development-identification-novel-subpopulation-human-neutrophil

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes <em>In Vitro</em>

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

Technion-Israel Insitute of Technology

The structure of Escherichia coli core RNA polymerase (RNAP) was determined by cryo-electron microscopy and image processing of helical crystals to a nominal resolution of 15 A. Because of the high sequence conservation between the core RNAP subunits, we were able to interpret the E. coli structure in relation to the high-resolution x-ray structure of Thermus aquaticus core RNAP. A very large conformational change of the T. aquaticus RNAP x-ray structure, corresponding to opening of the main DNA/RNA channel by nearly 25 A, was required to fit the E. coli map. This finding reveals, at least partially, the range of conformational flexibility of the RNAP, which is likely to have functional implications for the initiation of transcription, where the DNA template must be loaded into the channel.

Conformational flexibility of bacterial RNA polymerase.

The objective of the present study was to compare the effects of high stimulus rate and click position in the train on monaurally and binaurally evoked activities in the human auditory brainstem and suggest their possible physiological mechanism.

The cumulative effect of high click rate on monaural and binaural processing in the human auditory brainstem.

To study, in humans, the effects of sound source azimuth and elevation on primary auditory cortex binaural activity associated with sound localization.

Electrophysiological correlates of azimuth and elevation cues for sound localization in human middle latency auditory evoked potentials.

Our objective was to study the effects of sound source direction and elevation on human brainstem electrical activity associated with sound localization. The subjects comprised 15 normal-hearing and symmetrically hearing adults Auditory brainstem evoked potentials (ABEPs) were recorded from three channels, in response to alternating-polarity clicks, presented at a rate of 21.1/s, at nine virtual spatial locations with different direction and elevation attributes Equivalent dipoles of the binaural interaction components (BICs) of ABEPs were derived by subtracting the response to binaural clicks at each spatial location from the algebraic sum of monaural responses to stimulation of each ear in turn. The BICs included two major components corresponding in latency to the vertex-neck-recorded components V and VI of ABEP. A significant decrease of the first BIC's equivalent dipole magnitude was observed for clicks in the horizontal-frontal position (no elevation) in the midsagittal plane, and for clicks in the left-horizontal (no elevation) and right diagonally above the head (medium elevation) positions in the coronal plane, compared to clicks positioned directly above the head. Significant effects on equivalent dipole latencies of this component were found for front-back positions in the midsagittal plane and left-right positions in the coronal plane, compared to clicks positioned directly above the head. The most remarkable finding was a significant change in equivalent dipole orientations across stimulus conditions. We conclude that the changes in BIC equivalent dipole latency, amplitude and orientation across stimulus conditions reflect differences in the distribution of binaural pontine activity evoked by sounds in different spatial locations.

Electrophysiologic correlates of direction and elevation cues for sound localization in the human brainstem.

To study effects of forward masking and rapid stimulation on human monaurally- and binaurally-evoked brainstem potentials and suggest their relation to synaptic fatigue and recovery and to neuronal action potential refractoriness.

The combined effects of forward masking by noise and high click rate on monaural and binaural human auditory nerve and brainstem potentials.

Obstructive sleep apnea (OSA), characterized by intermittent hypoxia/reoxygenation (IHR), is associated with atherosclerosis. Polymorphonuclear leukocytes (PMNs) are implicated in atherogenesis by producing oxidizing radicals and proteolytic enzymes during PMN-endothelium interactions. PMN apoptosis is a fundamental, injury-limiting mechanism, which prevents their destructive potential.

Delayed neutrophil apoptosis in patients with sleep apnea.

Obstructive sleep apnea (OSA), a highly prevalent breathing disorder in sleep, characterized by intermittent and recurrent pauses in respiration, has emerged as an independent risk factor for cardiovascular morbidity and mortality. Accumulated evidence implicates Leukocyte-endothelial cell activation and adhesion as critical components that induce inflammation and injury to the vasculature resulting in the development of cardiovascular complications. Similar cellular interactions were described in conditions of ischemia/reperfusion, and various components of the metabolic syndrome as hypercholesterolemia and hypertension. The hallmark of sleep apnea--the multiple cycles of hypoxia/reoxygenation--promote oxidative stress and inflammation. These facilitate increased interactions of blood cells with endothelial cells, resulting in endothelial cell injury and dysfunction. Such events can promote atherosclerosis and the development of cardiovascular morbidities in OSA. However, inter-individual differences in response to intermittent hypoxia and activation of anti-inflammatory cytokine profiles in T lymphocytes can serve as protective mechanisms.

Biology of peripheral blood cells in obstructive sleep apnea--the tip of the iceberg.

