SEARCH

SEARCH BY CITATION

Keywords:

  • neutrophil extracellular traps;
  • myeloid derived suppressor cells;
  • chemokine ligand 1;
  • chemokine ligand 2;
  • C-X-C motif-chemokine receptor 2 (CXCR2)

Summary

  1. Top of page
  2. Summary
  3. Some basic (new) facts about neutrophils
  4. Neutrophil migration
  5. The lobulated nucleus – how and why?
  6. A fresh look at classical killing mechanisms
  7. Novel defence mechanisms – Neutrophil Extracellular Traps (NETs)
  8. The dark side of the force – neutrophils as promotors of disease
  9. Concluding remarks
  10. Acknowledgements
  11. References

For a long time neutrophil granulocytes were considered simply as terminally differentiated cells with a limited life span and pathogen killing by phagocytosis and chemical toxicity being the sole mode of action. However, work during the last 10 years has started to change this view fundamentally. Modern understanding is that neutrophils have an enormous complexity of functions. This review discusses very recent findings on how neutrophils can control the spread of pathogens and mediate their killing by mechanisms such as formation of DNA nets, how they influence tumour growth and adaptive immune responses and how they manoeuvre inside the diverse compartments of the body. It will also describe how the normally protective functions of neutrophils can have deleterious consequences if they occur in an uncontrolled fashion. These exciting novel findings are likely to completely and permanently change our view of this central leucocyte population.


Some basic (new) facts about neutrophils

  1. Top of page
  2. Summary
  3. Some basic (new) facts about neutrophils
  4. Neutrophil migration
  5. The lobulated nucleus – how and why?
  6. A fresh look at classical killing mechanisms
  7. Novel defence mechanisms – Neutrophil Extracellular Traps (NETs)
  8. The dark side of the force – neutrophils as promotors of disease
  9. Concluding remarks
  10. Acknowledgements
  11. References

Neutrophil granulocytes are the most frequent human leucocyte type. Classical measurements done in the 1970′s determined mean total bone marrow neutrophil numbers of 7·7 × 109 cells/kg body weight with 5·6 × 109/kg residing in the post mitotic pool. Thereby the marrow produces roughly 0·9 × 109 neutrophils/kg body weight per day (Dancey et al, 1976) and the transit time through the bone marrow of cells after entering the post mitotic pool is in the range of 6 days (Dancey et al, 1976; Pillay et al, 2010). Interestingly, using the transfer of cells labelled ex vivo with tritiated thymidine to measure their blood half-life, Dancey et al (1976) concluded that human neutrophils reside in the blood for around 8 h, and this value has since been reported in the scientific literature on this subject. However, it is well known that the ex vivo treatment of cells can dramatically influence their behaviour in vivo (Hasenberg et al, 2011a) and may lead to premature depletion of transferred cells from the circulation, thereby feigning a much shorter half-life of the cells than actually present.

A recent study has overcome these technical limitations by studying the blood half-lives of neutrophils in mouse and man using an in vivo labelling technique with 2H2O (Pillay et al, 2010). Interestingly, while the bone marrow transit times in this study matched very well with published data, the authors demonstrated that the circulatory half-life of neutrophils in mice was 18 and 90 h in humans, which is more than 10 times longer than previously thought. In addition, the values strongly differ between mouse and man, while they were previously thought to be equivalent (Pillay et al, 2010). These data have provoked enormous interest in the field but also cast some serious doubts (Li et al, 2011; Tofts et al, 2011) with the discussion on-going (Tak et al, 2013). Thus, when the new data can be independently confirmed, future work on the function of neutrophils should take these grossly different values into account. Such very long residence times in the blood would, for example, support novel concepts of neutrophils as regulators of T cell responses, that require prolonged direct physical interactions of T cells and neutrophils (Pillay et al, 2012), potentially already within the circulation.

Neutrophil migration

  1. Top of page
  2. Summary
  3. Some basic (new) facts about neutrophils
  4. Neutrophil migration
  5. The lobulated nucleus – how and why?
  6. A fresh look at classical killing mechanisms
  7. Novel defence mechanisms – Neutrophil Extracellular Traps (NETs)
  8. The dark side of the force – neutrophils as promotors of disease
  9. Concluding remarks
  10. Acknowledgements
  11. References

Neutrophils are produced from haematopoietic progenitors in the bone marrow, from where they permanently migrate to the circulation under steady state conditions. However, in case of emergency, up to 10 times higher levels of neutro-phils can be produced from the bone marrow in a process termed ‘danger mobilization’ (Köhler et al, 2011). A potent mediator of danger mobilization is the haematopoietic cytokine granulocyte colony-stimulating factor (G–CSF). It rapidly induces motility in bone marrow resident neutrophils mediated via the increased production of chemokine (C-X-C motif) ligand 1 (CXCL1) and CXCL2, from endothelial cells and megakaryocytes (Köhler et al, 2011). These factors constitute the most important neutrophil-recruiting chemokines in mice and act via the receptor CXCR2, (Köhler et al, 2011). A human analogue for CXCL1 and 2 would be Interleukin (IL) 8. Accompanying the action of CXCR2-chemokines are mechanisms that help to release neutrophils from their tethers to bone marrow stroma, which are made up of membrane bound CXCL12 (also known as SDF-1) in stromal cells binding to CXCR4 on the neutrophil (Eash et al, 2009).

