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The inflamed bronchial mucosal surface is a profoundly hypoxic environment. Neutrophilic airway inflammation and neutrophil-derived proteases have been linked to disease progression in conditions such as COPD and cystic fibrosis, but the effects of hypoxia on potentially harmful neutrophil functional responses such as degranulation are unknown.Following exposure to hypoxia (0.8% oxygen, 3 kPa for 4 h), neutrophils stimulated with inflammatory agonists (granulocyte-macrophage colony stimulating factor or platelet-activating factor and formylated peptide) displayed a markedly augmented (twofold to sixfold) release of azurophilic (neutrophil elastase, myeloperoxidase), specific (lactoferrin) and gelatinase (matrix metalloproteinase-9) granule contents. Neutrophil supernatants derived under hypoxic but not normoxic conditions induced extensive airway epithelial cell detachment and death, which was prevented by coincubation with the antiprotease α-1 antitrypsin; both normoxic and hypoxic supernatants impaired ciliary function. Surprisingly, the hypoxic upregulation of neutrophil degranulation was not dependent on hypoxia-inducible factor (HIF), nor was it fully reversed by inhibition of phospholipase C signalling. Hypoxia augmented the resting and cytokine-stimulated phosphorylation of AKT, and inhibition of phosphoinositide 3-kinase (PI3K)γ (but not other PI3K isoforms) prevented the hypoxic upregulation of neutrophil elastase release.Hypoxia augments neutrophil degranulation and confers enhanced potential for damage to respiratory airway epithelial cells in a HIF-independent but PI3Kγ-dependent fashion.

Introduction and objectives Neutrophils (PMNs) are a key component of the innate immune response to invading pathogens. They accumulate at sites of inflammation and infection, which are typically characterised by low oxygen tensions (e.g. in the acute respiratory distress syndrome (ARDS)). Human PMNs undergo constitutive apoptosis, their survival contingent upon pro-survival and pro-apoptotic signals derived from their microenvironment. Hypoxia profoundly delays PMN apoptosis, resulting in persistence of PMNs at inflammatory foci and this may perpetuate hypoxia-mediated lung injury. Given the importance of phosphoinositide 3-kinase (PI3-K) signalling in cytokine-mediated neutrophil survival, we hypothesised that hypoxia-induced PMN survival may also involve PI3-K-mediated signalling. Methods Highly pure PMNs isolated from healthy volunteers were incubated for 20 h under normoxic (20 kPa) and physiologically relevant hypoxic (3 kPa) conditions with a pan-PI3-K inhibitor (LY294002 at 10 μM), a novel pan-Class I PI3-K inhibitor (ZSTK474 at 1 μM, 3 μM and 10 μM) or novel PI3-K Class I isoform-selective inhibitors (PI3-Kδ at 1 μM; PI3-Kγ at 3 μM and 10 μM, or PI3-Kδγ at 3 μM). PMNs were also incubated in normoxia and hypoxia in the presence of GM-CSF (1 ng/ml) with the same panel of inhibitors, allowing comparison with GM-CSF mediated survival, which is largely PI3-K dependent. PMN apoptosis was assessed using two complementary techniques – morphology and flow cytometry following annexin V-FITC and propidium iodide staining. Results Compared with normoxia, hypoxia promoted PMN survival (mean% ± SEM apoptotic cells at 20 h; 30.9 ± 1.9 vs. 59.0 ± 1.8 respectively, p Conclusions Our results indicate that hypoxia-induced PMN survival is PI3-K dependent. Targeting this pathway may accelerate PMN apoptosis, resulting in resolution of inflammation.

Neutrophil recruitment into the bronchoalveolar space is central to the pathogenesis of acute respiratory distress syndrome injury (ARDS), and occurs via interaction with the lung microvascular endothelium. Tumour Necrosis Factor (TNF) is a key mediator in these processes, activating endothelial cells and inducing changes in microvascular permeability, as well as priming neutrophils (a pre-requisite for neutrophil-mediated tissue damage) and modulating neutrophil lifespan. TNF signals through two cell surface receptors, TNFR1 and TNFR2 initiating distinct signalling pathways and cellular responses. In a human in vivo model of ARDS, selective TNFR1 antagonism attenuated pulmonary inflammation (O’Kane et al , Thorax 2013; 63:A50). Using TNF receptor specific muteins and a novel highly selective TNFR1 antagonist, we investigated the role of differential TNFR signalling on neutrophil-pulmonary microvascular endothelial cell interactions. TNF-induced alterations in the expression of the neutrophil cell surface molecules CD11b, CD62L, TNFR1 and TNFR2 were all modulated via TNFR1. TNFR1 was also the dominant receptor mediating reactive oxygen species generation by TNF-primed, fMLP-stimulated neutrophils. We further examined the role of TNF receptors in modulating neutrophil apoptosis; whilst engagement of both TNFR1 and 2 was required to induce early neutrophil apoptosis, TNFR1 antagonism reversed TNF-induced late survival to constitutive levels of apoptosis. TNFR1 antagonsim of human pulmonary microvascular endothelial monolayers significantly reduced TNF-induced production of IL-1beta, IL-6 and IL-8 (p Collectively, these results suggest that TNFR1 regulates multiple components of neutrophil-endothelial interactions. Selective TNFR1 antagonism may offer a novel therapeutic approach in ARDS; phase II clinical trials of this therapy are scheduled.

