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The release of neutrophil extracellular traps (NETs) is a major immune mechanism intended to capture pathogens. These histone- and protease-coated DNA structures are released by neutrophils in response to a variety of stimuli, including respiratory pathogens, and have been identified in the airways of patients with respiratory infection, cystic fibrosis, acute lung injury, primary graft dysfunction, and chronic obstructive pulmonary disease. NET production has been demonstrated in the lungs of mice infected with Staphylococcus aureus, Klebsiella pneumoniae, and Aspergillus fumigatus. Since the discovery of NETs over a decade ago, evidence that "NET evasion" might act as an immune protection strategy among respiratory pathogens, including group A Streptococcus, Bordetella pertussis, and Haemophilus influenzae, has been growing, with the majority of these studies being published in the past 2 years. Evasion strategies fall into three main categories: inhibition of NET release by down-regulating host inflammatory responses; degradation of NETs using pathogen-derived DNases; and resistance to the microbicidal components of NETs, which involves a variety of mechanisms, including encapsulation. Hence, the evasion of NETs appears to be a widespread strategy to allow pathogen proliferation and dissemination, and is currently a topic of intense research interest. This article outlines the evidence supporting the three main strategies of NET evasion-inhibition, degradation, and resistance-with particular reference to common respiratory pathogens.

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.

Background: Critically ill patients with impaired neutrophil phagocytosis have significantly increased risk of nosocomial infection. Granulocyte-macrophage colony-stimulating factor (GM-CSF) improves phagocytosis by neutrophils ex vivo. This study tested the hypothesis that GM-CSF improves neutrophil phagocytosis in critically ill patients in whom phagocytosis is known to be impaired. Methods: This was a multicentre, phase IIa randomised, placebo-controlled clinical trial. Using a personalised medicine approach, only critically ill patients with impaired neutrophil phagocytosis were included. Patients were randomised 1:1 to subcutaneous GM-CSF (3 μg/kg/day) or placebo, once daily for 4 days. The primary outcome measure was neutrophil phagocytosis 2 days after initiation of GM-CSF. Secondary outcomes included neutrophil phagocytosis over time, neutrophil functions other than phagocytosis, monocyte HLA-DR expression and safety. Results: Thirty-eight patients were recruited from five intensive care units (17 randomised to GM-CSF). Mean neutrophil phagocytosis at day 2 was 57.2% (SD 13.2%) in the GM-CSF group and 49.8% (13.4%) in the placebo group, p=0.73. The proportion of patients with neutrophil phagocytosis≥50% at day 2, and monocyte HLA-DR, appeared significantly higher in the GM-CSF group. Neutrophil functions other than phagocytosis did not appear significantly different between the groups. The most common adverse event associated with GM-CSF was fever. Conclusions: GM-CSF did not improve mean neutrophil phagocytosis at day 2, but was safe and appeared to increase the proportion of patients with adequate phagocytosis. The study suggests proof of principle for a pharmacological effect on neutrophil function in a subset of critically ill patients.

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 Decreases in circulating neutrophils (polymorphonuclear leukocytes, PMNs) have been reported in patients treated with the anti-interleukin-6 receptor (IL-6R) antibody tocilizumab (TCZ); the mechanism for this is unclear. We hypothesize that TCZ reduces circulating neutrophils by affecting margination and / or bone marrow trafficking without affecting neutrophil function or apoptosis. Materials and methods 18 healthy subjects were randomized to single intravenous dose of TCZ 8 mg/kg (n = 12) or placebo (n = 6) on day 0. On day 4, each subject had autologous indium-111-labeled neutrophils re-injected, and their kinetics quantified with longitudinal profiling in a whole body gamma-counter. TCZ-treated subjects were divided into two groups according to the extent of reduction in neutrophil count. Results Mean day 4 neutrophil counts, as % baseline, were 101.9%, 68.3% and 44.2% in the placebo, TCZ-PMN-’high’ and TCZ-PMN-’low’ groups, respectively (p < 0.001). Following TCZ, neutrophil function, activation and apoptosis ex vivo were all unaffected. In vivo, there were no differences in early blood recovery or margination to liver / spleen and bone marrow; however, later neutrophil re-distribution to bone marrow was markedly reduced in the TCZ-PMN-low group (peak pelvic count as % day 4 count on: day 5, 188% placebo vs 127% TCZ-PMN-low, p < 0.001; day 10, 180% placebo vs 132% TCZ-PMN-low, p < 0.01), with a trend towards higher liver / spleen neutrophil retention. Conclusions We have demonstrated for the first time in humans that IL-6R blockade affects neutrophil trafficking to the bone marrow without influencing neutrophil functional capacity. This article is protected by copyright. All rights reserved.