The above findings have all been made in mice and thus were crucial to establish the general concept. Nevertheless, the conditions appear to be very similar in the human system. For many years G-CSF has been successfully used in patients with febrile neutropenia as a long-term neutrophil enhancing cytokine due to its action on haematopoietic stem cells of the bone marrow (Bennett et al, 2013). It is only recently that the acute activity of G-CSF to mobilize neutrophils and stem cells has also been exploited. Based on mouse studies, the finding that an artificial CXCR4-blocker, AMD3100 (Plerixafor®, Genzyme Corp., Cambridge, MA, USA), acts in a synergistic fashion with G-CSF to mobilize neutrophils and also stem cells from the bone marrow (Flomenberg et al, 2005; Cashen et al, 2008) in humans can now be functionally explained. It is probably mediated by the combined action of G-CSF, which raises CXCR2-binding chemokines, as well as AMD3100, which brings about the release of the cells from stromal tethers (Köhler et al, 2011).

Once inside the circulation, neutrophils are able to recognize the activated endothelium in inflammatory regions of the body and start to effectively extravasate at such sites (Kim et al, 2009). The extravasation follows a typical three-step model initially proposed by Springer (1994). From uncontrolled flow in the free blood stream, neutrophils first tether to and roll on the surface of endothelia via selectin-based semi-sticky interactions. Following triggering responses by inflammatory chemokines, rolling neutrophils come to a stop and finally transmigrate between (paracellular route) or right through (transcellular route) endothelial cells by means of integrins (Kolaczkowska & Kubes, 2013), whereby pericytes around venules seem to support them (Proebstl et al, 2012). To this end, integrins are inactive on freely flowing neutrophils and only become activated by the chemokine signals in inflammatory areas (Kolaczkowska & Kubes, 2013). A recent paper has identified novel membrane processes, ‘slings’, that help neutrophils to slow down on the endothelial surface from the high speed of free blood vessel transportation, even under strong shear forces, as a preparatory phase for final extravasation (Sundd et al, 2012).

Intravital imaging of basement membranes (BM) in cremaster muscle venules has shown preformed areas of reduced extracellular matrix protein density along blood vessels. These low-density BM spots coexist with gaps between the vessel-surrounding pericytes. Interestingly, it is primarily at these regions of low BM concentration that neutrophils mainly extravasate. The gaps are also widened under inflammatory conditions, potentially via protease activity of transmigrating neutrophils and thus facilitate effective extravasation of subsequent cells (Wang et al, 2006).

Once within tissues, neutrophils can follow gradients made up of chemokines in a process termed chemotaxis. This makes sure that the cells enter the core of inflammatory areas rapidly. These gradients can be produced by resident cells in the affected area, such as mast cells. We have shown that mast cells play an important role in guiding neutrophils to a site of inflammation, via the production of gradients made of CXCR2 chemokines (De Filippo et al, 2013). An interesting characteristic of neutrophils that only became known with the advent of intravital microscopy is their tendency to follow individual pioneering cells that have identified a stimulating structure, such as a pathogen, in large numbers and with highly directional movement resulting in clusters of neutrophils at “interesting” sites (Peters et al, 2008; Bruns et al, 2010; McDonald et al, 2010; Köhler et al, 2011). A recent elegant study has shown, in a model of sterile inflammation, that this mechanism is based on the receptor for the chemotactic lipid Leukotriene B4 (LTB4) (Lämmermann et al, 2013). The authors could demonstrate that, after recognition of tissue damage by some pioneering cells, these scouts start to produce LTB4. LTB4 from this source generates a gradient which immediately increases the speed of neutrophils in its diffusive reach (up to 200 μm from the core) and transforms their random walk into a highly chemotactic movement into the centre of the damage. Consequently, neutrophils that lack the receptor for LTB4 cannot follow this gradient and those that cannot produce LTB4 cannot kick-start the swarming response (Lämmermann et al, 2013).

Thus, the navigation of neutrophils inside inflamed tissues is a combination of chemotactic cues produced by resident local cells as well as the invading neutrophils themselves. This leads to an effective and very rapid enrichment of large numbers of neutrophils at critical sites. Interestingly, older studies had shown that activated neutrophils interacting with platelets could use the arachidonate from the platelets to transform it into LTB4 (Fiore & Serhan, 1990). Thus, similar mechanisms as those described for migration of neutrophils in tissues might also be active in recruiting the cells to sites of blood vessel injury via the interaction of pioneer cells with platelets.

The lobulated nucleus – how and why?

  1. Top of page
  2. Summary
  3. Some basic (new) facts about neutrophils
  4. Neutrophil migration
  5. The lobulated nucleus – how and why?
  6. A fresh look at classical killing mechanisms
  7. Novel defence mechanisms – Neutrophil Extracellular Traps (NETs)
  8. The dark side of the force – neutrophils as promotors of disease
  9. Concluding remarks
  10. Acknowledgements
  11. References

A morphologically defining characteristic of neutrophil granulocytes is their highly lobulated nucleus, which is well known to any haematologist and easily identified even by first year students looking at stained blood smears under the microscope. However, until today there still is no convincing teleological explanation for why the nucleus looks as it does (Brinkmann & Zychlinsky, 2012).

