Regulation of ICAM-1 in human neutrophils.

Intercellular cell adhesion molecule 1 (ICAM-1) is a cell surface glycoprotein with a vital role in the immune response to pathogens. The expression pattern of ICAM-1 is wide-ranging, encompassing endothelial cells, epithelial cells and neutrophils. Recent work has characterized the role of ICAM-1 in murine neutrophils, but the function of human neutrophil ICAM-1 is incompletely understood. Herein, we investigated the expression and role of ICAMs in human neutrophils in vitro and in vivo. Our findings show clear expression of ICAM-1, -3 and -4 on peripheral blood-derived neutrophils and demonstrate that the pathogen-associated molecular pattern (PAMP) lipoteichoic acid (LTA) is an inducer of ICAM-1 expression in vitro. In vivo, neutrophils obtained from the pleural cavity of patients with a parapneumonic effusion display enhanced expression of ICAM-1 compared to peripheral blood- and oral cavity-derived neutrophils. Moreover, migration of peripheral blood-derived neutrophils across endothelial cells can upregulate neutrophil ICAM-1 expression. These findings indicate that PAMPs and/or cytokines, alongside transmigration, enhance neutrophil ICAM-1 expression at sites of inflammation. Mechanistically we observed that ICAM-1high neutrophils display elevated S. aureus phagocytic capacity. However, unlike murine neutrophils, ICAM-1 intracellular signaling in human neutrophils was not essential for phagocytosis of S. aureus and reactive oxygen species (ROS) generation. Taken together, these results have important implications for the regulation of neutrophil-mediated pathogen clearance.

Elucidating mechanisms of genetic cross-disease associations at the PROCR vascular disease locus.

Many individual genetic risk loci have been associated with multiple common human diseases. However, the molecular basis of this pleiotropy often remains unclear. We present an integrative approach to reveal the molecular mechanism underlying the PROCR locus, associated with lower coronary artery disease (CAD) risk but higher venous thromboembolism (VTE) risk. We identify PROCR-p.Ser219Gly as the likely causal variant at the locus and protein C as a causal factor. Using genetic analyses, human recall-by-genotype and in vitro experimentation, we demonstrate that PROCR-219Gly increases plasma levels of (activated) protein C through endothelial protein C receptor (EPCR) ectodomain shedding in endothelial cells, attenuating leukocyte-endothelial cell adhesion and vascular inflammation. We also associate PROCR-219Gly with an increased pro-thrombotic state via coagulation factor VII, a ligand of EPCR. Our study, which links PROCR-219Gly to CAD through anti-inflammatory mechanisms and to VTE through pro-thrombotic mechanisms, provides a framework to reveal the mechanisms underlying similar cross-phenotype associations.

Measurement of Eosinophil Kinetics In Vivo.

Radiolabeled leukocyte scans are used in nuclear medicine to detect sites of infection and inflammation. We have previously demonstrated the use of clinical grade immunomagnetic beads to isolate autologous eosinophils and image their distribution in healthy volunteers. Here we describe the use of radiolabeled eosinophils coupled to single-photon emission computed tomography (SPECT) to quantify eosinophil uptake in the lungs of healthy volunteers, patients with asthma, and patients with focal eosinophilic inflammation.

Cell type-specific novel long non-coding RNA and circular RNA in the BLUEPRINT hematopoietic transcriptomes atlas.

Transcriptional profiling of hematopoietic cell subpopulations has helped to characterize the developmental stages of the hematopoietic system and the molecular bases of malignant and non-malignant blood diseases. Previously, only the genes targeted by expression microarrays could be profiled genome-wide. High-throughput RNA sequencing, however, encompasses a broader repertoire of RNA molecules, without restriction to previously annotated genes. We analyzed the BLUEPRINT consortium RNA-sequencing data for mature hematopoietic cell types. The data comprised 90 total RNA-sequencing samples, each composed of one of 27 cell types, and 32 small RNA-sequencing samples, each composed of one of 11 cell types. We estimated gene and isoform expression levels for each cell type using existing annotations from Ensembl. We then used guided transcriptome assembly to discover unannotated transcripts. We identified hundreds of novel non-coding RNA genes and showed that the majority have cell type-dependent expression. We also characterized the expression of circular RNA and found that these are also cell type-specific. These analyses refine the active transcriptional landscape of mature hematopoietic cells, highlight abundant genes and transcriptional isoforms for each blood cell type, and provide a valuable resource for researchers of hematologic development and diseases. Finally, we made the data accessible via a web-based interface: https://blueprint.haem.cam.ac.uk/bloodatlas/.

