Neutrophils are the most abundant circulating and first-responding innate myeloid cells and have so far been underestimated in the context of multiple sclerosis (MS). MS is the most frequent, immune-mediated, inflammatory disease of the central nervous system. MS is treatable but not curable and its cause(s) and pathogenesis remain elusive. The involvement of neutrophils in MS pathogenesis has been suggested by the use of preclinical animal disease models, as well as on the basis of patient sample analysis. In this review, we provide an overview of the possible mechanisms and functions by which neutrophils may contribute to the development and pathology of MS. Neutrophils display a broad variety of effector functions enabling disease pathogenesis, including (1) the release of inflammatory mediators and enzymes, such as interleukin-1β, myeloperoxidase and various proteinases, (2) destruction and phagocytosis of myelin (as debris), (3) release of neutrophil extracellular traps, (4) production of reactive oxygen species, (5) breakdown of the blood–brain barrier and (6) generation and presentation of autoantigens. An important question relates to the issue of whether neutrophils exhibit a predominantly proinflammatory function or are also implicated in the resolution of chronic inflammatory responses in MS.

Neutrophils are the most abundant leukocytes in human blood and the first cells responding to infection and injury. Due to their limited ex vivo lifespan and the impossibility to cryopreserve or expand them in vitro, neutrophils need to be purified from fresh blood for immediate use in experiments. Importantly, neutrophil purification methods may artificially modify the phenotype and functional characteristics of the isolated cells. The aim of this study was to expose the effects of ‘classical’ density-gradient purification versus the more expensive but faster immunomagnetic isolation on neutrophil phenotype and functionality. We found that in the absence of inflammatory stimuli, density-gradient-derived neutrophils showed increased polarization responses as well as enhanced release of reactive oxygen species (ROS), neutrophil extracellular traps (NETs) and granular proteins compared to cells derived from immunomagnetic isolation, which yields mostly quiescent neutrophils. Upon exposure to pro-inflammatory mediators, immunomagnetic isolation-derived neutrophils were significantly more responsive in polarization, ROS production, phagocytosis, NETosis and degranulation assays, in comparison to density-gradient-derived cells. We found no difference in chemotactic response in Multiscreen and under-agarose migration assays, but Boyden assays showed reduced chemotaxis of immunomagnetic isolation-derived neutrophils. Finally, we confirmed that density-gradient purification induces artificial activation of neutrophils, evidenced by e.g. higher expression of CD66b, formyl peptide receptor 1 (FPR1) and CD35, and the appearance of a separate neutrophil population expressing surface molecules atypical for neutrophils (e.g. CXCR3, MHC-II and CD14). Based on these results, we recommend using immunomagnetic separation of neutrophils for studying neutrophil polarization, phagocytosis, ROS production, degranulation and NETosis, whereas for Boyden chemotaxis assays, the density-gradient purification is more suitable.

Infection, sterile injury, and chronic inflammation trigger the acute phase response in order to re-establish homeostasis. This response includes production of positive acute phase proteins in the liver, such as members of the serum amyloid A (SAA) family. In humans the major acute phase SAAs comprise a group of closely related variants of SAA1 and SAA2. SAA1 was proven to be chemotactic for several leukocyte subtypes through activation of the G protein-coupled receptor FPRL1/FPR2. Several other biological activities of SAA1, such as cytokine induction, reported to be mediated via TLRs, have been debated recently. Especially commercial SAA1, recombinantly produced in Escherichia coli, was found to be contaminated with bacterial products confounding biological assays performed with this rSAA1. We purified rSAA1 by RP-HPLC to homogeneity, removing contaminants such as lipopolysaccharides, lipoproteins and formylated peptides, and re-assessed several biological activities attributed to SAA1 (chemotaxis, cytokine induction, MMP-9 release, ROS generation, and macrophage differentiation). The homogeneous rSAA1 (hrSAA1) lacked most cellactivating properties, but its leukocyte-recruiting capacity in vivo and it's in vitro synergy with other leukocyte attractants remained preserved. Furthermore, hrSAA1 maintained the ability to promote monocyte survival. This indicates that pure hrSAA1 retains its potential to activate FPR2, whereas TLR-mediated effects seem to be related to traces of bacterial TLR ligands in the E. coli-produced human rSAA1.

Background Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by recurrent airway infection and inflammation. There is no cure for PCD and to date there are no specific treatments available. Neutrophils are a crucial part of the immune system and are known to be dysfunctional in many inflammatory diseases. So far, the role of the neutrophils in PCD airways is largely unknown. The purpose of this study was to investigate the phenotype and function of airway neutrophils in PCD, and compare them to blood neutrophils. Methods Paired peripheral blood and spontaneously expectorated sputum samples from patients with PCD (n = 32) and a control group of patients with non-PCD, non-cystic fibrosis bronchiectasis (n = 5) were collected. The expression of neutrophil-specific surface receptors was determined by flow cytometry. Neutrophil function was assessed by measuring the extent of actin polymerization, production of reactive oxygen species (ROS) and release of neutrophil extracellular traps (NETs) in response to activating stimuli. Results Sputum neutrophils displayed a highly activated phenotype and were unresponsive to stimuli that would normally induce ROS production, actin polymerization and the expulsion of NETs. In addition, PCD sputum displayed high activity of neutrophil elastase, and impaired the efferocytosis by healthy donor macrophages. Conclusions Sputum neutrophils in PCD are dysfunctional and likely contribute to ongoing inflammation in PCD airways. Further research should focus on anti-inflammatory therapies and stimulation of efferocytosis as a strategy to treat PCD.

The serum amyloid A (SAA) gene family is highly conserved and encodes acute phase proteins that are upregulated in response to inflammatory triggers. Over the years, a considerable amount of literature has been published attributing a wide range of biological effects to SAAs such as leukocyte recruitment, cytokine and chemokine expression and induction of matrix metalloproteinases. Furthermore, SAAs have also been linked to protumorigenic, proatherogenic and anti-inflammatory effects. Here, we investigated the biological effects conveyed by murine SAA3 (mu rSAA3) recombinantly expressed in Escherichia coli. We observed the upregulation of a number of chemokines including CCL2, CCL3, CXCL1, CXCL2, CXCL6 or CXCL8 following stimulation of monocytic, fibroblastoid and peritoneal cells with mu rSAA3. Furthermore, this SAA variant displayed potent in vivo recruitment of neutrophils through the activation of TLR4. However, a major problem associated with proteins derived from recombinant expression in bacteria is potential contamination with various bacterial products, such as lipopolysaccharide, lipoproteins and formylated peptides. This is of particular relevance in the case of SAA as there currently exists a discrepancy in biological activity between SAA derived from recombinant expression and that of an endogenous source, i.e. inflammatory plasma. Therefore, we subjected commercial recombinant mu rSAA3 to purification to homogeneity via reversed-phase high-performance liquid chromatography (RP-HPLC) and re-assessed its biological potential. RP-HPLC-purified mu rSAA3 did not induce chemokines and lacked in vivo neutrophil chemotactic activity, but retained the capacity to synergize with CXCL8 in the activation of neutrophils. In conclusion, experimental results obtained when using proteins recombinantly expressed in bacteria should always be interpreted with care.