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Abstract
This work describes the investigation of interferon-gamma (IFN-$\gamma$)-initiated molecular events in macrophages at two different stages of differentiation. To test the hypothesis that the response to IFN-$\gamma$ is developmentally regulated in cells of the macrophage lineage, two murine macrophage cell lines, RAW 264.7 and WEHI-3, were characterized. Initial studies demonstrated that the two cell lines differed with respect to cell tumoricidal activity. Further phenotypic analyses, assessing surface molecule and gene expression, revealed that the two cell lines represent two distinct stages of macrophage development, with the RAW 264.7 cell line representing a mature macrophage, and the WEHI-3 cell line representing an immature macrophage. Subsequent analysis of the transcriptional response demonstrated that the two cell lines differ at multiple levels in response to IFN-$\gamma$ stimulation. both cell lines responded to IFN-$\gamma$ treatment with an increased mRNA expression for several genes that are known to be transcriptionally controlled by this cytokine. However, genes regulated by an Interferon-Stimulated Response Element (ISRE) or a Gamma Activated Sequence (GAS) were introduced by IFN-$\gamma$ only in the Raw 264.7 cell line. Transient transfection assays revealed that promotor constructs containing either the ISRE or GAS regulatory motif were transcriptionally inactive in the WEHI-3 cell line. The expression of two members of the STAT (Signal Transduction and Activators of Transcription) transcription factor family that can mediate ISRE or GAS utilization, STAT1 and STAT5, was assessed. Both factors were detected in both cell lines using Western blot and Quantitative Competitive RT-PCR analysis. However, differences were evident in both the level of STAT1 expression and the STAT5 isoforms expressed. The results of these studies indicate that cis-acting DNA response element utilization, and the subsequent profile of IFN-$\gamma$-induced gene expression, differs in macrophages at different stages of maturation. These data also suggest that differential expression of STAT transcription factor levels and isoforms may play a role in the development control of the IFN-$\gamma$-induced response in macrophages.