From a molecular point of view the analysis of the rare dominant genetic disorder Pelger-Huёt anomaly (PHA) has, at least, helped our understanding of how nuclear lobulation is brought about. PHA is caused by a defect in the gene encoding the Lamin B receptor (LBR) (Hoffmann et al, 2002) and neutrophils of patients with this disorder have bi-lobulated or round nuclei (Rowat et al, 2013). The LBR is highly expressed in neutrophils and leads to an increased nuclear envelope surface (Rowat et al, 2013), which appears to produce the lobulation as a side effect. The LBR is essential for connecting heterochromatin with the nuclear envelope, which leads to repression of transcription in this genomic area (Hirano et al, 2012). Hence, the increase in LBR expression might indicate the necessity of a mature neutrophil to exclude large proportions of its chromatin from transcription (Zhang et al, 2004), which would explain the need for a larger inner surface of the nuclear membrane.

In contrast, old concepts, already generated by the discoverer of the neutrophil, Eli Metchnikoff, of the lobulated nucleus being important for helping neutrophils to migrate efficiently through small pores and blood vessel walls (Hirsch, 1959), have not been convincingly confirmed. An extensive analysis of neutrophils from dogs carrying the mutation that produces PHA showed no detectable defects in random or directed migration in vitro or immigration into inflamed sites in vivo (Latimer et al, 1989).

Along the same line, a recent molecular study in HL60 cells that were induced to develop into mature neutrophils showed that the knockdown of LBR in these cells reproduced the round phenotype of PHA nuclei but did not have any impact on the ability of the cells to cross through pores as narrow as 3 μm (Rowat et al, 2013). However, the overexpression of Lamin A, a component of the nuclear envelope that increases its stiffness (Stewart et al, 2007) also produced a round nucleus, this time however strongly impairing transmigration through narrow pores (Rowat et al, 2013). Hence, rather than the lobulation, it appears to be the nuclear stiffness mediated by the composition of the nuclear protein matrix that is a decisive factor in the ability of neutrophils to cross narrow pores. Consequently, upon their development from haematopoietic precursors, neutrophils strongly downregulate Lamin A, probably to reduce nuclear rigidity (Rowat et al, 2013).

Thus, our currently available knowledge provides no convincing explanation for the evolutionary advances of a lobulated nucleus in neutrophils other than allowing them to have a larger inner surface. This is reminiscent of alveoli in the lung, which are polyhedral rather than round to provide a maximum surface at a given diameter (Hasenberg et al, 2013). Given the fact that at least heterozygous PHA is essentially without a severe phenotype (Hoffmann et al, 2002), a lobulated nucleus might really only be a non-functional side effect of changes in nuclear envelope composition that occurs during the development of neutrophils from their precursors.

A fresh look at classical killing mechanisms

  1. Top of page
  2. Summary
  3. Some basic (new) facts about neutrophils
  4. Neutrophil migration
  5. The lobulated nucleus – how and why?
  6. A fresh look at classical killing mechanisms
  7. Novel defence mechanisms – Neutrophil Extracellular Traps (NETs)
  8. The dark side of the force – neutrophils as promotors of disease
  9. Concluding remarks
  10. Acknowledgements
  11. References

Neutrophils are essential for early immune responses against microbial pathogens (Köhler et al, 2011). Thereby they feature two well-known main mechanisms of defence: phagocytosis (Behnsen et al, 2007) and intra- as well as extra-cellular destruction by chemical means (Segal, 2005; Nauseef, 2007; Winterbourn & Kettle, 2013).

Phagocytosis by neutrophils is a highly dynamic process in which (up to) high numbers of individual pathogenic elements are bound on the cell surface and also collected by individual neutrophils via dynamic migration in vitro (Behnsen et al, 2007; Sasse et al, 2013) and in vivo (Peters et al, 2008; Bruns et al, 2010). Interestingly, not every contact with a pathogenic surface leads to a successful phagocytosis event but instead can also result in the loss of the pathogenic element from the neutrophil surface (Behnsen et al, 2007). Furthermore, in a direct comparison, neutrophils appear much more efficient in particle phagocytosis than, for example, alveolar macrophages (Behnsen et al, 2007). Molecularly, phagocytosis requires a very complex orchestration of diverse events. It has been shown that phagocytes are able to distinguish soluble constituents of a fungal cell wall from its cell wall-associated counterparts in an intact spore. Only the spore is able to induce a phagocytic event as it mediates the formation of a so-called ‘phagocytic synapse’ that triggers a complex intracellular cascade via the extracellular receptor Dectin-1 and the kinase SYK culminating in the phagocytic process (Goodridge et al, 2011).

During phagocytosis particles are enclosed in a phagosome and killed inside this structure by a number of processes (see below). However, the recognition of a pathogenic particle on the cell surface or the presence of a particle in the phagosome trigger a multitude of additional responses in the phagocyte, such as production of calcium flushes (Nunes et al, 2012), pro-inflammatory cytokines or chemokines that can recruit other immune cells (summarized in Underhill & Goodridge, 2012).

