Christine Fiddler

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.

Viscoelastic Properties of Differentiating Blood Cells Are Fate- and Function-Dependent

Although cellular mechanical properties are known to alter during stem cell differentiation, understanding of the functional relevance of such alterations is incomplete. Here, we show that during the course of differentiation of human myeloid precursor cells into three different lineages, the cells alter their viscoelastic properties, measured using an optical stretcher, to suit their ultimate fate and function. Myeloid cells circulating in blood have to be advected through constrictions in blood vessels, engendering the need for compliance at short time-scales (<seconds). Intriguingly, only the two circulating myeloid cell types have increased short time scale compliance and flow better through microfluidic constrictions. Moreover, all three differentiated cell types reduce their steady-state viscosity by more than 50% and show over 140% relative increase in their ability to migrate through tissue-like pores at long time-scales (>minutes), compared to undifferentiated cells. These findings suggest that reduction in steady-state viscosity is a physiological adaptation for enhanced migration through tissues. Our results indicate that the material properties of cells define their function, can be used as a cell differentiation marker and could serve as target for novel therapies.

Mechanical deformation induces depolarization of neutrophils

In vivo–mimicking mechanical deformations quickly depolarize neutrophils—a mechanism potentially failing in acute lung injury. The transition of neutrophils from a resting state to a primed state is an essential requirement for their function as competent immune cells. This transition can be caused not only by chemical signals but also by mechanical perturbation. After cessation of either, these cells gradually revert to a quiescent state over 40 to 120 min. We use two biophysical tools, an optical stretcher and a novel microcirculation mimetic, to effect physiologically relevant mechanical deformations of single nonadherent human neutrophils. We establish quantitative morphological analysis and mechanical phenotyping as label-free markers of neutrophil priming. We show that continued mechanical deformation of primed cells can cause active depolarization, which occurs two orders of magnitude faster than by spontaneous depriming. This work provides a cellular-level mechanism that potentially explains recent clinical studies demonstrating the potential importance, and physiological role, of neutrophil depriming in vivo and the pathophysiological implications when this deactivation is impaired, especially in disorders such as acute lung injury.

Hypoxia causes IL‐8 secretion, Charcot Leyden crystal formation, and suppression of corticosteroid‐induced apoptosis in human eosinophils

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.

The Aminopeptidase CD13 Induces Homotypic Aggregation in Neutrophils and Impairs Collagen Invasion.

Aminopeptidase N (CD13) is a widely expressed cell surface metallopeptidase involved in the migration of cancer and endothelial cells. Apart from our demonstration that CD13 modulates the efficacy of tumor necrosis factor-α-induced apoptosis in neutrophils, no other function for CD13 has been ascribed in this cell. We hypothesized that CD13 may be involved in neutrophil migration and/or homotypic aggregation. Using purified human blood neutrophils we confirmed the expression of CD13 on neutrophils and its up-regulation by pro-inflammatory agonists. However, using the anti-CD13 monoclonal antibody WM-15 and the aminopeptidase enzymatic inhibitor bestatin we were unable to demonstrate any direct involvement of CD13 in neutrophil polarisation or chemotaxis. In contrast, IL-8-mediated neutrophil migration in type I collagen gels was significantly impaired by the anti-CD13 monoclonal antibodies WM-15 and MY7. Notably, these antibodies also induced significant homotypic aggregation of neutrophils, which was dependent on CD13 cross-linking and was attenuated by phosphoinositide 3-kinase and extracellular signal-related kinase 1/2 inhibition. Live imaging demonstrated that in WM-15-treated neutrophils, where homotypic aggregation was evident, the number of cells entering IL-8 impregnated collagen I gels was significantly reduced. These data reveal a novel role for CD13 in inducing homotypic aggregation in neutrophils, which results in a transmigration deficiency; this mechanism may be relevant to neutrophil micro-aggregation in vivo.

P255 The Effects of Hypoxia on Neutrophil Degranulation

Tissues such as the skin and intestinal epithelium experience physiological hypoxia whereas pathological hypoxia occurs at inflammatory sites. Neutrophils are recruited to infective/inflamed areas and are thus required to operate under low oxygen tensions. We have shown previously that hypoxia delays neutrophil apoptosis (JEM 2005; 201:105) and impairs bacterial killing (J Immunol 2011; 186:453) and have now studied the effect of hypoxia on the release of histotoxic neutrophil proteases. Neutrophils isolated from healthy volunteers were subjected to normoxia or hypoxia (3 kPa). Superoxide anion release was measured by the reduction of cytochrome. C Elastase release was quantified by the cleavage of labelled elastin. Hypoxic incubation for 4 hours resulted in a 3- fold reduction in superoxide anion release from cells stimulated with GM-CSF and fMLP. In contrast, elastase release from the azurophilic granules was augmented almost 3-fold under hypoxia. The release of MMP-9 and lactoferrin was similarly up-regulated, suggesting a more generalised increase in degranulation under hypoxia. In addition to this electron microscopy showed that hypoxia induced a more activated phenotype (e.g. increased membrane ruffling and cell spreading). We show that hypoxia can induce a more destructive neutrophil phenotype, with enhanced degranulation and release of potentially histotoxic proteases, impaired bacterial killing, and delayed apoptosis. These data suggest that hypoxia aversively affects neutrophil function and may augment neutrophil mediated tissue destruction.

