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Neutrophil cannibalism – a back up when the macrophage clearance system is insufficient
Kristina Rydell-T?rm?nen, Lena Uller, Jonas S Erjef?lt
Respiratory Research , 2006, DOI: 10.1186/1465-9921-7-143
Abstract: Intranasal lipopolysaccharide administration was used to induce lung inflammation in mice. The animals were sacrificed at seven time points following administration, bronchoalveolar lavage was performed and tissue samples obtained. Electron microscopy and histochemistry was used to assess neutrophil phagocytosis.Electron microscopic studies revealed that phagocytosing neutrophils was common, at 24 h after LPS administration almost 50% of the total number of neutrophils contained phagosomes, and the engulfed material was mainly derived from other neutrophils. Histochemistry on bronchoalvolar lavage cells further showed phagocytosing neutrophils to be frequently occurring.Neutrophils are previously known to phagocytose invading pathogens and harmful particles. However, this study demonstrates that neutrophils are also able to engulf apoptotic neutrophils or cell debris resulting from secondary necrosis of neutrophils. Neutrophils may thereby contribute to clearance and resolution of inflammation, thus acting as a back up system in situations when the macrophage clearance system is insufficient and/or overwhelmed.Neutrophils are short lived immune cells who invade tissues in response to a variety of stimuli, for example viral and bacterial infections [1,2]. They are professional phagocytes and contribute to resolution of inflammation by removing infectious and inflammatory stimuli [1,2]. Apart from being present during acute infections, neutrophils are also found to a variable degree during airway diseases such as COPD, asthma and ARDS/ALI [3,4]. Neutrophils have a high turnover and are normally rapidly cleared by apoptosis, followed by macrophage phagocytosis [2,5]. During infection a large number of neutrophils are present in order to efficiently clear the infection, and studies have shown that ingestion of bacteria may delay neutrophil apoptosis [2], thereby causing very large number of cells accumulating in the same area. In such cases, the normally rapid clearance
Neutrophil elastase downmodulates native G-CSFR expression and granulocyte-macrophage colony formation
Melissa G Piper, Pam R Massullo, Megan Loveland, Lawrence J Druhan, Tamila L Kindwall-Keller, Jing Ai, Alexander Copelan, Belinda R Avalos
Journal of Inflammation , 2010, DOI: 10.1186/1476-9255-7-5
Abstract: Human peripheral blood PMN isolated from healthy donors were incubated with NE. Expression of the G-CSFR was analyzed by flow cytometry and western blot analyses. Detection of G-CSFR cleavage products from the culture supernatants was also performed. Human bone marrow mononuclear cells were also cultured in the presence or absence of NE to determine its effects on the proliferation of granulocyte-macrophage colony forming units (CFU-GM).Treatment of PMN with NE induced a time-dependent decrease in G-CSFR expression that correlated with its degradation and the appearance of proteolytic cleavage fragments in conditioned media. Immunoblot analysis confirmed the G-CSFR was cleaved at its amino-terminus. Treatment of progenitor cells with NE prior to culture inhibited the growth of granulocyte-macrophage colony forming units.These findings indicate that in addition to transcriptional controls and ligand-induced internalization, direct proteolytic cleavage of the G-CSFR by NE also downregulates G-CSFR expression and inhibits G-CSFR-mediated granulopoiesis in vitro. Our results suggest that NE negatively regulates granulopoiesis through a novel negative feedback loop.Granulocyte colony-stimulating factor (G-CSF) is the major regulator of granulopoiesis and supports the survival, proliferation, and maturation of myeloid progenitor cells along the neutrophil (PMN) lineage [1]. G-CSF also activates certain functions of mature PMN and stimulates hematopoietic stem cell mobilization [2-6]. The growth of neutrophilic granulocytes in vitro from progenitor cells committed to neutrophils and monocytes (CFU-GM) is absolutely dependent upon G-CSF and sigmoidally increases with increasing G-CSF concentrations [2,5,7,8]. A critical role for G-CSF in regulating granulopoiesis in vivo has been demonstrated in G-CSF null mice who have chronic neutropenia and severely impaired granulopoietic responses to infection [6].The biological activities of G-CSF are mediated by the G-CSFR receptor (
