Elsevier

Journal of Proteomics

Volume 73, Issue 3, 3 January 2010, Pages 552-561
Journal of Proteomics

Review
Application of proteomics to neutrophil biology

https://doi.org/10.1016/j.jprot.2009.06.013Get rights and content

Abstract

Polymorphonuclear leukocytes or neutrophils are a primary effector cell of the innate immune system and contribute to the development of adaptive immunity. Neutrophils participate in both the initiation and resolution of inflammatory responses through a series of highly coordinated molecular and phenotypic changes. To accomplish these changes, neutrophils express numerous receptors and use multiple overlapping and redundant signal transduction pathways. Dysregulation of the activation or resolution pathways plays a role in a number of human diseases. A comprehensive understanding of the regulation of neutrophil responses can be provided by high throughput proteomic technologies and sophisticated computational analysis. The first steps in the application of proteomics to understanding neutrophil biology have been taken. Here we review the application of expression, structural, and functional proteomic studies to neutrophils. Although defining the complex molecular events associated with neutrophil activation is in the early stages, the data generated to date suggest that proteomic technologies will dramatically enhance our understanding of 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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