Involvement of Hypoxia-Inducible Factor-1 in the Inflammatory Responses of Human LAD2 Mast Cells and Basophils

We recently showed that hypoxia-inducible factor 1 (HIF-1) plays a crucial role in the pro-allergic functions of human basophils by transcriptional control of energy metabolism via glycolysis as well as directly triggering expression of the angiogenic cytokine vascular endothelium growth factor (VEGF). Here, we investigated HIF-1 involvement in controlling the synthesis of angiogenic and inflammatory cytokines from various human effector cells stimulated by IgE-dependent or innate immune triggers. Purified primary human basophils, LAD2 human mast cells and THP-1 human myeloid cells were used for investigations of FcεRI and Toll-like receptor (TLR) ligand-induced responses. In contrast to basophils, LAD2 mast cells expressed background levels of HIF-1α, which was largely independent of the effects of stem cell factor (SCF). Both mast cells and basophils expressed TLR2 and 4, albeit weakly compared to THP-1 cells. Cytokine production in mast cells following TLR ligand stimulation was markedly reduced by HIF-1α knockdown in LAD2 mast cells. In contrast, although HIF-1 is involved in IgE-mediated IL-4 secretion from basophils, it is not clearly induced by peptidoglycan (PGN). HIF-1α accumulation is critical for sustaining human allergic effector cell survival and function. This transcription complex facilitates generation of both pro-angiogenic and inflammatory cytokines in mast cells but has a differential role in basophil stimulation comparing IgE-dependent triggering with innate immune stimuli

Effects of Stem Cell Factor on Hypoxia-Inducible Factor 1 Alpha Accumulation in Human Acute Myeloid Leukaemia and LAD2 Mast Cells

Stem cell factor (SCF) is a hematopoietic growth factor that exerts its activity by signalling through the tyrosine kinase receptor known as Kit or CD117. SCF-Kit signalling is crucial for the survival, proliferation and differentiation of hematopoietic cells of myeloid lineage. Furthermore, since myeloid leukaemia cells express the Kit receptor, SCF may play an important role in myeloid leukaemia progression too. However, the mechanisms of this pathophysiological effect remain unclear. Recent evidence shows that SCF triggers accumulation of the inducible alpha subunit of hypoxia-inducible factor 1 (HIF-1) in hematopoietic cells—a transcription complex that plays a pivotal role in cellular adaptation to low oxygen availability. However, it is unknown how SCF impacts on HIF-1α accumulation in human myeloid leukaemia and mast cells. Here we show that SCF induces HIF-1α accumulation in THP-1 human myeloid leukaemia cells but not in LAD2 mast cells. We demonstrated that LAD2 cells have a more robust glutathione (GSH)-dependent antioxidative system compared to THP-1 cells and are therefore protected against the actions of ROS generated in an SCF-dependent manner. BSO-induced GSH depletion led to a significant decrease in HIF-1α prolyl hydroxylase (PHD) activity in THP-1 cells and to near attenuation of it in LAD2 cells. In THP-1 cells, SCF-induced HIF-1α accumulation is controlled via ERK, PI3 kinase/PKC-δ/mTOR-dependent and to a certain extent by redox-dependent mechanisms. These results demonstrate for the first time an important cross-talk of signalling pathways associated with HIF-1 activation—an important stage of the myeloid leukaemia cell life cycle

Myeloid cell death associated with Toll-like receptor 7/8-mediated inflammatory response. Implication of ASK1, HIF-1alpha, IL-1beta and TNF-alpha

Programmed cell death or apoptosis is an important part of the host innate immune defence, especially against ssRNA viruses (influenza virus, HIV-1, ebola virus, hepatitis C virus and many others). Viral ssRNA is recognised by endosomal Toll-like receptors 7 and 8 (TLR7/8) which induce further stages of immune defence against these pathogens. Some of the immune cells die because of inflammatory stress allowing for the selection of those cells which are resistant to stress-induced apoptosis and which are used in further stages of the host immune response. On the other hand, apoptosis could be used as an instrument to suppress the function of activated inflammatory cells. However, the mechanisms underlying death of the inflammatory cells associated with stress induced by ligands of TLR7/8 remain unclear. In this study we have found that programmed death of human myeloid cells from different cell lines associated with ligand-induced TLR7/8-mediated inflammatory stress depends on activation of apoptosis signal-regulating kinase 1 (ASK1). This enzyme is, however, not required for the production of pro-inflammatory cytokines – TNF-? and IL-1?. We have found that released IL-1? and TNF-? are involved in apoptosis of myeloid cells associated with TLR7/8-mediated inflammatory stress. The pro-apoptotic effect of released TNF-? in this case is much lower compared to that of IL-1?