Lesson of the month: novel method to quantify neutrophil uptake in early lung cancer using SPECT-CT.

Neutrophils play an important role in the lung tumour microenvironment. We hypothesised that radiolabelled neutrophils coupled to single-photon emission CT (SPECT) may non-invasively quantify neutrophil uptake in tumours from patients with non-small cell lung cancer. We demonstrated increased uptake of radiolabelled neutrophils from the blood into tumours compared with non-specific uptake using radiolabelled transferrin. Moreover, indium-111-neutrophil activity in the tumour biopsies also correlated with myeloperoxidase (MPO)-positive neutrophils. Our data support the utility of imaging with In-111-labelled neutrophils and SPECT-CT to quantify neutrophil uptake in lung cancer.

Effect of the CXCR4 antagonist plerixafor on endogenous neutrophil dynamics in the bone marrow, lung and spleen.

Treatment with the CXCR4 antagonist, plerixafor (AMD3100), has been proposed for clinical use in patients with WHIM (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome and in pulmonary fibrosis. However, there is controversy with respect to the impact of plerixafor on neutrophil dynamics in the lung, which may affect its safety profile. In this study, we investigated the kinetics of endogenous neutrophils by direct imaging, using confocal intravital microscopy in mouse bone marrow, spleen, and lungs. Neutrophils are observed increasing their velocity and exiting the bone marrow following plerixafor administration, with a concomitant increase in neutrophil numbers in the blood and spleen, while the marginated pool of neutrophils in the lung microvasculature remained unchanged in terms of numbers and cell velocity. Use of autologous radiolabeled neutrophils and SPECT/CT imaging in healthy volunteers showed that plerixafor did not affect GM-CSF-primed neutrophil entrapment or release in the lungs. Taken together, these data suggest that plerixafor causes neutrophil mobilization from the bone marrow but does not impact on lung marginated neutrophil dynamics and thus is unlikely to compromise respiratory host defense both in humans and mice.

Use of autologous 99mTechnetium-labelled neutrophils to quantify lung neutrophil clearance in COPD.

RATIONALE: There is a need to develop imaging protocols which assess neutrophilic inflammation in the lung. AIM: To quantify whole lung neutrophil accumulation in (1) healthy volunteers (HV) following inhaled lipopolysaccharide (LPS) or saline and (2) patients with COPD using radiolabelled autologous neutrophils and single-photon emission computed tomography/CT (SPECT/CT). METHODS: 20 patients with COPD (Global initiative for chronic obstructive lung disease (GOLD) stages 2-3) and 18 HVs were studied. HVs received inhaled saline (n=6) or LPS (50 µg, n=12) prior to the injection of radiolabelled cells. Neutrophils were isolated using dextran sedimentation and Percoll plasma gradients and labelled with 99mTechnetium (Tc)-hexamethylpropyleneamine oxime. SPECT was performed over the thorax/upper abdomen at 45 min, 2 hours, 4 hours and 6 hours. Circulating biomarkers were measured prechallenge and post challenge. Blood neutrophil clearance in the lung was determined using Patlak-Rutland graphical analysis. RESULTS: There was increased accumulation of 99mTc-neutrophils in the lungs of patients with COPD and LPS-challenged subjects compared with saline-challenged subjects (saline: 0.0006±0.0003 mL/min/mL lung blood distribution volume [mean ±1 SD]; COPD: 0.0022±0.0010 mL/min/mL [p<0.001]; LPS: 0.0025±0.0008 mL/min/mL [p<0.001]). The accumulation of labelled neutrophils in 10 patients with COPD who underwent repeat radiolabelling/imaging 7-10 days later was highly reproducible (0.0022±0.0010 mL/min/mL vs 0.0023±0.0009 mL/min/mL). Baseline interleukin (IL)-6 levels in patients with COPD were elevated compared with HVs (1.5±1.06 pg/mL [mean ±1 SD] vs 0.4±0.24 pg/mL). LPS challenge increased the circulating IL-6 levels (7.5±2.72 pg/mL) 9 hours post challenge. CONCLUSIONS: This study shows the ability to quantify 'whole lung' neutrophil accumulation in HVs following LPS inhalation and in subjects with COPD using autologous radiolabelled neutrophils and SPECT/CT imaging. Moreover, the reproducibility observed supports the feasibility of using this approach to determine the efficacy of therapeutic agents aimed at altering neutrophil migration to the lungs.