The Neutrophil's Eye-View: Inference and Visualisation of the Chemoattractant Field Driving Cell Chemotaxis In Vivo  [PDF]
Visakan Kadirkamanathan, Sean R. Anderson, Stephen A. Billings, Xiliang Zhang, Geoffrey R. Holmes, Constantino C. Reyes-Aldasoro, Philip M. Elks, Stephen A. Renshaw
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0035182
Abstract: As we begin to understand the signals that drive chemotaxis in vivo, it is becoming clear that there is a complex interplay of chemotactic factors, which changes over time as the inflammatory response evolves. New animal models such as transgenic lines of zebrafish, which are near transparent and where the neutrophils express a green fluorescent protein, have the potential to greatly increase our understanding of the chemotactic process under conditions of wounding and infection from video microscopy data. Measurement of the chemoattractants over space (and their evolution over time) is a key objective for understanding the signals driving neutrophil chemotaxis. However, it is not possible to measure and visualise the most important contributors to in vivo chemotaxis, and in fact the understanding of the main contributors at any particular time is incomplete. The key insight that we make in this investigation is that the neutrophils themselves are sensing the underlying field that is driving their action and we can use the observations of neutrophil movement to infer the hidden net chemoattractant field by use of a novel computational framework. We apply the methodology to multiple in vivo neutrophil recruitment data sets to demonstrate this new technique and find that the method provides consistent estimates of the chemoattractant field across the majority of experiments. The framework that we derive represents an important new methodology for cell biologists investigating the signalling processes driving cell chemotaxis, which we label the neutrophils eye-view of the chemoattractant field.
Prolonged, granulocyte–macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha
Greg Parsonage, Andrew Filer, Magdalena Bik, Debbie Hardie, Sian Lax, Katherine Howlett, Leigh D Church, Karim Raza, See-Heng Wong, Emily Trebilcock, Dagmar Scheel-Toellner, Mike Salmon, Janet M Lord, Christopher D Buckley
Arthritis Research & Therapy , 2008, DOI: 10.1186/ar2406
Abstract: IL-17-expressing cells in the rheumatoid synovium, and IL-17-expressing cells in the peripheral blood, and synovial fluid were examined by confocal microscopy and flow cytometry, respectively. Peripheral blood neutrophils were cocultured either with rheumatoid arthritis synovial fibroblasts (RASF) or with conditioned medium from RASF that had been pre-exposed to recombinant human IL-17, TNFα or a combination of the two cytokines. Neutrophils were harvested and stained with the vital mitochondrial dye 3,3'-dihexyloxacarbocyanine iodide before being enumerated by flow cytometry.TH17-expressing CD4+ cells were found to accumulate within rheumatoid synovial tissue and in rheumatoid arthritis synovial fluid. RASF treated with IL-17 and TNFα (RASFIL-17/TNF) effectively doubled the functional lifespan of neutrophils in coculture. This was entirely due to soluble factors secreted from the fibroblasts. Specific depletion of granulocyte–macrophage colony-stimulating factor from RASFIL-17/TNF-conditioned medium demonstrated that this cytokine accounted for approximately one-half of the neutrophil survival activity. Inhibition of phosphatidylinositol-3-kinase and NF-κB pathways showed a requirement for both signalling pathways in RASFIL-17/TNF-mediated neutrophil