Neda Farahi

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.

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.

Use of 111-Indium-labelled autologous eosinophils to establish the in vivo kinetics of human eosinophils in healthy subjects

Eosinophils are the major cellular effectors of allergic inflammation and represent an important therapeutic target. Although the genesis and activation of eosinophils have been extensively explored, little is known about their intravascular kinetics or physiological fate. This study was designed to determine the intravascular life span of eosinophils, their partitioning between circulating and marginated pools, and sites of disposal in healthy persons. Using autologous, minimally manipulated 111-Indium-labeled leukocytes with blood sampling, we measured the eosinophil intravascular residence time as 25.2 hours (compared with 10.3 hours for neutrophils) and demonstrated a substantial marginated eosinophil pool. γ camera imaging studies using purified eosinophils demonstrated initial retention in the lungs, with early redistribution to the liver and spleen, and evidence of recirculation from a hepatic pool. This work provides the first in vivo measurements of eosinophil kinetics in healthy volunteers and shows that 111-Indium-labeled eosinophils can be used to monitor the fate of eosinophils noninvasively.

Eotaxin-1/CC Chemokine Ligand 11: A Novel Eosinophil Survival Factor Secreted by Human Pulmonary Artery Endothelial Cells

Airway eosinophilia plays a major role in the pathogenesis of asthma with the inhibition of apoptosis by GM-CSF and IL-5 proposed as a mechanism underlying prolonged eosinophil survival. In vivo and ex vivo studies have indicated the capacity of interventions that drive human eosinophil apoptosis to promote the resolution of inflammation. Far less is known about the impact of transendothelial migration on eosinophil survival, in particular, the capacity of endothelial cell-derived factors to contribute toward the apoptosis-resistant phenotype characteristic of airway-resident eosinophils. We examined the effects of conditioned medium from human pulmonary artery endothelial cells (HPAEC-CM) on eosinophil apoptosis in vitro. HPAEC-CM inhibited eosinophil, but not neutrophil apoptosis. This effect was specific to HPAECs and comparable in efficacy to the survival effects of GM-CSF and IL-5. The HPAEC survival factor was shown, on the basis of GM-CSF, IL-5, and IL-3 detection assays, Ab neutralization, and sensitivity to PI3K inhibition, to be clearly discrete from these factors. Gel filtration of HPAEC-CM revealed a peak of eosinophil survival activity at 8–12 kDa, and PCR confirmed the presence of mRNA for CCL5, CCL11, CCL24, CCL26, and CCL27 in the HPAECs. The CCR3 antagonist GW782415 caused a major inhibition of the HPAEC-CM-induced survival effect, and Ab neutralization of individual CCR3 chemokines revealed CCL11 as the major survival factor present in the HPAEC-CM. Furthermore, chemokine Ab arrays demonstrated up-regulation of CCL11 in HPAEC-CM. These data demonstrate the capacity of HPAECs to generate CCR3 agonists and the ability of CCL11 to inhibit human eosinophil apoptosis.

Characterization of the survival effect of tumour necrosis factor-alpha in human neutrophils

Granulocyte apoptosis has been proposed as a fundamental, injury-limiting granulocyte-clearance mechanism. As such, inhibition of this process may prevent the resolution of inflammation. Our previous studies have shown that TNFalpha (tumour necrosis factor-alpha) has a bi-modal influence on the rate of constitutive neutrophil apoptosis in vitro, causing early acceleration and late inhibition of this process. The pro-apoptotic effect is uniquely TNFR1 (TNF receptor 1) and TNFR2-dependent and the latter survival process is mediated via phosphoinositide 3-kinase and NF-kappaB (nuclear factor-kappaB) activation. In the present study, we show that, in contrast with GM-CSF (granulocyte/macrophage colony-stimulating factor), the delayed addition (i.e. at 6 h) of TNFalpha increases its survival effect despite substantial loss of neutrophil TNFR1 and TNFR2 at that time. This paradox was resolved using PBMC (peripheral blood mononuclear cell)-deplete and 5% PBMC-replete neutrophil cultures, where the enhanced survival effect observed after delayed TNFalpha addition was shown to be PBMC-dependent. TNFR2-blocking antibodies had no effect on the late survival effect of TNFalpha, implying a TNFR1-dependent process. Finally, I-kappaBalpha (inhibitory kappaB-alpha) and NF-kappaB time-course studies demonstrated that the survival effects of both GM-CSF and TNFalpha could be explained by maintenance of functional NF-kappaB.

Effects of the cyclin-dependent kinase inhibitor R-roscovitine on eosinophil survival and clearance

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.

Granulocyte/macrophage colony-stimulating factor causes a paradoxical increase in the BH3-only pro-apoptotic protein Bim in human neutrophils.

