Andrew S. Cowburn

Overexpression of leukotriene C4 synthase in bronchial biopsies from patients with aspirin-intolerant asthma.

Aspirin causes bronchoconstriction in aspirin-intolerant asthma (AIA) patients by triggering cysteinyl-leukotriene (cys-LT) production, probably by removing PGE2-dependent inhibition. To investigate why aspirin does not cause bronchoconstriction in all individuals, we immunostained enzymes of the leukotriene and prostanoid pathways in bronchial biopsies from AIA patients, aspirin-tolerant asthma (ATA) patients, and normal (N) subjects. Counts of cells expressing the terminal enzyme for cys-LT synthesis, LTC4 synthase, were fivefold higher in AIA biopsies (11.5+/-2.2 cells/mm2, n = 10) than in ATA biopsies (2.2+/-0.7, n = 10; P = 0. 0006) and 18-fold higher than in N biopsies (0.6+/-0.4, n = 9; P = 0. 0002). Immunostaining for 5-lipoxygenase, its activating protein (FLAP), LTA4 hydrolase, cyclooxygenase (COX)-1, and COX-2 did not differ. Enhanced baseline cys-LT levels in bronchoalveolar lavage (BAL) fluid of AIA patients correlated uniquely with bronchial counts of LTC4 synthase+ cells (rho = 0.83, P = 0.01). Lysine-aspirin challenge released additional cys-LTs into BAL fluid in AIA patients (200+/-120 pg/ml, n = 8) but not in ATA patients (0. 7+/-5.1, n = 5; P = 0.007). Bronchial responsiveness to lysine-aspirin correlated exclusively with LTC4 synthase+ cell counts (rho = -0.63, P = 0.049, n = 10). Aspirin may remove PGE2-dependent suppression in all subjects, but only in AIA patients does increased bronchial expression of LTC4 synthase allow marked overproduction of cys-LTs leading to bronchoconstriction.

Hypoxia-induced neutrophil survival is mediated by HIF-1α-dependent NF-κB activity

Neutrophils are key effector cells of the innate immune response and are required to migrate and function within adverse microenvironmental conditions. These inflammatory sites are characterized by low levels of oxygen and glucose and high levels of reductive metabolites. A major regulator of neutrophil functional longevity is the ability of these cells to undergo apoptosis. We examined the mechanism by which hypoxia causes an inhibition of neutrophil apoptosis in human and murine neutrophils. We show that neutrophils possess the hypoxia-inducible factor (HIF)-1α and factor inhibiting HIF (FIH) hydroxylase oxygen-sensing pathway and using HIF-1α–deficient myeloid cells demonstrate that HIF-1α is directly involved in regulating neutrophil survival in hypoxia. Gene array, TaqMan PCR, Western blotting, and oligonucleotide binding assays identify NF-κB as a novel hypoxia-regulated and HIF-dependent target, with inhibition of NF-κB by gliotoxin or parthenolide resulting in the abrogation of hypoxic survival. In addition, we identify macrophage inflammatory protein-1β as a novel hypoxia-induced neutrophil survival factor.

Advances in Neutrophil Biology: Clinical Implications

Many lung diseases are characterized by neutrophil-dominated inflammation; therefore, an understanding of neutrophil function is of considerable importance to respiratory physicians. This review will focus on recent advances in our understanding of how neutrophils are produced, how these cells leave the circulation, the molecular events regulating neutrophil activation and, ultimately, how these cells die and are removed. The neutrophil is now recognized as a highly versatile and sophisticated cell with significant synthetic capacity and an important role in linking the innate and adaptive arms of the immune response. One of the key challenges in conditions such as COPD, bronchiectasis, cystic fibrosis, and certain forms of asthma is how to manipulate neutrophil function in a way that does not compromise antibacterial and antifungal capacity. The possession by neutrophils of a unique repertoire of surface receptors and signaling proteins may make such targeted therapy possible.

