly than that seen with IFN-gamma/IL-2 treatment. The cooperative activation of NF kappa B was dose dependent for both IFN-gamma and IL-2. The activation of NF kappa B by IFN-gamma/IL-2 did not depend upon protein synthesis. These results suggest that the induction of cytokine gene expression in macrophages by the combination of IFN-gamma and IL-2 may involve the activation of NF kappa B.