Our studies show that HIF-1 generally plays an important role in supporting the ability of allergic effector cells to generate inflammatory and immunomodulatory cytokines. In contrast, degranulation of mast cells and basophils does not seem to be affected by HIF-1. However, comparing mast cells to basophils and also THP-1 cells we discovered crucial differences in HIF-1 input. Whereas, in basophils, HIF-1 is strikingly induced by IgE-dependent triggers, in mast cells the protein is clearly expressed constitutively but only weakly induced by stimulation. One might have expected this to be due to the presence of SCF in LAD2 mast cell cultures but, surprisingly, LAD2 cells cultured for 24–72 h in the absence of SCF still expressed substantial levels of HIF-1α. Similarly, IgE sensitization only weakly increased constitutive HIF-1α expressions. This supports the notion that mast cells are highly dependent on cytosolic oxygen metabolism 
, which is controlled by HIF-1 complex on the transcriptional level 
. However, while this is supported in LAD2 mast cell lines, which resemble the general characteristics and functions of primary tissue mast cells more closely than other cell lines, it is important to recognise that primary human mast cells are heterogeneous and may differ in function depending on their tissue location. Importantly, the malignant phenotype of the LAD2 cell line might also influence constitutive HIF-1α, thereby increasing the sensitivity of these cells to its loss. It would therefore be interesting to further investigate the crucial role of HIF-1 in primary mast cells and using in vivo
It has long been appreciated that mast cells and basophils not only express FcεRI but also TLRs2/4 
. These receptors recognise molecular patterns shared by Gram-positive and Gram-negative bacteria, therefore potentially amplifying the pro-allergic (Th2) responses of these allergic effector cells 
. However, the cross-interactions between these two systems remain unclear. In the present study we found that LAD2 human mast cells and primary human basophils express detectable amounts of TLR2 and TLR4, although the levels of these receptors in both cell types are much lower compared to those in THP-1 human myeloid cells (). IgE-mediated histamine, VEGF and TNF-α releases in mast cells were not affected or potentiated by PGN/Pam3Cys or LPS.
As with IgE-dependent triggering, TLR2/4 ligands (PGN/Pam3Cys and LPS respectively) were unable to induce clear increases in HIF-1α accumulation in LAD2 mast cells. Both ligands, however, upregulated TNF-α production, although much less so compared to THP-1 cells. However, TLR ligands failed to induce VEGF production or histamine release in mast cells. IgE-mediated stimulation in mast cells also failed to significantly upregulate HIF-1α accumulation. This was supported by the lack of increases in IgE-induced VEGF mRNA expression, though VEGF protein was released. These results suggest that the level of constitutive HIF-1α expression may be sufficient to meet the stress adaptation requirements given the fact that the levels of the intracellular ATP were not affected.
Employment of silencing RNA to knock-down HIF-1α expression confirmed that, while HIF-1α is only poorly induced by stimulating mast cells with a variety of secretagogues, it still plays a fundamental role in mast cell survival and activity. HIF-1α knock-down significantly reduced the ability of mast cells to express VEGF and to produce TNF-α in response to stimulation with TLR2/4 or FcεRI ligands, although it had no effect on degranulation. The levels of intracellular ATP were also significantly reduced, which correlated with caspase 3 activation in ligand-stimulated LAD2 cells. The observed decrease in viability of HIF-1α knockdown LAD2 mast cells further indicates that their adaptation to any kind of inflammatory stress or their proper functioning in principle becomes problematic in the absence of this protein and further highlights their reliance on glycolysis. In addition, it is also possible that IgE-dependent histamine releases may be more affected by longer treatment periods with HIF-1α siRNA due to decreased cell viability.
In basophils TLR2/4 ligands were unable to induce significant increases in HIF-1α accumulation on their own whereas anti-IgE induced HIF-1α accumulation, which was consistent with our previously published data 
. As with mast cells, PGN or LPS failed to induce histamine release in basophils. LPS was unable to induce IL-4 or VEGF release, while PGN induced IL-4 but not VEGF release in basophils and potentiated IgE-induced IL-4 release. In this case one could suggest that TLR2 in basophils displays higher inflammatory activity compared to LAD2 mast cells where TLR4 seems to be more active (). Basophil TLR4 did not display any signs of pro-inflammatory activity, which is consistent with recently published observations 
. PGN-induced IL-4 production in basophils does not seem to recruit HIF-1. This is most likely associated with the fact that PGN does not induce any histamine release and thus energy consuming major alterations of cytoskeleton associated with degranulation do not take place.
Our results show that pro-allergic IgE-dependent responses are not significantly potentiated by TLR2/4 ligands. This suggests that, in cases of bacterial infection in asthmatic airways, where mast cells and basophils may be stimulated by both TLR2/4 ligands and by IgE-dependent mechanisms, it is unlikely that IgE-dependent responses are potentiated by pathogen-associated TLR2/4 ligands 
In THP-1 human myeloid cells anti-IgE stimulation induced the release of TNF-α and VEGF. Slightly increased release of IL-6 was also detected. IgE-dependent stimulation did not lead to a potentiation of TLR2/4-mediated TNF-α or IL-6 release. LPS or PGN-induced VEGF production in THP-1 cells was, however, potentiated by anti-IgE, in stark contrast to mast cells or basophils.
Taken together our results demonstrate that both LAD2 mast cells and basophils respond differentially to co-stimulation with pro-allergic stimuli and pathogen-derived TLR2/4 ligands. Both cell types do not strongly respond to ligand-induced TLR2/4 activation in comparison to human myeloid innate immune cells. This could be a result of lower levels of expression as well as lower activity of these receptors in mast cells and basophils compared to myeloid cells. The presence of constitutive HIF-1 in LAD2 mast cells was revealed to be essential for them in order to respond to both IgE-dependent and TLR-mediated stimulation. However, in contrast to basophils, increases in HIF-1 expressions to these modes of activation were not pronounced. In basophils the protein also plays an essential role in IgE-dependent activation but less so for TLR2-mediated triggering. The results discussed above are summarised in the scheme presented in the .
Comparative analysis of HIF-1α involvement in inflammatory responses and their cross-interactions in LAD2 human mast cells, primary human basophils and THP-1 human myeloid cells.