Monocytes Latently Infected with Human Cytomegalovirus Evade Neutrophil Killing.

One site of latency of human cytomegalovirus (HCMV) in vivo is in undifferentiated cells of the myeloid lineage. Although latently infected cells are known to evade host T cell responses by suppression of T cell effector functions, it is not known if they must also evade surveillance by other host immune cells. Here we show that cells latently infected with HCMV can, indeed, be killed by host neutrophils but only in a serum-dependent manner. Specifically, antibodies to the viral latency-associated US28 protein mediate neutrophil killing of latently infected cells. To address this mechanistically, a full proteomic screen was carried out on latently infected monocytes. This showed that latent infection downregulates the neutrophil chemoattractants S100A8/A9, thus suppressing neutrophil recruitment to latently infected cells. The ability of latently infected cells to inhibit neutrophil recruitment represents an immune evasion strategy of this persistent human pathogen, helping to prevent clearance of the latent viral reservoir.

Metabolic Profiling of Human Eosinophils.

Immune cells face constant changes in their microenvironment, which requires rapid metabolic adaptation. In contrast to neutrophils, which are known to rely near exclusively on glycolysis, the metabolic profile of human eosinophils has not been characterized. Here, we assess the key metabolic parameters of peripheral blood-derived human eosinophils using real-time extracellular flux analysis to measure extracellular acidification rate and oxygen consumption rate, and compare these parameters to human neutrophils. Using this methodology, we demonstrate that eosinophils and neutrophils have a similar glycolytic capacity, albeit with a minimal glycolytic reserve. However, compared to neutrophils, eosinophils exhibit significantly greater basal mitochondrial respiration, ATP-linked respiration, maximum respiratory capacity, and spare respiratory capacity. Of note, the glucose oxidation pathway is also utilized by eosinophils, something not evident in neutrophils. Furthermore, using a colorimetric enzymatic assay, we show that eosinophils have much reduced glycogen stores compared to neutrophils. We also show that physiologically relevant levels of hypoxia (PO2 3 kPa), by suppressing oxygen consumption rates, have a profound effect on basal and phorbol-myristate-acetate-stimulated eosinophil and neutrophil metabolism. Finally, we compared the metabolic profile of eosinophils purified from atopic and non-atopic subjects and show that, despite a difference in the activation status of eosinophils derived from atopic subjects, these cells exhibit comparable oxygen consumption rates upon priming with IL-5 and stimulation with fMLP. In summary, our findings show that eosinophils display far greater metabolic flexibility compared to neutrophils, with the potential to use glycolysis, glucose oxidation, and oxidative phosphorylation. This flexibility may allow eosinophils to adapt better to diverse roles in host defense, homeostasis, and immunomodulation.

Radiolabelled leucocytes in human pulmonary disease.

Introduction: Radionuclides for leucocyte kinetic studies have progressed from non-gamma emitting cell-labelling radionuclides through gamma emitting nuclides that allow imaging of leucocyte kinetics, to the next goal of positron emission tomography (PET). Sources of data: Mostly the authors' own studies, following on from studies of the early pioneers. Areas of controversy: From early imaging studies, it appeared that the majority of the marginated granulocyte pool was located in the lungs. However, later work disputed this by demonstrating the exquisite sensitivity of granulocytes to ex vivo isolation and labelling, and that excessive lung activity is artefactual. Areas of agreement: Following refinement of labelling techniques, it was shown that the majority of marginated granulocytes are located in the spleen and bone marrow. The majority of leucocytes have a pulmonary vascular transit time only a few seconds longer than erythrocytes. The minority showing slow transit, ~5% in healthy persons, is increased in systemic inflammatory disorders that cause neutrophil priming and loss of deformability. Using a range of imaging techniques, including gamma camera imaging, whole-body counting and single photon-emission computerized tomography, labelled granulocytes were subsequently used to image pulmonary trafficking in lobar pneumonia, bronchiectasis, chronic obstructive pulmonary disease and adult respiratory distress syndrome. Growing points: More recently, eosinophils have been separated in pure form using magnetic bead technology for the study of eosinophil trafficking in asthma. Areas timely for developing research: These include advancement of eosinophil imaging, development of monocyte labelling, development of cell labelling with PET tracers and the tracking of lymphocytes.

