Alison M. Condliffe

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.

Neutrophil kinetics in health and disease

Neutrophils play a key role in the elimination of pathogens. They are remarkably short-lived with a circulating half life of 6–8 h and hence are produced at a rate of 5 × 1010–10 × 1010 cells/day. Tight regulation of these cells is vital because they have significant histotoxic capacity and are widely implicated in tissue injury. This review outlines our current understanding of how neutrophils are released from the bone marrow; in particular, the role of the CXC chemokine receptor 4/stromal-derived factor 1 axis, the relative size and role of the freely circulating and marginated (i.e. slowly transiting) pools within the vascular compartment, and the events that result in the uptake and removal of circulating neutrophils. We also review current understanding of how systemic stress and inflammation affect this finely balanced system.

Phosphoinositide 3-Kinase δ Gene Mutation Predisposes to Respiratory Infection and Airway Damage

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. Gene sequencing of unrelated patients with recurrent airway infections identifies a common underlying mutation. [Also see Perspective by Conley and Fruman] Genetic mutations cause primary immunodeficiencies (PIDs) that predispose to infections. Here, we describe activated PI3K-δ syndrome (APDS), a PID associated with a dominant gain-of-function mutation in which lysine replaced glutamic acid at residue 1021 (E1021K) in the p110δ protein, the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ), encoded by the PIK3CD gene. We found E1021K in 17 patients from seven unrelated families, but not among 3346 healthy subjects. APDS was characterized by recurrent respiratory infections, progressive airway damage, lymphopenia, increased circulating transitional B cells, increased immunoglobulin M, and reduced immunoglobulin G2 levels in serum and impaired vaccine responses. The E1021K mutation enhanced membrane association and kinase activity of p110δ. Patient-derived lymphocytes had increased levels of phosphatidylinositol 3,4,5-trisphosphate and phosphorylated AKT protein and were prone to activation-induced cell death. Selective p110δ inhibitors IC87114 and GS-1101 reduced the activity of the mutant enzyme in vitro, which suggested a therapeutic approach for patients with APDS.

Copy number of FCGR3B, which is associated with systemic lupus erythematosus, correlates with protein expression and immune complex uptake.

Copy number (CN) variation (CNV) has been shown to be common in regions of the genome coding for immune-related genes, and thus impacts upon polygenic autoimmunity. Low CN of FCGR3B has recently been associated with systemic lupus erythematosus (SLE). FcγRIIIb is a glycosylphosphatidylinositol-linked, low affinity receptor for IgG found predominantly on human neutrophils. We present novel data demonstrating that both in a family with FcγRIIIb-deficiency and in the normal population, FCGR3B CNV correlates with protein expression, with neutrophil uptake of and adherence to immune complexes, and with soluble serum FcγRIIIb. Reduced FcγRIIIb expression is thus likely to contribute to the impaired clearance of immune complexes, which is a feature of SLE, explaining the association between low FCGR3B CNV and SLE that we have confirmed in a Caucasian population. In contrast, antineutrophil cytoplasmic antibody–associated systemic vasculitis (AASV), a disease not associated with immune complex deposition, is associated with high FCGR3B CN. Thus, we define a role for FCGR3B CNV in immune complex clearance, a function that may explain why low FCGR3B CNV is associated with SLE, but not AASV. This is the first report of an association between disease-related gene CNV and variation in protein expression and function that may contribute to autoimmune disease susceptibility.

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.

The Neutrophil in Chronic Obstructive Pulmonary Disease. Too Little, Too Late or Too Much, Too Soon?

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide and has few effective therapies. It is characterized by anomalous and persistent inflammation, both local and systemic. Neutrophilic inflammation predominates in the COPD airway wall and lumen, but, despite the presence of abundant innate immune cells, the progressive clinical course of the disease is punctuated by recurrent infection-driven exacerbations. An extensive body of evidence (from cell culture to murine models and finally to the susceptibility of human patients with α1-antitrypsin deficiency to develop COPD) implicates neutrophil elastase and other neutrophil-derived proteases as key mediators of the tissue damage and relentless decline in lung function that occurs in this condition. In addition to the well recognized role of cytokines in modulating neutrophil function and survival, it has recently become apparent that hypoxia can influence neutrophil function, with impaired killing of pathogenic bacteria, enhanced release of proteases, and delayed apoptosis. This destructive neutrophil phenotype is predicted to be highly detrimental in the setting of the COPD microenvironment.