rescue.The increased number of neutrophils with an extended lifespan found in the rheumatoid synovial microenvironment is partly accounted for by IL-17 and TNFα activation of synovial fibroblasts. TH17-expressing T cells within the rheumatoid synovium are likely to contribute significantly to this effect.In established rheumatoid arthritis (RA), highly differentiated CD4+ T lymphocytes persist within synovial tissue, and are prevented from undergoing apoptosis by high local concentrations of type I interferons [1]. Simplistically, the preponderance of IFNγ-expressing T cells and the paucity of IL-4-expressing T cells, in situ and ex vivo, has led to the description of RA as an immune-mediated inflammatory disease that
Drift-Diffusion Analysis of Neutrophil Migration during Inflammation Resolution in a Zebrafish Model  [PDF]
Geoffrey R. Holmes,Giles Dixon,Sean R. Anderson,Constantino Carlos Reyes-Aldasoro,Philip M. Elks,Stephen A. Billings,Moira K. B. Whyte,Visakan Kadirkamanathan,Stephen A. Renshaw
Advances in Hematology , 2012, DOI: 10.1155/2012/792163
Abstract: Neutrophils must be removed from inflammatory sites for inflammation to resolve. Recent work in zebrafish has shown neutrophils can migrate away from inflammatory sites, as well as die in situ. The signals regulating the process of reverse migration are of considerable interest, but remain unknown. We wished to study the behaviour of neutrophils during reverse migration, to see whether they moved away from inflamed sites in a directed fashion in the same way as they are recruited or whether the inherent random component of their migration was enough to account for this behaviour. Using neutrophil-driven photoconvertible Kaede protein in transgenic zebrafish larvae, we were able to specifically label neutrophils at an inflammatory site generated by tailfin transection. The locations of these neutrophils over time were observed and fitted using regression methods with two separate models: pure-diffusion and drift-diffusion equations. While a model hypothesis test (the F-test) suggested that the datapoints could be fitted by the drift-diffusion model, implying a fugetaxis process, dynamic simulation of the models suggested that migration of neutrophils away from a wound is better described by a zero-drift, “diffusion” process. This has implications for understanding the mechanisms of reverse migration and, by extension, neutrophil retention at inflammatory sites. 1. Introduction The fate of neutrophils following completion of the inflammatory programme is of critical importance for the outcome of episodes of acute inflammation and can determine whether there is prompt healing of a wound or the development of chronic inflammation and tissue injury. Neutrophils recruited to sites of inflammation may leave the site or die in situ [1]. The most widely accepted mechanism of neutrophil disposal is the programmed cell death or apoptosis, of the neutrophil followed by macrophage uptake and clearance (reviewed in [2]). Recently, other routes have been proposed; neutrophils may move away from the inflamed site into the bloodstream (“reverse transmigration” [3]), by migration through other tissues (“retrograde chemotaxis” or “reverse migration” [4–6]), or be lost into the inflammatory exudate [7, 8]. Current understanding of the process of reverse migration is reviewed elsewhere [9]. The uncertainty as to the in vivo fates of individual cells relates in part to the difficulty in following individual cells during inflammation resolution in vivo. The transgenic zebrafish model is emerging as a key model for the study of vertebrate immunity [10] and allows direct
Implication of granulocyte-macrophage colony-stimulating factor induced neutrophil gelatinase-associated lipocalin in pathogenesis of rheumatoid arthritis revealed by proteome analysis
Masayoshi Katano, Kazuki Okamoto, Mitsumi Arito, Yuki Kawakami, Manae S Kurokawa, Naoya Suematsu, Sonoko Shimada, Hiroshi Nakamura, Yang Xiang, Kayo Masuko, Kusuki Nishioka, Kazuo Yudoh, Tomohiro Kato
Arthritis Research & Therapy , 2009, DOI: 10.1186/ar2587
