Differential utilization of IFN-γ-responsive elements in two maturationally distinct macrophage cell lines

We have characterized the transcriptional response to IFN-γ in two maturationally distinct macrophage populations: the mature RAW 264.7 cell line, phenotypically identical to thioglycollate-elicited peritoneal macrophages, and the less mature WEHI-3 cell line. We first investigated the use of two IFN-γ-responsive regulatory elements, the interferon-stimulated response element (ISRE) and the gamma-activated sequence (GAS), in these cells. Transient transfection assays revealed that synthetic promoter constructs containing either the ISRE or GAS regulatory motif fused to a luciferase reporter gene were transcriptionally inactive in the WEHI-3 cell line. We then analyzed the expression in the two cell lines of a panel of known IFN-γ-responsive genes that are transcriptionally controlled by different regulatory elements. RT-PCR analysis revealed that both cell lines responded to IFN-γ treatment by up-regulating genes that are transcriptionally controlled by κB or W box DNA binding motifs. However, genes regulated by ISRE or GAS elements were induced by IFN-γ only in the RAW 264.7 cell line. Kinetic analysis of the transcriptional activity of synthetic promoter constructs in the RAW 264.7 cell line showed rapid IFN-γ induction through both the ISRE and GAS motifs, indicating that both elements are utilized early after IFN-γ stimulation in mature macrophages. These results suggest that cis-acting DNA response element utilization, and the subsequent profiles of IFN-γ-induced gene expression, differ in macrophages at different stages of maturation.