The killing process itself is mediated by two principal mechanisms, O2-independent and O2-dependent killing. O2-independent killing is mediated by the constituents of the granules in the cytoplasm of neutrophils (Hurst, 2012). The granules fuse with phagosomes and thereby transport their contents into the small residual lumen that is left over between the pathogen and the phagosomal membrane. This enables very high concentrations of toxic proteins to be brought directly to the cell wall of the pathogen. There are three types of granules: (i) azurophil/primary granules, which contain mainly myeloperoxidase (MPO) as well as the neutral proteases elastase, cathepsin G and proteinase 3. Also the cell wall-cracking lysozyme is highly enriched in these granules. (ii) Gelatinase/secondary granules containing lactoferrin and the largest source of the available lysozyme in a neutrophil. (iii) Gelatinase/tertiary granules that may represent partly emptied secondary granules that do not contain lactoferrin. Importantly, all protein contents in granules are tightly associated in an inactive fashion with a space filling matrix composed of proteoglycans (Segal, 2005). The release of the toxic proteins from the matrix is thought to be mediated by a redox reaction where an electrochemical gradient is generated across the phagosomal membrane by action of the NAD(P)H oxidase enzyme complex (Nox). Under consumption of NAD(P)H, Nox pumps electrons over the phagosomal membrane which are followed by cations (Segal, 2005). The increased ionic strength in the phagosome is then thought to drive the release and activation of matrix-associated toxic proteins (Segal, 2005; Hurst, 2012).

O2-dependent killing exploits the ability of Nox to produce extremely aggressive reactive oxygen species (ROS) in the phagosome (Winterbourn & Kettle, 2013). ROS can be directly toxic via the destruction of bacterial proteins or the induction of DNA double-strand breaks (Hurst, 2012). The importance of Nox is evident from patients with chronic granulomatous disease (CGD), where Nox is defective or missing. These patients suffer from recurrent bacterial and fungal infections which can be treated in experimental approaches with the re-transfer of a functional Nox system in mice (Stein et al, 2013) and humans (Bianchi et al, 2009). In addition, MPO is able to halogenate the surface of pathogens in the presence of H2O2 generated from Nox products, a reaction that starts immediately after closure of the phagosome (Winterbourn & Kettle, 2013).

However, while ROS are indeed bacteriolytic in a test tube, Segal and colleagues pointed out that this might be entirely different under the conditions of a phagosome with its extreme concentrations of salt and proteins (Reeves et al, 2002; Segal, 2005). Instead they showed that the ROS pumped into a phagosome by Nox raised the very acid pH to slightly alkaline levels and that the negative charge generated by the electron flux across the membrane of the phagosome was compensated by an equivalent flux of K+ ions via large-conductance BK channels (Reeves et al, 2002). They, thus proposed that, rather than direct pathogen killing, the main function of ROS was this increase in phagosomal pH and the importation of a large number of K+ ions to allow the matrix bound proteases to be released/activated and bring about the killing of pathogens, at least of fungal and bacterial species (Reeves et al, 2002). This would explain why the lack of granular proteases renders mice highly susceptible to bacterial and fungal infections while mice and humans lacking MPO do not suffer from strongly increased susceptibility to infection.

However, this concept has been challenged for a number of reasons (summarized in Nauseef, 2007), the most important ones being that the lack of BK channels, which transport K+ ions across membranes and thus are vital to Segal's hypothesis, does not interfere with effective bacterial killing in neutrophils (Essin et al, 2007) and that such channels are absent in murine and human neutrophils (Femling et al, 2006; Essin et al, 2007). Collectively, the majority of independently obtained data appears to confirm a central role for oxidative killing by the Nox-ROS-MPO system in pathogen destruction by neutrophils.

In any case, rather than being absolutely essential, MPO might be more of a baseline mechanism to deal with very high pathogenic loads and its lack could be compensated against at a limited infective threat (Klebanoff et al, 2013; Winterbourn & Kettle, 2013). This might explain why even the complete lack of MPO seems to be compatible with a normal life span in the absence of any obvious infectious complications, at least in areas with a high standard of hygiene (Nauseef, 2007; Wang et al, 2013).

Importantly, neutrophils are also able to kill pathogens that are too large to be ingested. Particularly good examples for this are fungi that have managed to escape all initial immune cell attacks and started to germinate. Here, neutrophils can attach themselves to the surface of the pathogen in large numbers or even transport hyphal fragments in a cooperative fashion to spots with large clusters of other neutrophils (Bruns et al, 2010). It has been shown that in such clusters ROS are produced also outside of the neutrophils (Bonnett et al, 2006) and we showed an immediate recruitment of Nox to the site of hyphal contact in a neutrophil (Niesner et al, 2008), which would provide a potential mechanistic basis for the extracellular production of ROS. Whether this would provide a functional killing mechanism is subject for further study. Strikingly, platelets binding to the surface of fungal hyphae have been shown to be cytotoxic to the fungus themselves but also to enhance the killing capacity of co-associated polymorphonuclear cells by unknown mechanisms (Christin et al, 1998). This might provide a powerful mechanism to attack fungal elements that have managed to escape immune control in the lung and started to grow invasively into blood vessels.