P-Rex1 Regulates Neutrophil Function

Rac GTPases regulate cytoskeletal structure, gene expression, and reactive oxygen species (ROS) production. Rac2-deficient neutrophils cannot chemotax, produce ROS, or degranulate upon G protein-coupled receptor (GPCR) activation. Deficiency in PI3Kgamma, an upstream regulator of Rac, causes a similar phenotype. P-Rex1, a guanine-nucleotide exchange factor (GEF) for Rac, is believed to link GPCRs and PI3Kgamma to Rac-dependent neutrophil responses. We have investigated the functional importance of P-Rex1 by generating a P-Rex1(-/-) mouse. P-Rex1(-/-) mice are viable and healthy, with apparently normal leukocyte development, but with mild neutrophilia. In neutrophils from P-Rex1(-/-) mice, GPCR-dependent Rac2 activation is impaired, whereas Rac1 activation is less compromised. GPCR-dependent ROS formation is absent in lipopolysaccharide (LPS)-primed P-Rex1(-/-) neutrophils, but less affected in unprimed or TNFalpha-primed cells. Recruitment of P-Rex1(-/-) neutrophils to inflammatory sites is impaired. Surprisingly, chemotaxis of isolated neutrophils is only slightly reduced, with a mild defect in cell speed, but normal polarization and directionality. Secretion of azurophil granules is unaffected. In conclusion, P-Rex1 is an important regulator of neutrophil function by mediating a subset of Rac-dependent neutrophil responses. However, P-Rex1 is not an essential regulator of neutrophil chemotaxis and degranulation.

Gβγs and the Ras binding domain of p110γ are both important regulators of PI3Kγ signalling in neutrophils

Through their ability to regulate production of the key lipid messenger PtdIns(3,4,5)P3, the class I phosphatidylinositol-3-OH kinases (PI(3)Ks) support many critical cell responses. They, in turn, can be regulated by cell-surface receptors through signals acting on either their adaptor subunits (for example, through phosphotyrosine or Gβγs) or their catalytic subunits (for example, through GTP-Ras). The relative significance of these controlling inputs is undefined in vivo. Here, we have studied the roles of Gβγs, the adaptor p101, Ras and the Ras binding domain (RBD) in the control of the class I PI(3)K, PI(3)Kγ, in mouse neutrophils. Loss of p101 leads to major reductions in the accumulation of PtdIns(3,4,5)P3, activation of protein kinase B (PKB) and in migration towards G-protein activating ligands in vitro, and to an aseptically inflamed peritoneum in vivo. Loss of sensitivity of PI(3)Kγ to Ras unexpectedly caused similar reductions, but additionally caused a substantial loss in production of reactive oxygen species (ROS). We conclude that Gβγs, p101 and the Ras–RBD interaction all have important roles in the regulation of PI(3)Kγ in vivo and that they can simultaneously, but differentially, control distinct PI(3)Kγ effectors.

Priming differentially regulates neutrophil adhesion molecule expression/function.

Lung injury in a variety of disease states is critically dependent on neutrophil‐mediated inflammatory responses. Neutrophil recruitment to sites of infection or tissue damage requires co‐ordinated regulation of neutrophil adhesion and activation status. We have examined the effects of treatment of human peripheral blood neutrophils with priming agents [lipopolysaccharide (LPS), tumour necrosis factor‐α (TNF‐α) and platelet‐activating factor (PAF)] upon expression of CD11a, CD11b, CD11c, CD35 and CD62‐L and CD11b function to assess whether subtle regulation of neutrophil adhesion potential accompanies augmented formyl‐methionyl‐leucyl‐phenylalanine‐stimulated superoxide production. We have found that there are differential effects of ‘priming’ concentrations of these agents. For LPS, CD62L loss occurs in the absence of changes in CD11b, whereas for PAF, CD11b up‐regulation occurs in the absence of detectable loss of CD62‐L. However, for TNF‐α, decreased expression of CD62‐L occurs concomitantly with increased expression of CD11b. In addition, we have shown that priming agents augment CD11b functional activity in a manner that parallels the priming of the respiratory burst. Thus, priming agents may differentially regulate neutrophil adhesive capacity and data presented in this manuscript suggest that the increased effector cell function observed in primed cells may be associated with a distinct repertoire of potential adhesive interactions.

Nontuberculous mycobacteria in bronchiectasis: prevalence and patient characteristics

The aim of the current study was to investigate the prevalence and clinical associations of nontuberculous mycobacteria (NTM) in a well-characterised cohort of patients with adult-onset bronchiectasis. The sputum of all patients attending a tertiary referral bronchiectasis clinic between April 2002 and August 2003 was examined for mycobacteria as part of an extensive diagnostic work-up. NTM-positive patients subsequently had further sputa examined. A modified bronchiectasis scoring system was applied to all high-resolution computed tomography (HRCT) scans from NTM-positive patients, and a matched cohort without NTM. Out of 98 patients attending the clinic, 10 had NTM in their sputum on first culture; of those, eight provided multiple positive cultures. Three patients were treated for NTM infection. A higher proportion of NTM-positive than -negative patients were subsequently diagnosed with cystic fibrosis (two out of nine versus two out of 75). On HRCT scoring, more patients in the NTM-positive group had peripheral mucus plugging than in the NTM-negative group. In the current prospective study of a large cohort of patients with bronchiectasis, 10% cultured positive for nontuberculous mycobacteria in a random clinic sputum sample. Few clinical parameters were helpful in discriminating between groups, except for a higher prevalence of previously undiagnosed cystic fibrosis and of peripheral mucus plugging on high-resolution computed tomography in the nontuberculous mycobacteria group.