Endotoxin and interferon-γ differentially regulate the transcriptional levels of proto-oncogenes and cytokine genes during the differentiation of colony-stimulating factor type-1-derived macrophages

The expression of cytokine genes for interleukin-1 (IL-1) (α and β) and tumour necrosis factor-α (TNF-α), along with the proto-oncogenes c-fos, c-fms and c-myc, was examined by nuclear run-off and Northern blot analysis during in vitro differentiation of colony-stimulating factor type-1 (CSF-1)-derived bone marrow macrophages (BMDM). Constitutive transcription of c-myc was maximal on day 3 and decreased with differentiation. Constitutive transcription of c-fms and c-fos was similar at all times examined. In contrast, the steady-state mRNA levels were maximal on day 5 for c-myc and day 7 for c-fms and c-fos. Thirty minutes after endotoxin (lipopolysaccharide; LPS) stimulation, there was a rapid increase in run-off transcription rates for c-myc in day 3-day 9 BMDM, with maximal levels observed in day 7 BMDM. c-fms transcription was maximally induced within 1 hr by LPS in day 3 and day 5 BMDM. LPS induced transcription of c-fos to equivalent levels in day 3-day 9 BMDM. LPS stimulation augmented steady-state mRNA levels for c-myc, c-fms and c-fos. Maximal induction of c-myc was observed in day 3 BMDM. c-fos and c-fms were both maximally induced in day 5 and day 7 BMDM. Interferon-γ (IFN-γ) had no effect on transcription of the proto-oncogenes examined. In contrast to the proto-oncogenes, peak levels of run-off transcription for IL-1α and IL-1β genes were observed 1-2 hr after LPS stimulation for day 3, day 5 and day 7 BMDM. The kinetics of LPS-induced steady-state mRNA accumulation of IL-1α and IL-1β were similar to the kinetics of run-off transcription. Constitutive transcription of TNF-α was observed on all days of differentiation. LPS and IFN-γ both enhanced run-off transcription of the TNF-α gene; however, LPS had a more pronounced effect. The kinetics of induction of TNF-α transcription paralleled the kinetics of steady-state TNF-α mRNA accumulation. IFN-γ resulted in secretion of TNF-α in day 5, day 7, and day 9 BMDM after 4-8 hr of stimulation. Day 3 BMDM had little, if any, secreted TNF-α activity. These findings suggest that during macrophage development there is a regulated expression of IL-1α, IL-1β and TNF-α genes, which is intrinsically determined and can be altered by stimulation with either LPS or IFN-γ. Furthermore, the patterns for both transcription and steady-state levels for c-myc and c-fms change as the BMDM undergo differentiation.