Novel defence mechanisms – Neutrophil Extracellular Traps (NETs)

  1. Top of page
  2. Summary
  3. Some basic (new) facts about neutrophils
  4. Neutrophil migration
  5. The lobulated nucleus – how and why?
  6. A fresh look at classical killing mechanisms
  7. Novel defence mechanisms – Neutrophil Extracellular Traps (NETs)
  8. The dark side of the force – neutrophils as promotors of disease
  9. Concluding remarks
  10. Acknowledgements
  11. References

Few discoveries in the immunological field have gained as much attention in the last decade as the discovery that neutrophils are able to throw out their nuclear DNA in a final act of defence as a sticky and toxic net that is able to bind and kill pathogens (Brinkmann et al, 2004). These DNA nets have been termed Neutrophil Extracellular Traps (NET). Since its publication the initial paper has been cited almost 1000 times and the process has also been found to exist in diverse other cell types, such as mast cells (von Köckritz-Blickwede et al, 2008) and eosinophils (Yousefi et al, 2008).

Neutrophil Extracellular Traps are composed of a backbone of nuclear DNA decorated with a multitude of nuclear proteins as well as the contents of neutrophil granules, including MPO and elastase (Urban et al, 2009). They are found in normal pus (Brinkmann & Zychlinsky, 2012) and generated by dying neutrophils in a controlled process that lasts 3 h in vitro and which can be functionally and morphologically distinguished from necrosis or apoptosis (Fuchs et al, 2007). Therefore the term ‘NETosis’ has been coined to describe this novel form of cell death (Steinberg & Grinstein, 2007). NETosis is dependent on the function of Nox, which is why CGD patients cannot make NETs (Bianchi et al, 2009). Movies of NETting neutrophils show the initial stopping of a previously motile cell, then the gradual breakdown of the nuclear (and potentially also the granular) membranes and extensive mixing of the components inside of the cell. The final release of the DNA-protein mix is rapid and explosive (Bruns et al, 2010; Remijsen et al, 2011). The function of Nox is essential but not sufficient to induce NETosis. Other mechanisms, such as autophagy and citrullination of histones, also play a non-redundant role in the process. Furthermore, NETosis can be clearly differentiated from apoptosis as a normal mechanism of cell death by a number of features including its independence from caspase activity (summarized in Remijsen et al, 2011).

Interestingly, in a presumably homogenous population of isolated neutrophils, only a small fraction of cells performs NETosis despite the fact that all cells are stimulated by the same trigger or touch the same stimulating pathogenic surface (Bruns et al, 2010). Thrown out NETs form a tight contact with pathogens as diverse as fungi (Fig 1) (Urban et al, 2006; Hasenberg et al, 2011b), bacteria (Beiter et al, 2006) and protozoa (Guimaraes-Costa et al, 2009; Abi Abdallah et al, 2012), during which many of the pathogens are also killed.

image

Figure 1. Neutrophils make neutrophil extracellular traps after contact with fungal elements in vitro. Purified human neutrophils were incubated with conidia of A. fumigatus (A, B) or yeast cells of C. albicans (C–F) in vitro for 3 h, subsequently fixed and analysed by scanning electron microscopy. (A) Overview of two neutrophils (Nφ) contacting several conidia (artificially coloured in green). (B) Enlarged view of the area boxed in (A) showing the fine knobbly structure of single neutrophil extracellular trap (NET) fibres in close contact with the surface of a conidium. (C) Overview of four neutrophils making a large mesh and some fine NET fibres in contact with yeast cells of C. albicans. (D) Enlarged view of the area boxed in (C) showing tight contact of NET fibres with the surface of yeast cells. (E) Overview of a NET structure that appears like a thread of glue and sticks to yeast elements. (F) Enlarged view of the area boxed in (E). Dimensions: In these images conidia are 2 μm in diameter, yeast cells are 3 μm long.

Download figure to PowerPoint

However, as Mocsai (2013) recently pointed out, despite the massive response of the field towards this initial discovery and while the general concept of NETs as means to immobilize pathogens is now accepted, their role and importance in pathogen killing are still controversial (Menegazzi et al, 2012; Nauseef, 2012). Indeed, Nauseef (2012) compiled a ‘wish list’ of critical issues in the biology of NETs, the response to which he would consider essential to solidify the concept. Some of these points, such as the appearance of NETs in vivo, have since been convincingly addressed (see below) while others remain a problem, especially the contribution of NETs to the overall host protection. I would like to add two more points to this list, which have not been addressed thoroughly so far: (i) It is completely unclear why, in a homogenous population of neutrophils, some cells do and some (normally many more) do not make NETs. Thus, can neutrophils measure the successful generation of NETs from other cells and then stop their own programme when a certain amount of NETs is present, or is there a subpopulation of cells that are predisposed to make NETs? (ii) What happens to NETs after they have fulfilled their duty? Is there an organized way of NET removal? If so, by which mechanisms does it work and how is it regulated? First attempts to address this question showed that the presence of DNase1 in the serum was essential for disassembly of NETs (Hakkim et al, 2010) but no further analyses, e.g. including long-term intravital microscopy, have been undertaken so far to study this problem.