Apoptosis of polymorphonuclear cells (PMNs) is a fundamental mechanism to halt inflammation. It limits the lifespan of PMNs and thereby decreases tissue injury. In PMNs, unlike in other cells, hypoxia profoundly inhibits apoptosis. However, most studies investigating hypoxic effects on the functioning of PMN focus on acute or chronic sustained hypoxia. Thus, the mechanisms by which intermittent hypoxia (IH) affects PMN apoptosis are not known. Flow cytometry and Western blotting were used to evaluate mechanisms of constitutive and TNF-α-mediated PMN apoptosis in IH. The levels of NF-κB, p38 mitogen-activated protein kinase (MAPK), TNF receptor-2 (TNFR-2), intracellular IL-8 and its surface receptor CXCR2, were determined. Specific NF-κB (gliotoxin and parthenolide) and p38MAPK (SB202190) inhibitors were also used. TNF-α-mediated PMN apoptosis was concentration-dependent; low concentration increased PMN survival, whereas higher concentrations accelerated apoptosis. However, at all TNF-α concentrations, PMN survival was higher after four IH cycles than in normoxia. However, increasing the IH cycles to six abolished the pro-apoptotic/anti-apoptotic effects of TNF-α. Also, IH increased TNRF2 expression, nuclear NF-κB translocation, p38MAPK phosphorylation, and expression of IL-8 and CXCR2. The NF-κB inhibitors gliotoxin and parthenolide increased apoptosis and decreased IL-8 and CXCR2 expression. Also, the p38MAPK inhibitor SB202190 increased apoptosis and decreased IL-8 expression but had no effect on CXCR2 expression. Collectively, these findings provide insights into the mechanisms that prolong PMN survival after IH exposure and demonstrate the essential role played by NF-κB, the p38MAPK signaling pathway, and downstream genes in this process.

Molecular pathways of spontaneous and TNF-{alpha}-mediated neutrophil apoptosis under intermittent hypoxia.

Fibronectin (FN) is an extracellular matrix protein promoting cell proliferation, adhesion, and survival and is localized in the intimal layer of normal and atherosclerotic blood vessels. Dendritic cells (DCs) are potent antigen-presenting cells located in healthy and diseased intima, and thus may predispose arteries to atherosclerosis. Besides their pro-atherogenic activities DCs can promote neovascularization, by releasing pro-angiogenic mediators and/or by trans-differentiating into endothelial-like cells. Here, we investigated changes in morphology, function and angiogenic properties of monocyte-derived immature DCs (Mo-iDCs) after a short-term FN treatment and some of the signaling pathways involved in these processes. The cells were analyzed by time-lapse, confocal microscopy and flow cytometry. Within 90 min of re-plating, FN induced a swift morphologic transformation of most round iDCs into spindle-shaped iDCs (sp-iDCs). This was characterized by redistribution of mitochondria into dendritic spindles, decreased CD1c, and increased thrombomodulin (CD141) expression. Functionally, sp-iDCs acquired Ulex-europaeus-agglutinin-1 lectin binding, phagocytosis was decreased and intracellular (nuclear and cytosolic) vascular endothelial growth factor (VEGF) was increased. FN also induced ERK1/2 phosphorylation in round-iDCs, and p38MAPK phosphorylation in sp-iDCs. Inhibiting p38MAPK, but not ERK1/2, restrained the FN-induced transformation into sp-iDCs. Furthermore, FN-treatment of Mo-iDCs induced a paracrine angiogenic effect on endothelial tube formation, which was abolished by inhibiting ERK1/2 or VEGF. Inhibiting p38MAPK had no effect on endothelial tube formation. By contrast, in laminin-treated Mo-iDCs, which had round-shaped morphology, CD1c and CD141 expression was similar to control untreated cells, but intracellular VEGF levels were higher, and endothelial tube formation was an individual trait. We conclude that a short-term FN treatment induced angiogenic intracrine and paracrine properties in Mo-iDCs. This may act as an immediate protective mechanism to maintain vascular homeostasis. Moreover, inducing sp-iDCs by short term FN-treatment or ERK1/2 modulation might be considered as new approaches for regulating angiogenesis through the production/inhibition of pro-angiogenic mediators. Collectively, these findings may support a role for FN and Mo-iDCs in vascular function and angiogenesis.

Short-term fibronectin treatment induces endothelial-like and angiogenic properties in monocyte-derived immature dendritic cells: involvement of intracellular VEGF and MAPK regulation.

Prolonged neutrophil survival is evident in various cardiovascular and respiratory morbidities, in hypoxic conditions in-vitro and in patients with obstructive sleep apnea (OSA) characterized by nightly intermittent hypoxia (IH). This may lead to persistent inflammation, tissue injury and dysfunction. We therefore investigated by a translational approach the potential contribution of the intrinsic stress-induced mitochondrial pathway in extending neutrophil survival under IH conditions. Thus, neutrophils of healthy individuals treated with IH in-vitro and neutrophils of OSA patients undergoing nightly IH episodes in-vivo were investigated. Specifically, the balance between pro-apoptotic Bax and anti-apoptotic Mcl-1 protein expression, and the potential involvement of p38MAPK and ERK1/2 signaling pathways in the control of Mcl-1 expression were investigated.

Bax/Mcl-1 balance affects neutrophil survival in intermittent hypoxia and obstructive sleep apnea: effects of p38MAPK and ERK1/2 signaling.