Answers from Exomes Exome sequencing, which targets only the protein-coding regions of the genome, has the potential to identify the underlying genetic causes of rare inherited diseases. Angulo et al. (p. 866 , published online 17 October; see Perspective by Conley and Fruman ) performed exome sequencing of individuals from seven unrelated families with severe, recurrent respiratory infections. The patients carried the same mutation in the gene coding for the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ). The mutation caused aberrant activation of this kinase, which plays a key role in immune cell signaling. Drugs inhibiting PI3Kδ are already in clinical trials for other disorders.

RATIONALE: Acute respiratory distress syndrome is refractory to pharmacological intervention. Inappropriate activation of alveolar neutrophils is believed to underpin this disease's complex pathophysiology, yet these cells have been little studied. OBJECTIVES: To examine the functional and transcriptional profiles of patient blood and alveolar neutrophils compared with healthy volunteer cells, and to define their sensitivity to phosphoinositide 3-kinase inhibition. METHODS: Twenty-three ventilated patients underwent bronchoalveolar lavage. Alveolar and blood neutrophil apoptosis, phagocytosis, and adhesion molecules were quantified by flow cytometry, and oxidase responses were quantified by chemiluminescence. Cytokine and transcriptional profiling were used in multiplex and GeneChip arrays. MEASUREMENTS AND MAIN RESULTS: Patient blood and alveolar neutrophils were distinct from healthy circulating cells, with increased CD11b and reduced CD62L expression, delayed constitutive apoptosis, and primed oxidase responses. Incubating control cells with disease bronchoalveolar lavage recapitulated the aberrant functional phenotype, and this could be reversed by phosphoinositide 3-kinase inhibitors. In contrast, the prosurvival phenotype of patient cells was resistant to phosphoinositide 3-kinase inhibition. RNA transcriptomic analysis revealed modified immune, cytoskeletal, and cell death pathways in patient cells, aligning closely to sepsis and burns datasets but not to phosphoinositide 3-kinase signatures. CONCLUSIONS: Acute respiratory distress syndrome blood and alveolar neutrophils display a distinct primed prosurvival profile and transcriptional signature. The enhanced respiratory burst was phosphoinositide 3-kinase-dependent but delayed apoptosis and the altered transcriptional profile were not. These unexpected findings cast doubt over the utility of phosphoinositide 3-kinase inhibition in acute respiratory distress syndrome and highlight the importance of evaluating novel therapeutic strategies in patient-derived cells.

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.

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.

Human rhinoviruses (HRV) are a major cause of exacerbations of airways disease. Aspects of cell signalling responses to HRV infection remain unclear, particularly with regard to signalling via PI3K, and the PI3K-dependent pathway, autophagy. We investigated the roles of PI3K and autophagy in the responses of epithelial cells to major and minor group HRV infection. The PI3K inhibitor 3-MA, commonly used to inhibit autophagy, markedly reduced HRV-induced cytokine induction. Further investigation of potential targets of 3-MA and comparison of results using this inhibitor to a panel of general and class I-selective PI3K inhibitors showed that several PI3Ks cooperatively regulate responses to HRV. Targeting by siRNA of the autophagy proteins Beclin-1, Atg7, LC3, alone or in combination, or targeting of the autophagy-specific class III PI3K had at most only modest effects on HRV-induced cell signalling as judged by induction of proinflammatory cytokine production. Our data indicate that PI3K and mTOR are involved in induction of proinflammatory cytokines after HRV infection, and that autophagy has little role in the cytokine response to HRV or control of HRV replication.

