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Background Inflamed environments are typically hypercellular, rich in pro-inflammatory cytokines, and profoundly hypoxic. While the effects of hypoxia on neutrophil longevity and function have been widely studied, little is known about the consequences of this stimulus on eosinophils. Objective We sought to investigate the effects of hypoxia on several key aspects of eosinophil biology; namely secretion, survival, and their sensitivity to glucocorticosteroids (GCS), agents which normally induce eosinophil apoptosis. Methods Eosinophils derived from patients with asthma/atopy or healthy controls were incubated under normoxia and hypoxia, with or without glucocorticoids. Activation was measured by flow cytometry, ELISA of cultured supernatants and F-actin staining; apoptosis and efferocytosis by morphology and flow cytometry, and GCS efficacy by apoptosis assays and qPCR. Results Hypoxic incubation (3 kPa) caused: (i) stabilisation of HIF-2α and up-regulation of hypoxia regulated genes including BNIP3 (BCL2/adenovirus E1B 19 kDa protein-interacting protein 3) and GLUT1 (glucose transporter 1), (ii) secretion of pre-formed IL-8, and Charcot Leyden crystal (CLC) formation, that was most evident in eosinophils derived from atopic and asthmatic donors, (iii) enhanced F-actin formation, (iv) marked prolongation of eosinophil lifespan (via a NF-κB and Class I PI3-kinase-dependent mechanism), and (v) complete abrogation of the normal pro-apoptotic effect of dexamethasone and fluticasone furoate. This latter effect was evident despite preservation of GCS-mediated gene transactivation under hypoxia. Conclusion and Clinical Relevance These data indicate that hypoxia promotes an eosinophil pro-inflammatory phenotype by enhancing eosinophil secretory function, delaying constitutive apoptosis and importantly, antagonising the normal pro-apoptotic effect of GCS. Since eosinophils typically accumulate at sites that are relatively hypoxic, particularly during periods of inflammation, these findings may have important implications to understanding the behaviour these cells in vivo. This article is protected by copyright. All rights reserved.

GM‐CSF is important in regulating acute, persistent neutrophilic inflammation in certain settings, including lung injury. Ligand binding induces rapid internalization of the GM‐CSF receptor (GM‐CSFRα) complex, a process essential for signaling. Whereas GM‐CSF controls many aspects of neutrophil biology, regulation of GM‐CSFRα expression is poorly understood, particularly the role of GM‐CSFRα in ligand clearance and whether signaling is sustained despite major down‐regulation of GM‐CSFRα surface expression. We established a quantitative assay of GM‐CSFRα surface expression and used this, together with selective anti‐GM‐CSFR antibodies, to define GM‐CSFRα kinetics in human neutrophils, and in murine blood and alveolar neutrophils in a lung injury model. Despite rapid sustained ligand‐induced GM‐CSFRα loss from the neutrophil surface, which persisted even following ligand removal, pro‐survival effects of GM‐CSF required ongoing ligand‐receptor interaction. Neutrophils recruited to the lungs following LPS challenge showed initially high mGM‐CSFRα expression, which along with mGM‐CSFRβ declined over 24 hr; this was associated with a transient increase in bronchoalveolar lavage fluid (BALF) mGM‐CSF concentration. Treating mice in an LPS challenge model with CAM‐3003, an anti‐mGM‐CSFRα mAb, inhibited inflammatory cell influx into the lung and maintained the level of BALF mGM‐CSF. Consistent with neutrophil consumption of GM‐CSF, human neutrophils depleted exogenous GM‐CSF, independent of protease activity. These data show that loss of membrane GM‐CSFRα following GM‐CSF exposure does not preclude sustained GM‐CSF/GM‐CSFRα signaling and that this receptor plays a key role in ligand clearance. Hence neutrophilic activation via GM‐CSFR may play an important role in neutrophilic lung inflammation even in the absence of high GM‐CSF levels or GM‐CSFRα expression., GM‐CSF released during ALI stimulates neutrophil recruitment, induces down‐regulation of GM‐CSFR, and GM‐CSF consumption by neutrophils, yet sustained anti‐apoptotic signals require continued GM‐CSF stimulation.

We identified a novel, evolutionarily conserved receptor encoded within the human leukocyte receptor complex and syntenic region of mouse chromosome 7, named T cell–interacting, activating receptor on myeloid cells-1 (TARM1). The transmembrane region of TARM1 contained a conserved arginine residue, consistent with association with a signaling adaptor. TARM1 associated with the ITAM adaptor FcRγ but not with DAP10 or DAP12. In healthy mice, TARM1 is constitutively expressed on the cell surface of mature and immature CD11b+Gr-1+ neutrophils within the bone marrow. Following i.p. LPS treatment or systemic bacterial challenge, TARM1 expression was upregulated by neutrophils and inflammatory monocytes and TARM1+ cells were rapidly recruited to sites of inflammation. TARM1 expression was also upregulated by bone marrow–derived macrophages and dendritic cells following stimulation with TLR agonists in vitro. Ligation of TARM1 receptor in the presence of TLR ligands, such as LPS, enhanced the secretion of proinflammatory cytokines by macrophages and primary mouse neutrophils, whereas TARM1 stimulation alone had no effect. Finally, an immobilized TARM1-Fc fusion protein suppressed CD4+ T cell activation and proliferation in vitro. These results suggest that a putative T cell ligand can interact with TARM1 receptor, resulting in bidirectional signaling and raising the T cell activation threshold while costimulating the release of proinflammatory cytokines by macrophages and neutrophils.

