ANALYSIS OF THE IFN-GAMMA-SIGNALING PATHWAY IN MACROPHAGES AT DIFFERENT STAGES OF MATURATION

We previously demonstrated that the macrophage cell lines RAW 264.7 an d WEHI-3 exhibit distinct patterns of gene expression in response to I FN-gamma. This difference is controlled at the transcriptional level a nd results from a specific inability of the less mature WEHI-3 cells t o utilize either the IFN-stimulated response element or the gamma-acti vated sequence DNA regulatory element in response to stimulation with IFN-gamma, while other aspects of IFN-gamma gene induction remain inta ct. In the work described here, we examined the components of the IFN- gamma signal transduction pathway in RAW 264.7 and WEHI-3 cells to det ermine whether differences in pathway components or activity exist in WEHI-3 cells that could give rise to this difference in transcriptiona l response. Reverse transcriptase-PCR (RT-PCR) and how cytometric anal yses indicated that the levels of IFN-gamma 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 prese nt 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 IWW 264.7, but not in WEHI-3, cells after IFN-gamma stimula tion. These results demonstrate that the components of the IFN-gamma s ignal transduction pathway are intact in WEHI-3 cells, put stimulation of these cells by IFN-gamma does not result in STAT1 activation.