Neutrophil apoptosis is essential for the resolution of inflammation but is delayed by several inflammatory mediators. In such terminally differentiated cells it has been uncertain whether these agents can inhibit apoptosis through transcriptional regulation of anti-death (Bcl-X(L), Mcl-1, Bcl2A1) or BH3-only (Bim, Bid, Puma) Bcl2-family proteins. We report that granulocyte/macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF)-α prevent the normal time-dependent loss of Mcl-1 and Bcl2A1 in neutrophils, and we demonstrate that they cause an NF-κB-dependent increase in Bcl-X(L) transcription/translation. We show that GM-CSF and TNF-α increase and/or maintain mRNA levels for the pro-apoptotic BH3-only protein Bid and that GM-CSF has a similar NF-κB-dependent effect on Bim transcription and BimEL expression. The in-vivo relevance of these findings was indicated by demonstrating that GM-CSF is the dominant neutrophil survival factor in lung lavage from patients with ventilator-associated pneumonia, confirming an increase in lung neutrophil Bim mRNA. Finally GM-CSF caused mitochondrial location of Bim and a switch in phenotype to a cell that displays accelerated caspase-9-dependent apoptosis. This study demonstrates the capacity of neutrophil survival agents to induce a paradoxical increase in the pro-apoptotic proteins Bid and Bim and suggests that this may function to facilitate rapid apoptosis at the termination of the inflammatory cycle.

Interleukin-5 inhibits glucocorticoid-mediated apoptosis in human eosinophils

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 …

Effects of tocilizumab on neutrophil function and kinetics

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.

Use of technetium-99m-labeled eosinophils to detect active eosinophilic inflammation in humans.

To the Editor: The diagnosis of focal eosinophilic inflammation is notoriously difficult. Here we describe a novel technique using technetium-99m–labeled autologous eosinophils to image eosinophilic vasculitis affecting the lung and skin in a patient with granulomatosis with polyangiitis. Our patient, a 48-year-old male, presented with cough, purulent sputum, hemoptysis, lethargy, and painful ulcers affecting his hands and feet. His thoracic computed tomography (CT) showed bilateral upper zone nodules with cavity formation. Previously he had experienced recurrent anterior uveitis and small joint polyarthropathy. His medications were hydroxychloroquine and etodolac, which he took intermittently. Examination revealed periungual erythema and multiple deep ulcers affecting his fingers and right ankle (Figure 1A). His blood eosinophil count was 0.76 × 109/L, with normal renal and liver function and normal antinuclear antibody and rheumatoid factor. His antineutrophil cytoplasmic antibody (ANCA) immunofluorescence was positive with a (PR3) ANCA of 25 IU/ml (normal range < 0.2 IU/ml). He also had evidence of IgG4-related disease associated with his ANCA-positive vasculitis with a serum IgG4 of 4.1 g/L. Surgical lung biopsy revealed dense fibrosis, with scattered noncaseating granulomas, heavy eosinophil infiltration, a medium-small vessel vasculitis, and IgG4-staining plasma cells. Similar findings were observed in the skin (Figure 1B). Figure 1. Technetium-99m–labeled eosinophil scanning in granulomatosis with polyangiitis. (A) Photograph of vasculitic ulcers affecting the patient’s right ankle. (B) The corresponding histology (×20 magnification) shows active vasculitis ... Technetium-99m–labeled eosinophils (98.5% pure) were prepared using clinical grade anti-CD16 immuno-magnetic beads as described (1) and 16 × 106 cells (187 MBq) reinjected. Dynamic images were taken for 40 minutes (1) followed by single-photon emission CT (SPECT-CT) images at 45 minutes and 2, 4, and 6 hours. His legs were imaged using a γ camera (Figure 1C). The intravascular residence time (1.5 h) of the technetium-99m–labeled eosinophils was much shorter than that observed in healthy subjects (1) with only 4.9% of the injected cells remaining in the circulation at 30 minutes. This compares to an intravascular residence time of indium-111–labeled eosinophils in healthy volunteers of 28.1 ± 1.7 hours (mean ± SEM), with 14.5 ± 2.5% of radiolabeled cells remaining in the circulation at 30 minutes (1). Although the technetium-99m–labeled eosinophils accumulated as expected in the liver, spleen, and bone marrow (Figure 1D), SPECT-CT revealed additional uptake in some but not all of his lung nodules (Figures 1E–1G). Although radiolabeled leukocyte scanning is used routinely for localizing occult infection, this approach has not been used previously to demonstrate focal eosinophilic inflammation. Advances in immuno-magnetic bead technology have permitted the isolation of ultra-pure nonactivated eosinophils from blood, which are amenable to radiolabeling and behave physiologically when reinjected in healthy subjects (1). Alternative, indirect methods of detecting eosinophilic inflammation in humans, such as exhaled nitric oxide and analysis of breath condensates, are nonspecific and can only assess the inflammatory burden in the respiratory tract. In contrast, technetium-99m–labeled eosinophils plus SPECT-CT can localize focal eosinophilic inflammation directly in vivo. Furthermore, this technique is less invasive than performing bronchoscopy and transbronchial biopsy, which may be contraindicated in some patients and carry significant risk. This novel approach therefore offers the prospect of a noninvasive method to aid in the diagnosis, localization, and assessment of disease activity.