Our results describe for the first time the presence of the functionally active HIF-1α hydroxylase oxygen-sensing pathway in human peripheral blood neutrophils. The absence of HIF-2α expression demonstrates the differential expression of key members of this pathway between various myeloid lineage cells (35
). Because of the nonredundant function between different HIF α subunits (16
), this expression pattern may have a significant impact on how granulocytes sense and respond to changes in oxygenation.
The ability of hypoxia to regulate apoptotic thresholds is a cell-specific phenomenon. Hence, in neuronal cells, adenocarcinoma HT 29 cells, certain oncogenically transformed cells and cardiac myocytes hypoxia clearly acts to induce rather than inhibit apoptotic cell death (37
). This may reflect the different abilities of cells to adapt to anaerobic metabolism. In neutrophils, we show that the inhibition of apoptosis by hypoxia is accompanied by a time-dependent induction of transcripts for G3PDH and triosephosphate isomerse-1. The induction of these key glycolytic enzymes provides a mechanism for the continued generation of ATP, which is an essential requirement for neutrophil functional responses to inflammatory stimuli (39
). With respect to GAPDH, this also provides indirect evidence for HIF-1α transcriptional activation caused by the presence of functional hypoxia-responsive elements within the GAPDH promoter region (33
Initial work in the myeloid-targeted HIF-1α knockout mice has provided a further link between in vivo inflammatory responses and HIF-1α–dependent maintenance of intracellular energy homeostasis (18
). In addition to the ATP-dependent loss of function and diminished migratory potential, our data suggest that a second mechanism, namely decreased neutrophil survival, may also contribute to the impaired inflammatory response observed in these animals. Hence, we show a marked reduction in cell survival after anoxic challenge in murine bone marrow–derived neutrophils lacking HIF-1α. Because we see no modification of constitutive apoptosis in the HIF−/−
cells, the reduced survival would only be unmasked at sites of low oxygenation. This would be consistent with the normal total neutrophil count, but diminished inflammation seen in the HIF-1α myeloid-targeted knockout mice and the exaggerated inflammatory responses described in the VHL knockout animals (18
). Although murine bone marrow–derived neutrophils display different AV/PI staining properties and lower 20-h levels of constitutive cell death, the above data clearly support a role for HIF-1α the regulation of human neutrophil survival under hypoxic conditions. This conclusion is further supported by preliminary data obtained in patients with von Hippel-Lindau disease where enhanced survival of peripheral blood neutrophils is observed (unpublished data). Contaminating bone marrow monocytes are unlikely to influence these observations because human peripheral blood monocyte depletion does not modify the rate of apoptosis under either normoxia or hypoxia (40
). The involvement of a hydroxylase-dependent pathway is confirmed in human granulocytes by the antiapoptotic properties of the competitive hydroxylase inhibitor DMOG.
The mechanism by which HIF regulates neutrophil apoptosis remains to be fully characterised. However, the inhibition of hypoxic survival by gliotoxin and parthenolide implies that NF-κB is an important downstream effector of the HIF-1α–dependent response. Temporally, this is supported by the early stabilization of HIF-1α that is followed by the increase at 6 h in p65 transcript abundance and subsequent (12–20 h) reexpression of NF-κB and the persistence in NF-κB p65 and p50 DNA binding activity. The regulation of NF-κB by hypoxia is already described for different cell types, including J774.1 murine macrophage and RF/6A retinal lines (41
), yet interactions between HIF and NF-κB are poorly understood. Studies looking at the nonhypoxic induction of HIF have shown a number of indirect links between the HIF and NF-κB transcription pathways. In neuronal cells, a HIF-dependent up-regulation of erythropoietin has been described to prevent excitotoxin-induced apoptosis through Jak2 NF-κB cross-talk (43
), whereas in human embryonic kidney cells, TNFα stimulates the accumulation of ubiquitinated HIF via an NF-κB–dependent pathway (44
). Furthermore, the essential modulator of NF-κB NEMO/IKKγ has recently been shown to be a binding partner for HIF-2α (Peet, D.J., and C. Braken, personal communication). To date, no direct association between HIF and NF-κB has been reported after stimulation of cells with hypoxia. Using a series of real-time PCR reactions in HIF-1α knockout and wild-type neutrophils, we show for the first time the hypoxic induction of NF-κB transcription to be dependent on the presence of HIF-1α. Moreover, we show a HIF-1α–dependent up-regulation of the alternative NF-κB regulator IKKα. IKKα is recognized both to facilitate NF-κB transcriptional activity through the alternative pathway and itself be critical for the histone phosphorylation required for the activation of NF-κB–directed gene expression (45
). Thus HIF-1α not only facilitates an increase in NF-κB message but also facilitates the pathways required for enhanced transcriptional activity.
A key regulator of the NF-κB pathway in neutrophils is IκBα. After neutrophil stimulation with pro-inflammatory molecules, IκBα is degraded resulting in unopposed NF-κB activity (46
). Conversely, the nuclear localization of IκBα results in increased neutrophil apoptosis (48
). Given the persistent expression of IκBα protein with prolonged hypoxic culture and the lack of HIF-1α–dependent changes in murine transcript abundance, it is unlikely that HIF mediates the stimulation of NF-κB through the transcriptional inhibition of IκBα, although an indirect regulation of IκBα nuclear trafficking remains possible.
It is important to note that the inhibition of the PI3-kinase pathway has no effect on the direct inhibition of neutrophil apoptosis by hypoxia. This is in direct contrast to the survival effect of the supernatants derived from hypoxically cultured cells. Using heat inactivation and trypsin digestion, we first identify this factor to be a protein and possible cytokine. Luminex analysis, ELISA, and blocking antibody experiments subsequently support the identity of this cytokine as MIP-1β. Although the identification of MIP-1β as an oxygen-sensitive granulocyte survival factor is novel, MIP-1β is recognized to be up-regulated in Bacillus Calmette-Guerin–stimulated neutrophils along with MCP-1 and MIP-1α (49
). Interestingly, these cells were also noted to have diminished rates of apoptosis. In addition, MIP-1β secretion by murine alveolar macrophages has been shown to be oxygen sensitive (50
). These studies further support the role of MIP-1β as a novel hypoxia-stimulated granulocyte survival factor. In parallel, gene array analysis of human neutrophils identified the hypoxic induction of MIF mRNA. MIF has previously been described both as a neutrophil survival factor through its inhibition of Bax/Bid cleavage and caspase 3 activity (51
) and as a hypoxia-regulated cytokine (52
). However, we saw no induction of MIF protein release into the culture media of hypoxia-treated neutrophils. Although this does not exclude an intracellular up-regulation of MIF protein and the subsequent down-regulation of Bax–Bid pathways, this does exclude MIF as a transferable survival factor in our system.
In summary, we have revealed that the hypoxic inhibition of neutrophil apoptosis is regulated by the HIF-1α hydroxylase oxygen-sensing pathway and NF-κB reexpression, and indirectly regulated by the release of the novel survival factor MIP-1β. Although we have identified the transcriptional and functional activation of NF-κB after hypoxia and the HIF-1α–dependent regulation of NF-κB and Iκκα transcript, the precise mechanism of this interaction remains to be elucidated. HIF-1α would appear to be the critical upstream regulator of this hypoxic survival pathway because deletion of HIF-1α in murine neutrophils results in both a reduction in NF-κB and Iκκα message and anoxia-stimulated cell death.