S55 The Aminopeptidase CD13 Regulates Homotypic Aggregation of Neutrophils

Neutrophils are critical effector cells of the innate immune response and are recruited to sites of tissue injury in response to locally generated chemoattractants. Neutrophil recruitment is a highly regulated process involving complex interactions with the vascular endothelium and underlying tissue stroma. In addition to adhering to the endothelium, neutrophils can also self-associate, (a process known as homotypic aggregation - HA), which has been proposed to play a key role in disease states such as sepsis. Aminopepetidase N or CD13 is a widely expressed membrane-bound metallopeptidase involved in the migration and invasion of cancer and endothelial cells. Neutrophils express CD13 on their cell surface, which is upregulated by TNF-α, IL-8 and fMLP We have shown that inhibition of aminopeptidase activity enhances the efficacy of TNF-α-induced neutrophil apoptosis (Cowburn et al. J Biol Chem 2006; 281:12458). Cross-linking anti-CD13 monoclonal antibodies (mAb) have been shown to induce HA of monocytic cells through PI3K activation (Mina-Osorio et al. J Leuk Biol 2006; 79:719). We hypothesised that CD13 may be involved in neutrophil migration and HA. Using plasma-Percoll purified human neutrophils and a modified Boyden filtre assay we showed that IL-8 mediated neutrophil chemotaxis was not affected by either the CD13 mAb WM-15 or the aminopeptidase enzymatic inhibitor bestatin. In contrast, IL-8-mediated neutrophil migration through type 1 collagen gels was significantly impaired by WM-15 and MY7 mAbs, which both inhibit enzymatic activity and induce clustering of CD13. The non-clustering CD13 antibody WM-47 and bestatin had no effect. WM-15 and MY7 also promoted neutrophil aggregation as assessed by light microscopy. Phase-contrast video-microscopy demonstrated that in WM-15 treated neutrophils, where HA was evident, the percentage of cells entering collagen 1 gels in response to IL-8 was significantly reduced (26.9% vs 71.8% in non-HA cells). WM-15 does not prime neutrophils, as assessed by superoxide production and shape change, and the cell surface expression of CD11b, CD18 and CD66b were not altered. These data suggest a novel role for CD13 in the homotypic aggregation of neutrophils, which reduces chemoattractant-induced migration through collagen 1 matrix and may predispose to neutrophil micro-aggregation within the circulation.

S117 Effects of Hypoxia on Eosinophil Apoptosis, Efferocytosis and Sensitivity to Glucocorticosteroids

Many tissues including the skin, intestinal epithelium and potentially the airway operate at ‘physiological’ levels of tissue hypoxia with normal PO2 values below 3KPa. Both sterile and non-sterile inflammation exacerbates the degree of tissue hypoxia and predicates the need for granulocytes including eosinophils to operate efficiently under hypoxia. In these experiments we have examined the effects of hypoxia on eosinophil longevity and show that a PO2 below 3KPa severely attenuates the pro-apoptotic effect of dexamethasone. Human blood eosinophils were prepared from healthy donors using hetastarch-sedimentation and EasySep®-immunomagenetic beads, and cultured in RPMI + 10% autologous serum for 6–24 h. Apoptosis and efferocytosis were quantified using standard morphology, AnV/PI staining and myeloperoxidase counter-stain methods. Hypoxic incubation (H) (media PO2 2.9±0.1 KPa) caused a marked survival response in eosinophils compared to normoxia (N) (% apoptosis at 24 h: N 16.3±3.0%; H 1.2±0.2%, n=5), which was of similar magnitude to that observed with IL-5 (N 4.2±0.3%). This hypoxic survival effect was mimicked by the iron chelator/2-oxyglutarate analogue DFO (10 mM) and DMOG (1 mM) and blocked in a concentration-dependent manner by the protein synthesis inhibitor cycloheximide. In preliminary experiments hypoxia also independently reduced the capacity of monocyte-derived macrophages to phagocytose apoptotic eosinophils as seen previously with neutrophils. Most strikingly, hypoxic incubation also reduced the normal pro-apoptotic effect of dexamethasone (1 µM) (% apoptosis at 24 h: N + Dex 27.6±3.9%, H + Dex 11.2±2.9%). qPCR analysis of the glucocorticosteroid-dependent gene GILZ and the hypoxia-HIF-1α-dependent gene GLUT1 demonstrated that the above suppression of dex-induced eosinophil apoptosis was not a consequence of the inhibition of steroid-induced transcriptional activity (mRNA fold change at 16 h: GILZ N 12.89±3.6, GILZ H 23.9±9.2; GLUT1 H (control) 28.5±7.2, GLUT1 H (Dex) 45.5±13.5, n=3). These data suggest that hypoxia can cause a profound effect on eosinophil longevity and macrophage uptake, and render these cells partially resistant to the pro-apoptotic effects of dexamethasone. This may impede the effective resolution of eosinophilic inflammation in vivo.

Idiopathic pulmonary fibrosis in a Christmas Island nuclear test veteran

We describe the case of a 71-year-old man with idiopathic pulmonary fibrosis (usual interstitial pneumonia (UIP) pattern) diagnosed on clinical, radiological and lung function criteria, in accordance with the American Thoracic Society/European Respiratory Society consensus criteria (2000), who had been in close proximity to three atmospheric nuclear bomb blasts during military service in 1957. He does not have clubbing and clinically and radiologically his lung disease is stable. He also has bladder carcinoma and carotid arteriosclerosis, both recognised consequences of radiation injury. This is the first reported case of UIP in a nuclear test veteran. Awareness of this potential association is important given the current attempts of the British Nuclear Test Veterans Association to gain compensation for claimed injuries.