Dm. Lucas et al., ANALYSIS OF THE IFN-GAMMA-SIGNALING PATHWAY IN MACROPHAGES AT DIFFERENT STAGES OF MATURATION, The Journal of immunology, 160(9), 1998, pp. 4337-4342
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.