ENDOTOXIN AND INTERFERON-GAMMA DIFFERENTIALLY REGULATE THE TRANSCRIPTIONAL LEVELS OF PROTOONCOGENES AND CYTOKINE GENES DURING THE DIFFERENTIATION OF COLONY-STIMULATING FACTOR TYPE-1-DERIVED MACROPHAGES

The expression of cytokine genes for interleukin-1 (IL-1) (alpha and b eta) and tumour necrosis factor-alpha (TNF-alpha), along with the prot o-oncogenes c-fos, c-fms and c-myc, was examined by nuclear run-off an d Northern blot analysis during in vitro differentiation of colony-sti mulating factor type-1 (CSF-1)-derived bone marrow macrophages (BMDM). Constitutive transcription of c-myc was maximal on day 3 and decrease d 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 (lipopolysassharide; 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 t ranscription 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 da y 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 da y 3 BMDM. c-fos and c-fms were both maximally induced in day 5 and day 7 BMDM. Interferon-gamma (IFN-gamma) had no effect on transcription o f the proto-oncogenes examined. In contrast to the proto-oncogenes, pe ak levels of run-off transcription for IL-1 alpha and IL-1 beta 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 -1 alpha and IL-1 beta were similar to the kinetics of run-off transcr iption. Constitutive transcription of TNF-alpha was observed on all da ys of differentiation, LPS and IFN-gamma both enhanced run-off transcr iption of the TNF-alpha gene; however, LPS had a more pronounced effec t. The kinetics of induction of TNF-alpha transcription paralleled the kinetics of steady-state TNF-alpha mRNA accumulation. IFN-gamma resul ted in secretion of TNF-alpha in day 5, day 7, and day 9 BMDM after 4- 8 hr of stimulation. Day 3 BADM had little, if any. secreted TNF-alpha activity. These findings suggest that during macrophage development t here is a regulated expression of IL-1 alpha, IL-1 beta and TNF-alpha genes, which is intrinsically determined and can be altered by stimula tion with either LPS or IFN-. Furthermore, the patterns for both trans cription and steady-state levels for c-myc and c-fms change as the BMD M undergo differentiation.