Glucocorticoids (GCs) represent one of the most effective treatments for eosinophil-mediated inflammatory diseases such as asthma. GCs act through the GC receptor, leading to proinflammatory cytokine suppression and a reduction in the number of inflammatory cells including eosinophils and T cells.1 However, the benefits of GCs have been limited by their side effects and the presence of GC resistance. This led to the development of more selective GCs such as fluticasone propionate (FP) and fluticasone furoate (FF).2 Increased eosinophil survival has been proposed as a mechanism underlying tissue eosinophilia, and part of the anti-inflammatory effects of GCs has been attributed to their ability to promote eosinophil apoptosis. Interleukin 5 (IL-5) enhances eosinophil survival by inhibiting apoptosis, and increased IL-5 expression is reported in eosinophilic inflammation.3 We sought to address the ability of the ‘enhanced-affinity’ FF, alongside dexamethasone (DEX) and FP, to modulate eosinophil apoptosis and their potential to …

Introduction Neutrophils are an essential component of the innate immune response. However, their microbicidal mechanisms (generation of reactive oxygen species and release of proteolytic enzymes) may contribute to tissue injury. Neutrophil-mediated tissue damage is a cardinal feature in the pathogenesis/progression of COPD, cystic fibrosis and certain types of asthma. Apoptosis is the key determinant of tissue neutrophil longevity and is critical to the resolution of granulocyte inflammation; pharmacological acceleration of neutrophil apoptosis can promote resolution of inflammation in animal models. The cytokine GM-CSF drives the aberrant neutrophil survival phenotype observed in patients with ARDS, and recent studies suggest the phosphoinositide 3-kinase (PI3K)/AKT pathway is pivotal in signalling GM-CSF-mediated neutrophil survival. Hypothesis Given the emerging evidence that individual Class I PI3K isoforms (α, β, δ and γ) exert non-redundant signalling roles and represent promising therapeutic targets in inflammation, we hypothesised a distinct contribution of individual Class I PI3K isoforms in mediating constitutive neutrophil apoptosis and the GM-CSF cytoprotective effect. Methods We established techniques to isolate peripheral blood neutrophils from humans and from knockout/transgenic mice lacking functional PI3K isoforms to 95% purity, and used pan-PI3K inhibitor (LY294002), pan-Class I PI3K inhibitor (PI-103) and novel PI3K isoform-selective small molecule inhibitors (YM-024, TGX-221, IC87114 and AS605240) to determine precise involvement of Class I PI3K isoforms (α, β, δ and γ) in constitutive and GM-CSF-delayed neutrophil apoptosis. Apoptosis was quantified using morphology and annexin V-FITC/propidium iodide staining. Results GM-CSF-mediated neutrophil survival was reversed by pan-PI3K inhibition but not by individual PI3K isoform inhibition. Combinatorial experiments suggest there is near-complete functional redundancy amongst Class I PI3Ks with regard to GM-CSF-mediated inhibition of neutrophil apoptosis; additionally, neutrophils derived from double knockout PI3K-δ/γ and PI3K-β/δ mice had normal constitutive apoptosis and GM-CSF mediated survival, but were sensitised to inhibition of the remaining isoforms. Conclusions Thus Class I PI3Ks mediate GM-CSF survival of human and murine neutrophils but there is complete functional redundancy of the PI3K isoforms, necessitating multiple isoform inhibition to reverse GM-CSF-induced neutrophil survival. This finding informs our understanding of the mechanisms regulating neutrophil apoptosis and suggests neutrophil survival would be resilient to individual isoform-selective PI3K inhibitors.

Background Eosinophils are pro-inflammatory cells implicated in the pathogenesis of asthma and atopy. Apoptosis has been proposed as a potential mechanism underlying the resolution of eosinophilic inflammation and studies have indicated the ability of interventions that induce human eosinophil apoptosis to promote the resolution of eosinophilic inflammation. Recently, the cyclin-dependent kinase (CDK) inhibitor R-roscovitine was shown to enhance neutrophil apoptosis and promote the resolution of neutrophilic inflammation. Aim The purpose of this study was to examine the expression of CDKs in human blood eosinophils, the effects of R-roscovitine on eosinophil survival and phagocytosis in vitro and determine whether R-roscovitine could influence eosinophilic lung inflammation in vivo . Methods Eosinophils were isolated from human peripheral blood and the effects of R-roscovitine on apoptosis, degranulation and phagocytic uptake examined in vitro . The effects of R-roscovitine on eosinophilic lung inflammation in vivo were also assessed using an ovalbumin mouse model. Results Our data demonstrate that human eosinophils express five targets for R-roscovitine: CDK1, −2, −5, −7 and −9. R-roscovitine induced eosinophil apoptosis in a time- and concentration-dependent manner but also accelerated transition to secondary necrosis as assessed by light and electron microscopy, flow cytometry and caspase activation. In addition, we report that the pro-apoptotic effect of R-roscovitine is associated with suppression of Mcl-1L expression and that the apoptotic eosinophils are phagocytosed by human monocyte derived macrophages. R-roscovitine also induced apoptosis in mouse eosinophils purified from the bone-marrow, spleen and peripheral blood. Despite this, R-roscovitine did not modulate the tissue and lumen eosinophilia characteristic of the ovalbumin mouse model of airway eosinophilia. Conclusions These data demonstrate that R-roscovitine is capable of inducing rapid apoptosis and secondary necrosis in human eosinophils but does not affect the onset or resolution of eosinophilic airway inflammation in vivo .