Abstract: Neutrophils stimulated by GM-CSF were divided into four subcellular fractions: cytosol, membrane/organelle, nuclei, and cytoskeleton. Then, proteins were extracted from each fraction and digested by trypsin. The produced peptides were detected using matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-TOF MS).We detected 33 peptide peaks whose expression was upregulated by more than 2.5-fold in GM-CSF stimulated neutrophils and identified 11 proteins out of the 33 peptides using MALDI-TOF/TOF MS analysis and protein database searches. One of the identified proteins was neutrophil gelatinase-associated lipocalin (NGAL). We confirmed that the level of NGAL in SF was significantly higher in patients with RA than in those with osteoarthritis. We next addressed possible roles of the increased NGAL in RA. We analysed proteome alteration of synoviocytes from patients with RA by treatment with NGAL in vitro. We found that, out of the detected protein spots (approximately 3,600 protein spots), the intensity of 21 protein spots increased by more than 1.5-fold and the intensity of 10 protein spots decreased by less than 1 to 1.5-fold as a result of the NGAL treatment. Among the 21 increased protein spots, we identified 9 proteins including transitional endoplasmic reticulum ATPase (TERA), cathepsin D, and transglutaminase 2 (TG2), which increased to 4.8-fold, 1.5-fold and 1.6-fold, respectively. Two-dimensional electrophoresis followed by western blot analysis confirmed the upregulation of TERA by the NGAL treatment and, moreover, the western blot analysis showed that the NGAL treatment changed the protein spots caused by post-translational modification of TERA. Furthermore, NGAL cancelled out the proliferative effects of fibroblast growth factor (FGF)-2 and epidermal growth factor (EGF) on chondrocytes from a patient with RA and proliferative effect of FGF-2 on chondrosarcoma cells.Our results indicate that GM-CSF contributes to the pathogenesis
Deficiencies of the Lipid-Signaling Enzymes Phospholipase D1 and D2 Alter Cytoskeletal Organization, Macrophage Phagocytosis, and Cytokine-Stimulated Neutrophil Recruitment  [PDF]
Wahida H. Ali, Qin Chen, Kathleen E. Delgiorno, Wenjuan Su, Jason C. Hall, Tsunaki Hongu, Huasong Tian, Yasunori Kanaho, Gilbert Di Paolo, Howard C. Crawford, Michael A. Frohman
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0055325
Abstract: Cell migration and phagocytosis ensue from extracellular-initiated signaling cascades that orchestrate dynamic reorganization of the actin cytoskeleton. The reorganization is mediated by effector proteins recruited to the site of activity by locally-generated lipid second messengers. Phosphatidic acid (PA), a membrane phospholipid generated by multiple enzyme families including Phospholipase D (PLD), has been proposed to function in this role. Here, we show that macrophages prepared from mice lacking either of the classical PLD isoforms PLD1 or PLD2, or wild-type macrophages whose PLD activity has been pharmacologically inhibited, display isoform-specific actin cytoskeleton abnormalities that likely underlie decreases observed in phagocytic capacity. Unexpectedly, PA continued to be detected on the phagosome in the absence of either isoform and even when all PLD activity was eliminated. However, a disorganized phagocytic cup was observed as visualized by imaging PA, F-actin, Rac1, an organizer of the F-actin network, and DOCK2, a Rac1 activator, suggesting that PLD-mediated PA production during phagocytosis is specifically critical for the integrity of the process. The abnormal F-actin reorganization additionally impacted neutrophil migration and extravasation from the vasculature into interstitial tissues. Although both PLD1 and PLD2 were important in these processes, we also observed isoform-specific functions. PLD1-driven processes in particular were observed to be critical in transmigration of macrophages exiting the vasculature during immune responses such as those seen in acute pancreatitis or irritant-induced skin vascularization.