Nevertheless, NETs have now been unequivocally identified in a number of animal models. We and others (Urban et al, 2006) showed that they are produced after contact of neutrophils with Aspergillus fumigatus (Fig 1A,B) or Candida albicans (Fig 1C–F) in vitro or within 7 h after pulmonary infection with A. fumigatus by neutrophils invading the lung (Bruns et al, 2010; Hasenberg et al, 2011b,c, 2013). Clark et al (2007)identified the structures in mouse models of endotoxaemia and bacterial infection. By capturing pathogens from the blood stream NETs are able to protect the host from liver damage associated with bacterial (McDonald et al, 2012) and even viral infection (Jenne et al, 2013).

However, whilst able to limit pathogen spread, the production of NETs can also be increased by the simultaneous interaction of neutrophils with activated platelets leading to thrombosis as a side effect (Clark et al, 2007; Massberg et al, 2010). Even in sterile inflammation conditions the interaction of activated platelets with neutrophils can lead to NET production, the deposition of which can initiate events leading to deep vein thrombosis (DVT) (von Brühl et al, 2012). This is dependent on the enzyme peptidylarginine deiminase 4 (PAD4) (Martinod et al, 2013), which mediates the citrullination of histones leading to the decondensation of chromatin, an essential step before NETs can be released (Li et al, 2010). Mice lacking PAD4 are therefore largely resistant to experimental DVT (Martinod et al, 2013).

In the light of these findings, the concept of NETs has also facilitated a better understanding of a number of other disease settings and has thus opened novel options for diagnosis or treatment. For example, the presence of citrullinated proteins in NETs might provide a source of auto-antigens in rheumatoid arthritis (RA) that lead to the production of anti-citrullinated protein autoantibodies (ACPA), which are considered a key pathological factor in RA (Khandpur et al, 2013). Furthermore, NET production from activated host neutrophils following massive transfer of activated platelets during blood transfusion is associated with transfusion-related acute lung injury (TRALI) in mice and humans; interfering with platelet activation or destroying preformed NETs by DNAse injection was protective in an animal model of TRALI (Caudrillier et al, 2012). In the thrombotic microangiopathies a positive correlation was found between disease severity, the levels of circulating histones and MPO (Fuchs et al, 2012). Very interestingly, cancer-associated thrombosis might also be dependent on an increased propensity of neutrophils in cancer-carrying hosts to produce NETs, as was shown in animal models of leukaemia and solid tumours (Demers et al, 2012).

Knowledge of these connections obviously provides novel treatment avenues for clinicians that are worth testing, e.g. by interfering with platelet activation during blood collection and transfusion or by applying DNase to tumour patients to reduce the level of thrombosis formation. A potential application was recently published showing the effectiveness of a drug that inhibits Nox and thereby NETosis (Hosseinzadeh et al, 2012), which would, in principal, be suitable for use in humans.

However, not all connections that would seem logical in the context of NETs appear to be completely consistent. This is exemplified in the case of systemic lupus erythematosus (SLE). SLE patients show high serum levels of autoantibodies against nuclear antigens (including anti-self-DNA) and proteins from neutrophil granules. These autoantibodies constitute a major pathogenic factor in the disease but the source of auto-antigen in SLE has remained enigmatic. In this case, the discovery of NETs provided a convincing candidate. Indeed, researchers recently showed that neutrophils from SLE patients make more NETs and the DNA in NETs was able to trigger plasmacytoid dendritic cells to produce type I interferons, which in turn activated neutrophils to produce even more NETs (Garcia-Romo et al, 2011; Lande et al, 2011). This would result in a vicious circle and identify NETs and associated neutrophil proteins as a key source of auto-antigen in the disease (Garcia-Romo et al, 2011; Lande et al, 2011). However, when breeding a lupus-prone mouse to a Nox-deficient background, in which neutrophils are not able to produce NETs, the ensuing offspring did not show less but, in contrast, massively increased SLE signs. This finding led to the conclusion that NETosis does not contribute to SLE pathology (Campbell et al, 2012).

Thus, although a number of very interesting and convincing findings have been made in relation to NETs and their role in physiological and pathological defence, we should resist the move to try and explain everything based on this new concept and rather maintain a healthy criticism to novel findings in this field. On the other hand, the discovery of NETs has helped to explain a large number of previously enigmatic findings and thus is extremely helpful for a better understanding of innate immune mechanisms and their impact on general immunity.

The dark side of the force – neutrophils as promotors of disease

  1. Top of page
  2. Summary
  3. Some basic (new) facts about neutrophils
  4. Neutrophil migration
  5. The lobulated nucleus – how and why?
  6. A fresh look at classical killing mechanisms
  7. Novel defence mechanisms – Neutrophil Extracellular Traps (NETs)
  8. The dark side of the force – neutrophils as promotors of disease
  9. Concluding remarks
  10. Acknowledgements
  11. References

While neutrophils are clearly essential for normal host defence, discoveries in recent years have also indicated an important role of these cells in the induction or promotion of a diseased state. Some of these issues associated with the formation of NETs have been covered in the previous section on NET biology. Here I shall review additional problems related with other inflammatory responses of neutrophils that are very common to these cells.