Hypoxia Selectively Inhibits Respiratory Burst Activity and Killing of Staphylococcus aureus in Human Neutrophils

Neutrophils play a central role in the innate immune response and a critical role in bacterial killing. Most studies of neutrophil function have been conducted under conditions of ambient oxygen, but inflamed sites where neutrophils operate may be extremely hypoxic. Previous studies indicate that neutrophils sense and respond to hypoxia via the ubiquitous prolyl hydroxylase/hypoxia-inducible factor pathway and that this can signal for enhanced survival. In the current study, human neutrophils were shown to upregulate hypoxia-inducible factor (HIF)-1α–dependent gene expression under hypoxic incubation conditions (3 kPa), with a consequent substantial delay in the onset of apoptosis. Despite this, polarization and chemotactic responsiveness to IL-8 and fMLP were entirely unaffected by hypoxia. Similarly, hypoxia did not diminish the ability of neutrophils to phagocytose serum-opsonized heat-killed streptococci. Of the secretory functions examined, IL-8 generation was preserved and elastase release was enhanced by hypoxia. Hypoxia did, however, cause a major reduction in respiratory burst activity induced both by the soluble agonist fMLP and by ingestion of opsonized zymosan, without affecting expression of the NADPH oxidase subunits. Critically, this reduction in respiratory burst activity under hypoxia was associated with a significant defect in the killing of Staphylococcus aureus. In contrast, killing of Escherichia coli, which is predominantly oxidase independent, was fully preserved under hypoxia. In conclusion, these studies suggest that although the NADPH oxidase-dependent bacterial killing mechanism may be compromised by hypoxia, neutrophils overall appear extremely well adapted to operate successfully under severely hypoxic conditions.

The survival effect of TNF‐α in human neutrophils is mediated via NF‐κB‐dependent IL‐8 release

The capacity of cytokines to modulate neutrophil apoptosis is thought to be a major factor influencing the resolution of granulocytic inflammation. We have previously shown that the late survival effect of TNF‐α  in human neutrophils involves activation of both NF‐κB and phosphoinositide 3‐kinase (PI3‐kinase) pathways. In this study, we address how these pathways integrate to prevent cell death. In human neutrophils, TNF‐α  (200 U/ml) induced rapid IκB‐α  degradation, NF‐κB activation and IL‐8 release (31.8±5.4 pg/105 cells/2 h), whereas GM‐CSF (10 ng/ml) stimulated an equivalent IL‐8 release (26.5±4.5 pg/105 cells/2 h) without enhanced IκB‐α  degradation or NF‐κB activation compared to control. Importantly, inhibition of PI3‐kinase did not modify TNF‐α ‐induced IκB‐α  degradation, yet fully inhibited the survival effect of both cytokines. Inhibition of IκB‐α  phosphorylation, PI3‐kinase or ERK1/2 activation blocked IL‐8 release by both cytokines. Blocking IL‐8 activity by inhibiting its synthesis or by using a neutralizing antibody enhanced the early pro‐apoptotic effectof TNF‐α  and inhibited its late survival effect without affecting GM‐CSF‐induced survival. These data suggest that cross‐talk between NF‐κB and PI3‐kinase pathways in TNF‐α ‐stimulated neutrophils results from NF‐κB/ERK1/2‐dependent IL‐8 production which acts in an autocrine manner to drive PI3‐kinase‐dependent survival. In contrast, GM‐CSF‐mediated survival does not involve NF‐κB activation or IL‐8 release.

Prolonged survival of neutrophils from patients with ΔF508 CFTR mutations

Cystic fibrosis (CF) is an autosomal recessive “channelopathy” characterised by aberrant CFTR and ENaC function resulting in widespread epithelial cell dysfunction and persistent airway infection. Studies indicating that airway inflammation precedes infection1 and that patients with CF display exaggerated neutrophilic responses to pathogens have suggested a primary defect in innate immune responses in CF. Given the importance of apoptosis to the resolution of neutrophilic inflammation,2 we sought to determine whether circulating CF neutrophils display normal apoptotic capacity. Peripheral blood neutrophils were isolated from 12 clinically stable Pseudomonas colonised ΔF508 homozygote adult patients with CF and 12 age and sex matched healthy controls using discontinuous plasma/Percoll gradients.3 Blood neutrophil counts were in the normal range …

HIF-1α-PDK1 axis-induced active glycolysis plays an essential role in macrophage migratory capacity

In severely hypoxic condition, HIF-1α-mediated induction of Pdk1 was found to regulate glucose oxidation by preventing the entry of pyruvate into the tricarboxylic cycle. Monocyte-derived macrophages, however, encounter a gradual decrease in oxygen availability during its migration process in inflammatory areas. Here we show that HIF-1α-PDK1-mediated metabolic changes occur in mild hypoxia, where mitochondrial cytochrome c oxidase activity is unimpaired, suggesting a mode of glycolytic reprogramming. In primary macrophages, PKM2, a glycolytic enzyme responsible for glycolytic ATP synthesis localizes in filopodia and lammelipodia, where ATP is rapidly consumed during actin remodelling processes. Remarkably, inhibition of glycolytic reprogramming with dichloroacetate significantly impairs macrophage migration in vitro and in vivo. Furthermore, inhibition of the macrophage HIF-1α-PDK1 axis suppresses systemic inflammation, suggesting a potential therapeutic approach for regulating inflammatory processes. Our findings thus demonstrate that adaptive responses in glucose metabolism contribute to macrophage migratory activity.