Neutrophil Reverse Migration Becomes Transparent with Zebrafish  [PDF]
Taylor W. Starnes,Anna Huttenlocher
Advances in Hematology , 2012, DOI: 10.1155/2012/398640
Abstract: The precise control of neutrophil-mediated inflammation is critical for both host defense and the prevention of immunopathology. In vivo imaging studies in zebrafish, and more recently in mice, have made the novel observation that neutrophils leave a site of inflammation through a process called neutrophil reverse migration. The application of advanced imaging techniques to the genetically tractable, optically transparent zebrafish larvae was critical for these advances. Still, the mechanisms underlying neutrophil reverse migration and its effects on the resolution or priming of immune responses remain unclear. Here, we review the current knowledge of neutrophil reverse migration, its potential roles in host immunity, and the live imaging tools that make zebrafish a valuable model for increasing our knowledge of neutrophil behavior in vivo. 1. Introduction “Certain of the lower animals, transparent enough to be observed alive, clearly show in their midst a host of small cells with moving extensions. In these animals the smallest lesion brings an accumulation of these elements at the point of damage. In small transparent larvae, it can easily be shown that the moving cells, reunited at the damage point do often close over foreign bodies [1].” Ilya Mechnikov, one of the fathers of immunology, spoke these words at his Nobel Prize lecture in 1908. More than one hundred years after his seminal studies using transparent starfish larvae to illuminate a role for phagocytosis in immunity, we are again exploiting the power of transparent larvae for research on the immune system. Studies of neutrophils in both humans and mammalian model systems have brought great advances in our knowledge of their functions; however, zebrafish, a small tropical fish with transparent larvae, have demonstrated that direct observation of neutrophils in live animals can provide important insights that would have otherwise faced significant technical challenges using mice. Neutrophils are the most abundant leukocytes in both humans and zebrafish, and they are critical for defending the host against microbial infection [2]. In response to wounding, infection, or other inflammatory stimuli, neutrophils are rapidly recruited to perform their well-known effector functions: degranulation, phagocytosis, production of reactive oxygen species (ROS), secretion of proinflammatory cytokines, and extrusion of neutrophil extracellular traps (NETs) [3, 4]. These responses are acknowledged to kill and sequester microorganisms at their site of entry and promote the activation of the adaptive immune
Heat Shock Modulates Neutrophil Motility in Zebrafish  [PDF]
Pui-ying Lam, Elizabeth A. Harvie, Anna Huttenlocher
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0084436
Abstract: Heat shock is a routine method used for inducible gene expression in animal models including zebrafish. Environmental temperature plays an important role in the immune system and infection progression of ectotherms. In this study, we analyzed the impact of short-term heat shock on neutrophil function using zebrafish (Danio rerio) as an animal model. Short-term heat shock decreased neutrophil recruitment to localized Streptococcus iniae infection and tail fin wounding. Heat shock also increased random neutrophil motility transiently and increased the number of circulating neutrophils. With the use of the translating ribosome affinity purification (TRAP) method for RNA isolation from specific cell types such as neutrophils, macrophages and epithelial cells, we found that heat shock induced the immediate expression of heat shock protein 70 (hsp70) and a prolonged expression of heat shock protein 27 (hsp27). Heat shock also induced cell stress as detected by the splicing of X-box binding protein 1 (xbp1) mRNA, a marker for endoplasmic reticulum (ER) stress. Exogenous expression of Hsp70, Hsp27 and spliced Xbp1 in neutrophils or epithelial cells did not reproduce the heat shock induced effects on neutrophil recruitment. The effect of heat shock on neutrophils is likely due to a combination of complex changes, including, but not limited to changes in gene expression. Our results indicate that routine heat shock can alter neutrophil function in zebrafish. The findings suggest that caution should be taken when employing a heat shock-dependent inducible system to study the innate immune response.
RobCoP: A Matlab Package for Robust CoPlot Analysis  [PDF]
Yasemin Kayhan Atilgan, Erdinc Levent Atilgan
Open Journal of Statistics (OJS) , 2017, DOI: 10.4236/ojs.2017.71003
Abstract: The graphical representation method, Robust CoPlot, is a robust variant of the classical CoPlot method. CoPlot is an adaptation of multidimensional scaling (MDS), and is a practical tool for visual inspection and rich interpretation of multivariate data. CoPlot enables presentation of a multidimensional dataset in a two dimensions, in a manner that relations between both variables and observations to be analyzed together. It has also been used as a supplemental tool to cluster analysis, data envelopment analysis (DEA) and outlier detection methods in the literature. However, this method is very sensitive to outliers. When a multidimensional dataset contains outliers, this can lead to undesirable consequences such as the inaccurate representation of the variables. The motivation is to produce Robust CoPlot that is not unduly affected by outliers. In this study, we have presented a new MATLAB package RobCoP for generating robust graphical representation of a multidimensional dataset. This study serves a useful purpose for researchers studying the implementation of Robust CoPlot method by providing a description of the software package RobCoP; it also offers some limited information on the Robust CoPlot analysis itself. The package presented here has enough flexibility to allow a user to select an MDS type and vector correlation method to produce either classical or Robust CoPlot results.
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