Following a slow start before 2005 and a dramatic increase of published papers from 2007 onwards, investigations, particularly in the past 8 years, have identified and characterized a novel cell type, myeloid-derived suppressor cells (MDSC), that are derived from the myeloid/neutrophil lineage but are very potent suppressors of T cell responses (Gabrilovich & Nagaraj, 2009). The cells are present in mice and humans but remain in an inactivated state under baseline conditions. However, infections and tumours can strongly induce both their expansion and activation. In this state, MDSC can reduce immune responses, e.g. against chronic viral infections (Norris et al, 2013) or tumours (Ostrand-Rosenberg et al, 2012). Indeed, MDSC have also been identified in increased numbers in the blood of tumour patients and found to suppress T cell responses in vitro (Brandau et al, 2011). Thus, MDSC are a class of cells that might be beneficial in the suppression of overt immune responses but also have the potential to suppress protective host antitumoral and antiviral immunity (Ostrand-Rosenberg et al, 2012).

While neutrophils also have a number of antitumoral activities (Brandau et al, 2013), the role that they play in promoting tumour growth is becoming more and more obvious. A recent report showed that experimental breast cancers produce CXCL1 and 2 and that the neutrophils recruited by these chemokines promoted metastasis as well as resistance of tumours against chemotherapy by provision of S100A8/9 proteins (Acharyya et al, 2012). These are low molecular weight calcium-binding proteins (Manitz et al, 2003) that can also trigger the activation of tumour cells via Toll-like Receptor 4 (TLR4) and Receptor for Advanced Glycation Endproducts (RAGE) (Acharyya et al, 2012). Along these lines, massive neutrophil invasion mediated by the production of another CXCR2 ligand, CXCL5, has been found in hepatocellular carcinoma and the ensuing massive neutrophil infiltration was a factor constituting a poor prognosis in this tumour entity in humans (Zhou et al, 2012). Thus neutrophil invasion can promote the aggressive potential of tumours.

Neutrophil pathology is not restricted to tumour biology. In contrast, neutrophils can cause harm even in their most natural realm, pathogen defence. It has been shown that in Leishmania infection the parasites hijack skin-invading neutrophils and travel inside these cells into the centre of the body (Peters et al, 2008). In case of lung infection by influenza A virus (IAV) it was noted that the particularly lethal 1918 strain was characterized by inducing an early and massive infiltration of neutrophils into the lung, which mediated strong lung pathology (Kash et al, 2006). A molecular explanation for this finding was provided in a recent study, which showed that a mild IAV infection differed from a lethal one by the early and massive production of neutrophil recruiting chemokines, especially CXCL1 and 2, in the latter (Brandes et al, 2013). The major sources of these chemokines were invading neutrophils themselves, thus making up a feed forward inflammatory loop that mediated overt lung pathology. Consequently, the partial depletion of neutrophils had a protective effect in otherwise lethal IAV infections (Brandes et al, 2013).

Another example where neutrophils are a central element of a chronic inflammatory state is psoriasis. This skin inflammation can be elicited by a specific type of helper T cells that produce IL17 as a lineage defining cytokine and therefore are called Th17 cells (Weaver et al, 2007). Th17 cells and IL17 are essential for host defence at epithelial and mucosal surfaces, especially during fungal infections (Conti et al, 2009; Puel et al, 2011). To fulfill this function, Th17 cells also produce factors that increase the number of neutrophils (especially G-CSF) as well as CXC-chemokines that recruit neutrophils to sites of inflammation (Conti et al, 2009). Interestingly, systemic overexpression of IL17 alone induces a psoriasis-like phenotype in mice accompanied by massive neutrophilia (Haak et al, 2009), which is explained by the fact that the cytokine can also trigger the release of neutrophil-generating and -recruiting factors from a plethora of other cells including fibroblasts and endothelial cells (Kolls & Linden, 2004; Weaver et al, 2007). A direct clinical result from these findings is that the blockade of IL17 in patients is a highly effective treatment for psoriasis (Leonardi et al, 2012; Papp et al, 2012; Patel et al, 2013).

Importantly, the role of the IL17-neutrophil axis is not limited to psoriasis alone. In fact, the blockade of this mechanism has shown enormous potential in the treatment of other autoimmune disorders, such as RA, ankylosing spondylitis and autoimmune uveitis (Patel et al, 2013), suggesting that neutrophils also play a detrimental role here. Surprisingly, blocking IL17 as an attempt to treat Cohn's disease was unsuccessful or even detrimental in a clinical pilot study, indicating a protective function of this cytokine for the gut mucosa that might be dominant over pro-inflammatory side effects (Patel et al, 2013).

However, overt neutrophil activation is effectively counteracted in a functioning immune system by a number of mechanisms. Two recent papers suggested that neutrophil activity might be controlled by a CD11c+ cell type, presumably of dendritic cell (DC) origin (Autenrieth et al, 2012; Tittel et al, 2012). In a mouse model of pyelonephritis, neutrophils were found to be much more activated resulting in increased resistance to bacterial nephritis, when CD11c+ cells had been systemically removed via a diphtheria toxin-based system (Tittel et al, 2012). Similar findings were made in a comparable animal model for DC depletion, yet investigating the impact of systemic infection with Yersinia enterocolitica (Autenrieth et al, 2012). Thus, neutrophil activity is controlled by other immune cells, which can obviously limit the effector potential of these cells. Whether such a regulatory influence by DC is possibly missing or not effectively induced in the case of pathological IAV infection has not yet been investigated.

