ReviewApplication of proteomics to neutrophil biology
Introduction
As the primary effector cell of the innate immune system, polymorphonuclear leukocytes (neutrophils) provide the first line of defense against bacterial and fungal infections. The importance of this role is underscored by the high risk of infection in individuals with leukopenia. On the other hand, inappropriate or prolonged activation of neutrophils leads to tissue injury associated with a number of autoimmune and inflammatory diseases, including rheumatoid arthritis, Wegener's granulomatosus, systemic lupus erythematosus, multisystem organ failure associated with sepsis, and ischemia-reperfusion injury. In healthy individuals circulating neutrophils are poorly responsive to pro-inflammatory stimuli. Neutrophil activation results from a highly regulated series of phenotypic changes. A complex and interactive array of extracellular stimuli and intracellular signal transduction pathways regulate the transition of resting neutrophils to cells primed for enhanced responses and, finally, to fully activated cells. Resolution of inflammation is also an active process resulting in interruption of neutrophil activation, followed by neutrophil apoptosis and their engulfment by monocytes. As an introduction to the uses of proteomic technologies to study neutrophil biology, the processes leading to neutrophil participation in inflammation and to resolution of inflammation will be briefly described.
Section snippets
Neutrophil participation in inflammation
Neutrophils continually “sense” vascular endothelial cells for changes induced by underlying inflammation through a selectin-dependent process of loose adhesion called rolling. Release of pro-inflammatory cytokines from cells at a site of infection or tissue injury triggers vascular endothelial cells to increase expression of E-selectins, P-selectins, and intercellular adhesion molecules (ICAMs) and to generate another set of cytokines and chemokines. The increased selectin expression results
Neutrophil receptors and signal transduction pathways in activation and resolution of inflammation
As indicated above, the ability of neutrophils to respond to inflammatory stimuli in a coordinated manner requires plasma membrane expression of a large number of receptors. Toll-like receptors (TLRs) are a subfamily of pattern recognition receptors (PRRs) that initiate neutrophil responses by recognizing microbial products and damage-associated molecular pattern (DAMP) molecules released from injured cells [22]. Seven transmembrane-spanning G-protein coupled receptors (GPCRs) recognize
Application of proteomics to understanding neutrophil biology
Proteomic approaches can be grouped into three broad categories: expression, structural, and functional. The goal of expression proteomics is to define and quantify all proteins within a cell or subcellular organelle under normal, diseased, or treated conditions. Structural proteomics aims to determine the composition of protein complexes and to define protein–protein interactions. Functional proteomics aims to define the post-translational modifications of proteins that regulate their
Application of expression proteomics to neutrophils
Not all expressed genes are translated into proteins, highlighting the need to identify the protein composition of cells and subcellular organelles. The inconsistent correlation between mRNA and protein expression has proven true for neutrophils undergoing differentiation or during response to inflammatory stimuli [35], [36]. Piubelli et al. [37] performed a global analysis of rat neutrophil proteins using two-dimensional gel electrophoresis (2DE) to separate proteins from neutrophils isolated
Application of structural proteomics to neutrophils
Although transfection of cDNAs into neutrophils has been documented [58], [59], genetic manipulation of these terminally differentiated cells leading to high efficiency introduction of tagged proteins remains a challenge. Consequently, techniques to immunoprecipitate endogenous proteins have been developed to define the composition of protein complexes without altering the genetic composition of neutrophils. The applicability of immunoprecipitation of specific proteins from neutrophils to allow
Application of functional proteomics to neutrophils
Post-translational modifications are important regulators of individual protein functions. Given the sensitivity of current technology, most biologically relevant post-translational modifications, including phosphorylation, glycosylation, ubiquitination, methylation, acetylation, and isoprenylation, can be detected by mass spectrometry techniques. However, some post-translational modifications, including acetylation and citrullination, can impair detection of peptides by mass spectrometry due
Limitations
A number of limitations to the application of proteomics to neutrophils exist. A large number of proteases are contained in neutrophil granules. Release of some granules contents accompanies disruption of neutrophils for subcellular organelle isolation, making protease inactivation necessary to achieve accurate analysis by mass spectrometry. Studies of neutrophil subcellular organelles indicate that these organelles are complex and, thus, present problems related to the dynamic range of
Conclusions
Neutrophils play a major role in the initiation and resolution of the inflammatory response, and they clearly perform tasks in addition to killing microorganisms. Although once considered as terminally differentiated, static cells, neutrophils demonstrate significant transcriptional and translational activity. Understanding these changes at the protein level will advance understanding of neutrophil participation in health and disease. The promise of proteomics is that identifying all proteins
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MMP-12 polarizes neutrophil signalome towards an apoptotic signature
2022, Journal of ProteomicsCitation Excerpt :In MI wound healing, neutrophils undergo apoptosis starting at day 3 of MI in mice and are phagocytosed by macrophages [23]. Compared to necrosis, apoptosis subdues inflammation as it avoids release of danger associated molecular patterns (DAMPs), and further inactivates DAMPs such as high mobility group box protein 1 (HMGB1) [24,25]. Apoptotic cells are inactive but can decoy cytokines and scavenge receptors, aiding in inflammation resolution [26,27].
Biological Roles of Neutrophil-Derived Granule Proteins and Cytokines
2019, Trends in ImmunologyCitation Excerpt :Since neutrophils have been typically looked upon as terminally differentiated cells, proteomic studies on them have been rare, until recently. However, given changed views on neutrophil transcriptional activities, heterogeneity, and production, the particularities of neutrophils have been extensively studied by proteomics in recent years [13,14]. The proteome of neutrophils from various species has been investigated in depth from whole cells, as well as from various cellular compartments, including the plasma membrane [15,16], cytoskeleton [17], and granule subsets [18,19] (Figure 1, Key Figure).
Flavonoids as modulators of neutrophils’ oxidative burst: Structure-activity relationship
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2015, Systems Biology in Toxicology and Environmental HealthGM-CSF and TNFα modulate protein expression of human neutrophils visualized by fluorescence two-dimensional difference gel electrophoresis
2011, CytokineCitation Excerpt :An unbiased widely used tool to analyze the proteome of cells is 2-dimensional (2D) gel electrophoresis. Although only a limited number of papers describing neutrophil protein expression are published, the use of 2-D gel electrophoresis in neutrophil biology is increasing [14]. The neutrophil protein composition of the plasma membrane and secretory vesicles [15], neutrophil granules [16] or unstimulated neutrophils [17] have been analyzed by 2D gel electrophoresis.
All creatures great and small: Regulatory T cells in mice, humans, dogs and other domestic animal species
2011, International ImmunopharmacologyCitation Excerpt :Further work characterised a short-lived suppressor population – thought to be neutrophils – present in the peripheral blood of healthy dogs, mediating its effect in an indomethacin-independent manner by the release of a soluble factor [292]. These findings are harder to reconcile with the current literature, though recent work has suggested that neutrophils may indeed serve a regulatory role in some circumstances [293–295]. A study performed in the early 1990s described the inhibitory activity of spontaneous and induced suppressor lymphocyte populations – assumed to be T cells – in peripheral blood of dogs with atopic dermatitis [296].