Background Reactive oxygen species (ROS), generated via the phagocyte NADPH oxidase cytochrome b558, are essential for effective immune responses to common and serious pathogens. The phagocyte NADPH oxidase is a multisubunit protein complex and deficiency of either the membrane bound or cytoplasmic components leads to chronic granulomatous disease, a serious and often fatal illness characterised by recurrent infections and autoimmunity. Moreover, abnormal generation of ROS has been implicated in the pathogenesis of multigenic autoimmune diseases such as systemic lupus erythematosus. Eros (essential for reactive oxygen species), encoded by bc017643 , is a novel transmembrane protein that is highly expressed in the immune system and highly conserved in evolution but has no previously identified function. Eros is an orthologue of the plant protein Ycf4, necessary for expression of proteins of the photosynthetic photosystem 1 complex, an NADPH oxio-reductase complex. We elucidated its role in infection in mice. Methods ROS are essential for host defence against the serious bacterial pathogen Salmonella enterica serovar Typhimurium. We screened individual knockout mice (Wellcome Trust Knockout mouse project) for susceptibility to salmonella infection. Having identified mice deficient in Eros as being highly susceptible to salmonella, we used ex-vivo approaches including reactive oxygen burst assays and western blot, to characterise their defect further. Findings We found that Eros was essential for host defence to infection. Eros was crucial for generating reactive oxygen species through regulation of the essential NADPH oxidase components, gp91 and p22. Eros-deficient mice expressed almost no gp91 and p22 in neutrophils and macrophages secondary to accelerated degradation in the absence of Eros. As a result Eros-deficient mice died rapidly after infection with salmonella or listeria. Eros also regulated the ROS-dependent formation of neutrophil extracellular traps and melanoma metastases. Interpretation We have found a a key role for Eros in regulating host defence. The finding that Eros-deficient mice lack gp91 and p22 at the protein, though not mRNA, level shows how these key components of the reatcive oxygen burst are protected from degradation and furthers our understanding of reactive oxygen burst biology. Eros is highly conserved between mouse and man so it is likely that it also has a crucial role in human immunity. Funding Wellcome Trust, Academy of Medical Sciences starter grant.

We have investigated the contribution of individual phosphoinositide 3-kinase (PI3K) Class I isoforms to the regulation of neutrophil survival using (i) a panel of commercially available small molecule isoform-selective PI3K Class I inhibitors, (ii) novel inhibitors, which target single or multiple Class I isoforms (PI3K alpha, PI3K beta, PI3K delta, and PI3K gamma), and (iii) transgenic mice lacking functional PI3K isoforms (p110 delta(KO)gamma(KO) or p110 gamma(KO)). Our data suggest that there is considerable functional redundancy amongst Class I PI3Ks (both Class IA and Class IB) with regard to GM-CSF-mediated suppression of neutrophil apoptosis. Hence pharmacological inhibition of any 3 or more PI3K isoforms was required to block the GM-CSF survival response in human neutrophils, with inhibition of individual or any two isoforms having little or no effect. Likewise, isolated blood neutrophils derived from double knockout PI3K p110 delta(KO)gamma(KO) mice underwent normal time-dependent constitutive apoptosis and displayed identical GM-CSF mediated survival to wild type cells, but were sensitized to pharmacological inhibition of the remaining PI3K isoforms. Surprisingly, the pro-survival neutrophil phenotype observed in patients with an acute exacerbation of chronic obstructive pulmonary disease (COPD) was resilient to inactivation of the PI3K pathway.

International audience; Neutrophils are activated by immunoglobulin G (IgG)-containing immune complexes through receptors that recognize the Fc portion of IgG (Fc gamma Rs). Here, we used genetic and pharmacological approaches to define a selective role for the beta isoform of phosphoinositide 3-kinase (PI3K beta) in Fc gamma R-dependent activation of mouse neutrophils by immune complexes of IgG and antigen immobilized on a plate surface. At low concentrations of immune complexes, loss of PI3K beta alone substantially inhibited the production of reactive oxygen species (ROS) by neutrophils, whereas at higher doses, similar suppression of ROS production was achieved only by targeting both PI3K beta and PI3K delta, suggesting that this pathway displays stimulus strength-dependent redundancy. Activation of PI3K beta by immune complexes involved cooperation between Fc gamma Rs and BLT1, the receptor for the endogenous proinflammatory lipid leukotriene B-4. Coincident activation by a tyrosine kinase-coupled receptor (Fc gamma R) and a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (BLT1) may provide a rationale for the preferential activation of the beta isoform of PI3K. PI3K beta-deficient mice were highly protected in an Fc gamma R-dependent model of autoantibody-induced skin blistering and were partially protected in an Fc gamma R-dependent model of inflammatory arthritis, whereas combined deficiency of PI3K beta and PI3K delta resulted in near-complete protection in the latter case. These results define PI3K beta as a potential therapeutic target in inflammatory disease.

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.

We report a significant complication of acupuncture in a 50 year old woman who developed a pneumothorax shortly after receiving acupuncture needling to her scapulothoracic region in a lateral oblique direction. As acupuncture is increasingly being used in pain management, physicians need to be aware of its potential adverse effects. We discuss issues relating to appropriate counselling of patients receiving this form of therapy. The inner Bladder line should be needled obliquely towards the spine.

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.

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 .