The impact of SCF on HIF-1α accumulation and the GSH-dependent antioxidative system in human myeloid leukaemia cells.

<p>(<b>A</b>) THP-1 human myeloid leukaemia cells were stimulated for 24 h with 100 ng/ml SCF. HIF-1α accumulation, GSH and GSSG levels, GPx/GR activities and the quantity of the TBRS were then measured as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022502#s4" target="_blank">Materials and methods</a>. (<b>B</b>) THP-1 human myeloid leukaemia cells were cultured in the presence or absence of 100 ng/ml SCF for 24 h. HIF-1 DNA-binding activity was analysed using 10 mg/ml BSA as a negative control. (<b>C</b>) THP-1 cells were exposed for 4 h to 25, 50 and 100 ng/ml SCF. HIF-1α accumulation was then measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022502#s4" target="_blank">Materials and methods</a>. Quantitative data are mean values ± S.D. of at least three individual experiments. *P<0.01 vs control. Western blot data are shown from one representative experiment out of three that gave similar results.</p

Signalling pathways involved in SCF-induced HIF-1α accumulation in THP-1 human myeloid leukaemia cells.

<p>THP-1 cells were pre-treated for one hour with the indicated concentrations of the outlined inhibitors. In one case cells were transfected with dominant-negative form of ASK1 (ASK1-KM) as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022502#s4" target="_blank">Materials and methods</a>. After that THP-1 cells were exposed for 4 h to 100 ng/ml SCF followed by Western blot analysis of HIF-1α accumulation and ELISA assay of the VEGF release. Quantitative data are mean values ± S.D. of at least three individual experiments. *P<0.01 vs control. <b>^a</b> – differences are significant when comparing two indicated values (P<0.01). All Western blot data are from one experiment representative of three that gave similar results.</p

GSH depletion leads to a reduction of SCF-induced HIF-1α PHD activity in THP-1 cells.

<p>THP-1 human myeloid leukaemia cells were cultured (in fresh medium) for 24 h in the absence or presence of 100 ng/ml SCF ± 500 µM BSO. (<b>A</b>) HIF-1α accumulation/PHD activity and (<b>B</b>) annexin V/DAPI staining were performed as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022502#s4" target="_blank">Materials and methods</a>. Quantitative data are mean values ± S.D. of at least three individual experiments. *P<0.01 vs control. All Western blot and imaging data are from one representative experiment out of three that gave similar results.</p

Several pathways impact SCF-dependent HIF-1α accumulation <i>via</i> mTOR in THP-1 human myeloid cells.

<p>THP-1 cells were pre-treated for one hour with the indicated concentrations of the outlined inhibitors. In one case cells were transfected with dominant-negative form of ASK1 (ASK1-KM) as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022502#s4" target="_blank">Materials and methods</a>. THP-1 cells were then exposed for 4 h to 100 ng/ml SCF followed by Western blot analysis of HIF-1α and mTOR accumulation as well as S2448 phosphorylation. LAD2 cells were cultured for 24 h in the absence or presence of 100 ng/ml SCF. All Western blot data are from one experiment representative of three that gave similar results. Quantitative data are mean values ± S.D. of at least three individual experiments. *P<0.01 vs control. <b>^a</b> – differences are significant when comparing two indicated values (P<0.01).</p

GSH depletion leads to a strong decrease in SCF-dependent HIF-1α PHD activity in LAD2 cells.

<p>LAD2 cells were incubated for 24 h in fresh media ±100 ng/ml SCF and ±500 µM BSO. (<b>A</b>) HIF-1α accumulation and PHD activity were assessed as well as (<b>B</b>) Annexin V and DAPI staining as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022502#s4" target="_blank">Materials and methods</a>. Western blot and imaging data are from one representative experiment out of three. Quantitative data are mean values ± S.D. of at least three individual experiments. *P<0.01 vs control.</p

The impact of SCF withdrawal on HIF-1α accumulation and the GSH-dependent antioxidative system in LAD2 human mast cells.

<p>LAD2 cells (which were continuously cultured in the presence of 100 ng/ml SCF) were incubated in fresh media containing 100 ng/ml SCF for 24 h, while another fraction of these cells was subjected to SCF-free media for 24 h. (<b>A</b>) HIF-1α accumulation, GSH and GSSG levels, GPx/GR activities and the quantity of the TBRS were then measured as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022502#s4" target="_blank">Materials and methods</a>. HIF-1 DNA-binding activity was also analysed. (<b>B</b>) Quantitative data are mean values ± S.D. of at least three individual experiments. *P<0.01 vs control. Western blot data are shown from one representative experiment out of three that gave similar results.</p