We tested the hypothesis that in long-term culture conditions, some neutrophils remain viable and participate in debris clearance, and autophagy is involved in their prolonged survival. Neutrophils, classified as professional phagocytes, have the shortest half-life among leukocytes and are constitutively committed to apoptosis. Apoptotic neutrophils are actively removed by Mφ/DCs. However, early and acute inflammatory infiltrates primarily consist of neutrophils. Recently, neutrophils were suggested to facilitate debris clearance at inflammatory sites when the Mφ/DC system is insufficient. Here, purified CD15(+)/CD66b(+)/CD63(+) neutrophils were followed up to 7 days in culture using light, time-lapse, and confocal microscopy. After 3 days in culture, Annexin-V(-)/LC3B(+) large vacuolated cells, engulfing cellular residues, were noted among apoptotic neutrophils and cell debris. Thereafter, these cells were vastly enlarged and exhibited a neutrophilic phenotype (CD15(+)/CD63(+)/MPO(+)/CD66b(+)), phagocytosis, and oxidative burst activity. They also expressed CD68 scavenger receptors and internalized oxLDL. But, unlike in fresh neutrophils or cultured monocytes, oxLDL treatment increased their ROS production. Additionally, these phagocytes contained LC3B-coated vacuoles and LC3B aggregates, indicating the activation of autophagy. An intensive LC3B accumulation was also noted during oxLDL internalization. Importantly, the inhibition of autophagy by 3-MA or BafA1 prevented their development. In conclusion, the internalization of neutrophil remnants may induce activation of autophagic mechanisms in some neutrophil subsets or precursors. This may lead to cell adaptation and survival, resulting in their transformation into long-lived Gφ and potentially suggesting their involvement in inflammatory/anti-inflammatory processes.

The development of giant phagocytes in long-term neutrophil cultures.

Previously we identified, for the first time, a new small-size subset of neutrophil-derived giant phagocytes (Gϕ) which spontaneously develop in vitro without additional growth factors or cytokines. Gϕ are CD66b(+)/CD63(+)/MPO(+)/LC3B(+) and are characterized by extended lifespan, large phagolysosomes, active phagocytosis, and reactive oxygen species (ROS) production, and autophagy largely controls their formation. Hypoxia, and particularly hypoxia/reoxygenation, is a prominent feature of many pathological processes. Herein we investigated Gϕ formation by applying various hypoxic conditions. Chronic intermittent hypoxia (IH) (29 cycles/day for 5 days) completely abolished Gϕ formation, while acute IH had dose-dependent effects. Exposure to 24 h (56 IH cycles) decreased their size, yield, phagocytic ability, autophagy, mitophagy, and gp91-phox/p22-phox expression, whereas under 24 h sustained hypoxia (SH) the size and expression of LC3B and gp91-phox/p22-phox resembled Gϕ formed in normoxia. Diphenyl iodide (DPI), a NADPH oxidase inhibitor, as well as the PI3K/Akt and autophagy inhibitor LY294002 abolished Gϕ formation at all oxygen conditions. However, the potent antioxidant, N-acetylcysteine (NAC) abrogated the effects of IH by inducing large CD66b(+)/LC3B(+) Gϕ and increased both NADPH oxidase expression and phagocytosis. These findings suggest that NADPH oxidase, autophagy, and the PI3K/Akt pathway are involved in Gϕ development.

Intermittent Hypoxia Affects the Spontaneous Differentiation In Vitro of Human Neutrophils into Long-Lived Giant Phagocytes.

Few existing studies have dealt with cytogenetics in trematodes, largely due to the attendant technical difficulty of chromosome preparation. We performed a comparative analysis of chromosomes in five opistorchiid species, including Opisthorchis felineus Rivolta, 1884, Opisthorchis viverrini Poirier, 1886, Clonorchis sinensis Cobbold, 1875, Metorchis xanthosomus Creplin 1846, and Metorchis bilis (Braun, 1790) Odening, 1962. For some of these species, no detailed morphometric description of their karyotypes has yet been published; for the karyotype of Metorchis bilis this is the first-ever description. We found that opisthorchiids, like other trematodes, are characterized by karyotypic conservatism (N=6-7) and karyotype asymmetry, although comparison of chromosome morphometric traits did reveal differences between the karyotypes of the species. Moreover, to address certain a methodological issue in trematode chromosome preparation, we analyzed how the source of chromosomal material (partenitae or mature flukes) and the chromosome preparation techniques used (air-drying and cell suspension methods) affected chromosome spreading and size, concluding that the most reliable comparative method involves comparing relative parameters (relative length, arm ratio, centromeric index) of chromosomes prepared using the same technique.

Andrey Polyakov

The Lloyd Rigler Sleep Apnea Research Laboratory,
Unit of Anatomy and Cell Biology,
The Ruth and Bruce Rappaport Faculty of Medicine,
The Lloyd Rigler Sleep Apnea Research Laboratory, Unit of Anatomy and Cell Biology, The Ruth and Bruce Rappaport Faculty of Medicine

Technion-Israel Insitute of Technology

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro