Granulocyte-macrophage colony-stimulating factor increases 5-lipoxygenase gene transcription and protein expression in human neutrophils

Abstract

The complex process of neutrophil activation and accumulation is orchestrated by a cascade of cytokines and bioactive lipids produced at the site of inflammation. Neutrophils are a rich source of the potent inflammatory lipid leukotriene B4(LTB4). Granulocyte-macrophage colony- stimulating factor (GM-CSF) can activate neutrophils for an exponential increase in LTB4 production in response to a number of subsequent stimuli. In this report, we examined the temporal regulation, by GM-CSF, of the gene and protein expression of 5-lipoxygenase (5-LOX), a key enzyme of the LTB4 production pathway. Human neutrophils were exposed to 10 ng/mL of GM-CSF for various periods of time and 5-LOX mRNA was measured by Northern blot analysis. We observed no change in 5-LOX mRNA at early time points (0.25 to 3 hours); however, by 18 hours we observed a significant augmentation of 5- LOX-specific message (4.3- ± 1.7-fold increase; n = 6). Nuclear transcription assays indicated that the rate of 5-LOX gene transcription was augmented threefold in neutrophils incubated with GM-CSF, whereas the half- life of the message was not markedly changed. Parallel experiments indicated that the levels of 5-LOX protein were also increased by GM-CSF. The augmentation was observed within 30 minutes after stimulation and was maximal (5.23 ± 2.6; n = 4) at 18 hours. Incubation of GR4-CSF-stimulated neutrophils with protein synthesis inhibitors resulted in a time-dependent impairment of their ability to produce LTB4, with no inhibition seen during the first hours, a 75% decrease seen by 12 hours, and greater than 95% inhibition seen at 18 hours. Collectively, our data imply that GM-CSF can regulate LTB4 production by two distinct mechanisms: a short-term increase that is not related to increased 5-LOX mRNA expression and is independent of protein synthesis, and a sustained increase in LTB4 production that is associated with the transcriptional activation of the 5-LOX gene, increase in 5-LOX mRNA levels, and dependence on protein synthesis. Such transcriptional modulation of 5-LOX enzyme expression may provide new approaches for therapeutic intervention in protracted inflammatory conditions.