Analysis of the IFN-γ-Signaling Pathway in Macrophages at Different Stages of Maturation

We previously demonstrated that the macrophage cell lines RAW 264.7 and WEHI-3 exhibit distinct patterns of gene expression in response to IFN-γ. This difference is controlled at the transcriptional level and results from a specific inability of the less mature WEHI-3 cells to utilize either the IFN-stimulated response element or the γ-activated sequence DNA regulatory element in response to stimulation with IFN-γ, while other aspects of IFN-γ gene induction remain intact. In the work described here, we examined the components of the IFN-γ signal transduction pathway in RAW 264.7 and WEHI-3 cells to determine whether differences in pathway components or activity exist in WEHI-3 cells that could give rise to this difference in transcriptional response. Reverse transcriptase-PCR (RT-PCR) and flow cytometric analyses indicated that the levels of IFN-γ receptor mRNA accumulation and protein expression are comparable for RAW 264.7 and WEHI-3 cells. RT-PCR and immunoblot analyses revealed that the principal components of this signaling pathway, including JAK1, JAK2, and STAT1, are present in both RAW 264.7 and WEHI-3 cells. However, analysis of STAT1 DNA-binding activity by electrophoretic mobility shift assay and of STAT1 phosphorylation by immunoblot revealed that this DNA-binding factor is active in RAW 264.7, but not in WEHI-3, cells after IFN-γ stimulation. These results demonstrate that the components of the IFN-γ signal transduction pathway are intact in WEHI-3 cells, but stimulation of these cells by IFN-γ does not result in STAT1 activation.