In vivo imaging of hepatic neutrophil migration in severe alcoholic hepatitis with 111In-radiolabelled leucocytes.

The study's aim was to image severe alcoholic hepatitis (SAH) using 111In-labelled leucocytes with two objectives in mind: firstly for non-invasive diagnosis and secondly to provide a platform for experimental therapies aiming to inhibit intrahepatic neutrophil migration. 111In-leucocyte scintigraphy was performed 30 min and 24 h post-injection in 19 patients with SAH, 14 abstinent patients with alcohol-related cirrhosis and 11 normal controls. Eleven with SAH and seven with cirrhosis also had 99mTc-nanocolloid scintigraphy. Change in hepatic 111In radioactivity was expressed as decay-corrected 24 h:30 min count ratio and, in SAH, compared with histological grading of steatohepatitis and expression of granulocyte marker, CD15. Hepatic microautoradiography on biopsy specimens obtained 24 h post-injection of 111In-leucocytes was performed in one patient. Median 24 h:30 min hepatic 111In activity ratio was higher in SAH (2.5 (interquartile range (IQR): 1.7-4.0) compared with cirrhotics and normal controls (1.0 (0.8-1.1) and 0.8 (0.7-0.9) respectively, P<0.0001). In SAH, it correlated with CD15 expression (r = 0.62, P=0.023) and was higher in marked compared with mild/moderate steatohepatitis (4.0 (3.0-4.6) compared with 1.8 (1.5-2.6), P=0.006). Hepatic-to-splenic 99mTc count rate ratio was reduced in SAH (0.5 (0.4-1.4)) compared with cirrhotics (2.3( 0.6-3.0)) and three historic normal controls (4.2 (3.8-5.0); P=0.003), consistent with impaired hepatic reticuloendothelial function. Scintigraphic findings in SAH included prominent lung radioactivity at 30 min, likely the result of neutrophil primimg. Microautoradiography demonstrated cell-associated 111In in areas of parenchymal neutrophil infiltration. In conclusion, 111In-leucocyte scintigraphy can non-invasively diagnose SAH and could provide a platform for evaluation of novel treatments aiming to inhibit intrahepatic neutrophil migration.

Effects of tocilizumab on neutrophil function and kinetics.

BACKGROUND: Decreases in circulating neutrophils (polymorphonuclear leucocytes, 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: Eighteen 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-labelled 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.

Neutrophil-mediated IL-6 receptor trans-signaling and the risk of chronic obstructive pulmonary disease and asthma.

The Asp358Ala variant in the interleukin-6 receptor (IL-6R) gene has been implicated in asthma, autoimmune and cardiovascular disorders, but its role in other respiratory conditions such as chronic obstructive pulmonary disease (COPD) has not been investigated. The aims of this study were to evaluate whether there is an association between Asp358Ala and COPD or asthma risk, and to explore the role of the Asp358Ala variant in sIL-6R shedding from neutrophils and its pro-inflammatory effects in the lung. We undertook logistic regression using data from the UK Biobank and the ECLIPSE COPD cohort. Results were meta-analyzed with summary data from a further three COPD cohorts (7,519 total cases and 35,653 total controls), showing no association between Asp358Ala and COPD (OR = 1.02 [95% CI: 0.96, 1.07]). Data from the UK Biobank showed a positive association between the Asp358Ala variant and atopic asthma (OR = 1.07 [1.01, 1.13]). In a series of in vitro studies using blood samples from 37 participants, we found that shedding of sIL-6R from neutrophils was greater in carriers of the Asp358Ala minor allele than in non-carriers. Human pulmonary artery endothelial cells cultured with serum from homozygous carriers showed an increase in MCP-1 release in carriers of the minor allele, with the difference eliminated upon addition of tocilizumab. In conclusion, there is evidence that neutrophils may be an important source of sIL-6R in the lungs, and the Asp358Ala variant may have pro-inflammatory effects in lung cells. However, we were unable to identify evidence for an association between Asp358Ala and COPD.

Circulating granulocyte lifespan in compensated alcohol-related cirrhosis: a pilot study.