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. Objective The purpose of this study was to examine the expression of CDKs in human blood eosinophils, the effects of R-roscovitine on eosinophil survival in vitro and 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 known 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 microscopy, flow cytometry and caspase activation. In addition, we show that R-roscovitine can override the anti-apoptotic signals of GM-CSF and IL-5. We report that the pro-apoptotic effect of R-roscovitine is associated with suppression of Mcl-1L expression and that this compound enhanced phagocytic clearance of eosinophils by macrophages. Finally, we show that R-roscovitine induces apoptosis in murine peripheral blood and spleen-derived eosinophils; despite this, R-roscovitine did not modulate the tissue and lumen eosinophilia characteristic of the ovalbumin mouse model of airway eosinophilia. Conclusion and Clinical Relevance These data demonstrate that R-roscovitine is capable of inducing rapid apoptosis and secondary necrosis in eosinophils but does not affect the onset or improve the resolution of eosinophilic airway inflammation in vivo.

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.

Background Inappropriate accumulation or persistence of neutrophils (polymorphonuclear leucocytes [PMNs]), within the alveolar airspace is considered to be crucial to the pathogenesis of acute respiratory distress syndrome (ARDS). Previous work has established that alveolar PMNs from patients with ARDS have a pro-survival phenotype, and that bronchoalveolar lavage fluid (BALF) in ARDS suppresses constitutive PMN apoptosis in vitro. However, the functional activity of alveolar-sequestered PMNs in ARDS is unknown, partly due to the technical challenges in obtaining and purifying alveolar PMNs. Proinflammatory mediators in the ARDS alveolar milieu conceivably prime or activate key PMN functions in vivo, including the generation of toxic reactive oxygen species (ROS). This research evaluates agonist-stimulated ROS generation in alveolar and blood PMNs from the same patient with ARDS compared with healthy volunteer blood PMNs. Methods Alveolar and blood PMNs from mechanically ventilated patients who fulfilled the Berlin criteria of ARDS were studied in parallel. Patients underwent fibreoptic bronchoscopy and bronchoalveolar lavage within 36 h of ARDS onset, and the recovered BALF was processed immediately. Alveolar PMNs were isolated to 90% (SE 2·5) purity with a rapid semi-automated negative-selection immunomagnetic technique (Robosep, Stemcell Technologies, Vancouver, Canada). BALF supernatants and serum were stored at −80°C for subsequent cytokine analysis. Patients undergoing bronchoalveolar lavage for other clinical indications (sarcoidosis, tracheomalacia, and haemoptysis) served as controls. Blood PMNs were isolated using dextran sedimentation and plasma Percoll gradients. Neutrophil apoptosis was detected by flow cytometry using annexin V and propidium iodide. Kinetics of the PMN oxidative burst in response to ex-vivo stimulation with N-formyl-methionyl-leucylphenylalanine was determined with real-time horseradish peroxidase–luminol-dependent chemiluminescence detection. Findings The BALF from patients with ARDS consisted predominantly of alveolar PMNs (65%, SE 5), whereas alveolar macrophages constituted the major cell type (92%, SE 5) in BALF from control individuals. Nearly 70% of ARDS alveolar PMNs showed hypersegmented nuclear morphology and had a reduced rate of apoptosis after 20 h in culture (22% apoptotic PMNs, SE 10) compared with blood PMNs from control individuals (68% apoptotic PMNs, SE 7·2). ROS production by untreated alveolar neutrophils was 1·5 times higher compared with optimally (tumour necrosis factor α) ex vivo-primed ARDS blood PMNs from the same ARDS patient and control blood PMNs. This finding could reflect the alveolar cytokine milieu since concentrations of the proinflammatory mediators interleukin (IL) 1, IL6, IL8, IL10, IL12, and tumour necrosis factor α were significantly increased in BALF and serum from patients with ARDS compared with controls. Interpretation These findings indicate that ARDS alveolar PMNs have altered morphology, enhanced survival, and augmented capacity for ROS generation, which might contribute to PMN-dependent tissue injury. Funding NIHR Cambridge Biomedical Research Centre, GlaxoSmithKline.

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 …

Management of electrolyte abnormalities is challenging in older people as the sensation of thirst, renal function and hormonal modulators of the milieu interior are often impaired. Furthermore, the complex effects of ageing upon these homeostatic mechanisms are often superimposed upon a background of chronic disease, malnutrition and co-existent medications. Hyponatraemia is one of the commonest electrolyte abnormalities, occurring in approximately 7% of healthy elderly persons. Hyponatraemia may only come to light when some other ailment prompts investigations or hospital admission. Drug-induced hyponatraemia is common in older people and is most commonly associated with diuretics and SSRI/SNRI antidepressants, but has also been reported with a wide range of other drugs. We believe this is the first case report of hyponatraemia due to tolterodine.

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 .