HIF isoforms in the skin differentially regulate systemic arterial pressure

Significance The differential expression of the hypoxia-inducible factor-alpha (HIF-α) isoforms in the skin of mice influences vascular resistance and is correlated with homeostatic regulation of nitric oxide synthesis. A correlation between HIF isoform expression and hypertension was found in skin biopsies from human subjects, and may indicate a mechanism in the etiology of idiopathic hypertension. Vascular flow through tissues is regulated via a number of homeostatic mechanisms. Localized control of tissue blood flow, or autoregulation, is a key factor in regulating tissue perfusion and oxygenation. We show here that the net balance between two hypoxia-inducible factor (HIF) transcription factor isoforms, HIF-1α and HIF-2α, is an essential mechanism regulating both local and systemic blood flow in the skin of mice. We also show that balance of HIF isoforms in keratinocyte-specific mutant mice affects thermal adaptation, exercise capacity, and systemic arterial pressure. The two primary HIF isoforms achieve these effects in opposing ways that are associated with HIF isoform regulation of nitric oxide production. We also show that a correlation exists between altered levels of HIF isoforms in the skin and the degree of idiopathic hypertension in human subjects. Thus, the balance between HIF-1α and HIF-2α expression in keratinocytes is a control element of both tissue perfusion and systemic arterial pressure, with potential implications in human hypertension.

Cystic fibrosis neutrophils have normal intrinsic reactive oxygen species generation

Previous studies have identified abnormalities in the oxidative responses of the neutrophil in cystic fibrosis (CF), but it is unclear whether such changes relate to loss of membrane cystic fibrosis transmembrane conductance regulator (CFTR) or to the inflammatory environment present in this disease. The aim of the present study was to determine whether neutrophils from CF patients demonstrate an intrinsic abnormality of the respiratory burst. The respiratory burst activity of neutrophils isolated from stable ΔF508 homozygote CF patients and matched healthy controls was quantified by both chemiluminscence and cytochrome C reduction. Expression of NADPH oxidase components and CFTR was determined by Western blotting and RT-PCR. The oxidative output from neutrophils from CF in response to receptor-linked and particulate stimuli did not differ from that of controls. Expression of NADPH oxidase components was identical in CF and non-CF neutrophils. While low levels of CFTR mRNA could be identified in the normal human neutrophil, we were unable to detect CFTR protein in human neutrophil lysates or immunoprecipitates. CFTR has no role in controlling neutrophil oxidative activity; previously reported differences in neutrophil function between CF and non-CF subjects most likely relate to the inflammatory milieu from which the cells were isolated.

Aminopeptidase N (CD13) Regulates Tumor Necrosis Factor-α-induced Apoptosis in Human Neutrophils

Neutrophil apoptosis plays a central role in the resolution of granulocytic inflammation. We have shown previously that tumor necrosis factor-α (TNFα) enhances the rate of neutrophil apoptosis at early time points via a mechanism involving both TNF receptor (TNFR) I and TNFRII. Here we reveal a marked but consistent variation in the magnitude of the pro-apoptotic effect of TNFα in neutrophils isolated from healthy donors, and we show that inhibition of cell surface aminopeptidase N (APN) using actinonin, bestatin, or inhibitory peptides significantly enhanced the efficacy of TNFα-induced killing. Notably, an inverse correlation is shown to exist between neutrophil APN activity and the sensitivity of donor cells to TNFα-induced apoptosis. Inhibition of cell surface APN appears to interfere with the shedding of TNFRI, and as a consequence results in augmented TNFα-induced apoptosis, cell polarization, and TNFα-primed, formyl-methionyl-leucyl-phenylalanine-stimulated respiratory burst. Of note, actinonin and bestatin had no effect on TNFRII expression under resting or TNFα-stimulated conditions and did not alter CXCRI or CXCRII expression. These data suggest significant variation in the activity of APN/CD13 on the cell surface of neutrophils in normal individuals and reveal a novel mechanism whereby APN/CD13 regulates TNFα-induced apoptosis via inhibition of TNFRI shedding. This has therapeutic relevance for driving neutrophil apoptosis in vivo.