Although granulocyte dysfunction is known to occur in cirrhosis, in vivo studies of granulocyte lifespan have not previously been performed. The normal circulating granulocyte survival half-time (G - t½), determined using indium-111 ((111)In)-radiolabeled granulocytes, is ~7 h. In this pilot study, we aimed to measure the in vivo G - t½ in compensated alcohol-related cirrhosis. Sequential venous blood samples were obtained in abstinent subjects with alcohol-related cirrhosis over 24 h post injection (PI) of minimally manipulated (111)In-radiolabeled autologous mixed leukocytes. Purified granulocytes were isolated from each sample using a magnetic microbead-antibody technique positively selecting for the marker CD15. Granulocyte-associated radioactivity was expressed relative to peak activity, plotted over time, and G - t½ estimated from data up to 12 h PI This was compared with normal neutrophil half-time (N - t½), determined using a similar method specifically selecting neutrophils in healthy controls at a collaborating center. Seven patients with cirrhosis (six male, aged 57.8 ± 9.4 years, all Child-Pugh class A) and seven normal controls (three male, 64.4 ± 5.6 years) were studied. Peripheral blood neutrophil counts were similar in both groups (4.6 (3.5 - 5.5) × 10(9)/L vs. 2.8 (2.7 - 4.4) × 10(9)/L, respectively, P = 0.277). G - t½ in cirrhosis was significantly lower than N - t½ in controls (2.7 ± 0.5 h vs. 4.4 ± 1.0 h, P = 0.007). Transient rises in granulocyte and neutrophil-associated activities occurred in four patients from each group, typically earlier in cirrhosis (4-6 h PI) than in controls (8-10 h), suggesting recirculation of radiolabeled cells released from an unidentified focus. Reduced in vivo granulocyte survival in compensated alcohol-related cirrhosis is a novel finding and potentially another mechanism for immune dysfunction in chronic liver disease. Larger studies are needed to corroborate these pilot data and assess intravascular neutrophil residency in other disease etiologies.

Clinical application of autologous technetium-99m-labelled eosinophils to detect focal eosinophilic inflammation in the lung.

The detection of focal eosinophilic inflammation by non-invasive means may aid the diagnosis and follow-up of a variety of pulmonary pathologies. All current methods of detection involve invasive sampling, which may be contraindicated or too high-risk to be performed safely. The use of injected autologous technetium-99m (Tc-99m)-labelled eosinophils coupled to single-photon emission computed tomography (SPECT) has been demonstrated to localise eosinophilic inflammation in the lungs of a patient with antineutrophil cytoplasmic antibody-positive vasculitis. Here, we report on the utility of this technique to detect active eosinophilic inflammation in a patient with focal lung inflammation where a biopsy was contraindicated.

Measurement of eosinophil kinetics in healthy volunteers.

Radiolabelled leukocyte scans are widely used in nuclear medicine to locate sites of infection and inflammation. Radiolabelling of leukocyte subpopulations can also yield valuable information on cell trafficking and kinetics in vivo, but care must be taken to minimize inadvertent cell activation ex vivo. Here, we describe the use of autologous indium(111)-labelled eosinophils to measure eosinophil intravascular life-span and monitor their distribution and fate using gamma camera imaging in healthy non-atopic individuals.

Biomarkers of eosinophilic inflammation in asthma.

Eosinophils are mediators of allergic inflammation and are implicated in the pathogenesis of numerous conditions including asthma, parasitic infections, neoplasms, hyper-eosinophilic syndromes, vasculitic disorders, and organ-specific conditions. Assessing eosinophilic inflammation is therefore important in establishing a diagnosis, in monitoring and assessing response to treatment, and in testing novel therapeutics. Clinical markers of atopy and eosinophilic inflammation include indirect tests such as lung function, exhaled breath condensate analysis, fractional exhaled nitric oxide, serum immunoglobulin E levels and serum periostin. Direct measures, which quantify but do not anatomically localise inflammation include blood eosinophil counts, serum or plasma eosinophil cationic protein and sputum eosinophil levels. Cytology from bronchoalveolar lavage and histology from endobronchial and transbronchial biopsies are better at localising inflammation but are more invasive. Novel approaches using radiolabelled eosinophils with single-photon emission computed tomography, offer the prospect of non-invasive methods to localise eosinophilic inflammation.