Interestingly, neutrophils also provide mechanisms on their own to balance the activity of other neutrophils or macrophages. This is, for example, mediated by the release of small membrane ensheathed vesicles, so-called microparticles, that activated neutrophils exocytose and which can stimulate the secretion of the inhibitory cytokine transforming growth factor-β from macrophages (Gasser & Schifferli, 2004). Microparticles also directly dampen the activity of neutrophils by exposing the membrane component annexin to the ALX receptor of neutrophils (Dengler et al, 2013). However, microparticles can also have proinflammatory effects, e.g. on endothelial cells. In addition, they can induce thrombus formation by their binding to and activation of platelets (Dengler et al, 2013).

Concluding remarks

  1. Top of page
  2. Summary
  3. Some basic (new) facts about neutrophils
  4. Neutrophil migration
  5. The lobulated nucleus – how and why?
  6. A fresh look at classical killing mechanisms
  7. Novel defence mechanisms – Neutrophil Extracellular Traps (NETs)
  8. The dark side of the force – neutrophils as promotors of disease
  9. Concluding remarks
  10. Acknowledgements
  11. References

Neutrophils have long been considered as not much more than ‘dumb suicide killers’ (Mocsai, 2013). Consequently, the interest in and amazement about the investigation of these cells has remained limited for a long time, at least among professional immunologists. Probably triggered by the appearance of intravital imaging (Chtanova et al, 2008) with mouse lines that highlight neutrophils as well as such striking observations as the production of NETs (Brinkmann et al, 2004) or the mechanisms of group motility (Lämmermann et al, 2013), the interest in these enormously fascinating cells has risen again and they are now back on track for some headlines. It is hoped that this momentum can be maintained for a while to obtain a most comprehensive understanding of the role neutrophils play in health and disease. While ideas of neutrophils expressing functional T cell receptors (Puellmann et al, 2006) might be a bit far-fetched or at least still require the solid proof of in vivo relevance, other concepts mentioned in this review have already found their way into common knowledge. Future work will show whether these novel ideas will finally lead to new avenues for diagnosis and therapy in humans. The clinical success of IL17 blockade already provides a prominent example of how such developments can thrive after key discoveries have been made in basic studies.

Acknowledgements

  1. Top of page
  2. Summary
  3. Some basic (new) facts about neutrophils
  4. Neutrophil migration
  5. The lobulated nucleus – how and why?
  6. A fresh look at classical killing mechanisms
  7. Novel defence mechanisms – Neutrophil Extracellular Traps (NETs)
  8. The dark side of the force – neutrophils as promotors of disease
  9. Concluding remarks
  10. Acknowledgements
  11. References

M.G. was sponsored by the German research foundation [DFG, GU769/4-2 (SPP1468), GU769/5-1, SFB854], the International Leibniz Research School for Microbial and Biomolecular Interactions (ILRSJena) which is a part of the ‘Jena School for Microbial Communication’ (JSMC), by the Mercator Research Center Ruhr and the German ministry for education and research (BMBF, PROTumor). I thank Christine Winterbourn (Christchurch, New Zealand) for helpful discussions.

References

  1. Top of page
  2. Summary
  3. Some basic (new) facts about neutrophils
  4. Neutrophil migration
  5. The lobulated nucleus – how and why?
  6. A fresh look at classical killing mechanisms
  7. Novel defence mechanisms – Neutrophil Extracellular Traps (NETs)
  8. The dark side of the force – neutrophils as promotors of disease
  9. Concluding remarks
  10. Acknowledgements
  11. References
  • Abi Abdallah, D.S., Lin, C., Ball, C.J., King, M.R., Duhamel, G.E. & Denkers, E.Y. (2012) Toxoplasma gondii triggers release of human and mouse neutrophil extracellular traps. Infection and Immunity, 80, 768777.
  • Acharyya, S.S., Oskarsson, T., Vanharanta, S., Malladi, S., Kim, J., Morris, P.G., Manova-Todorova, K., Leversha, M., Hogg, N., Seshan, V.E., Norton, L., Brogi, E. & Massague, J. (2012) A CXCL1 paracrine network links cancer chemoresistance and metastasis. Cell, 150, 165178.
  • Autenrieth, S.E., Warnke, P., Wabnitz, G.H., Lucero, E.C., Pasquevich, K.A., Drechsler, D., Gunter, M., Hochweller, K., Novakovic, A., Beer-Hammer, S., Samstag, Y., Hammerling, G.J., Garbi, N. & Autenrieth, I.B. (2012) Depletion of dendritic cells enhances innate anti-bacterial host defense through modulation of phagocyte homeostasis. PLoS Pathogens, 8, e1002552.
  • Behnsen, J., Narang, P., Hasenberg, M., Gunzer, F., Bilitewski, U., Klippel, N., Rohde, M., Brock, M., Brakhage, A.A. & Gunzer, M. (2007) Environmental dimensionality controls the interaction of phagocytes with the pathogenic fungi Aspergillus fumigatus and Candida albicans. PLoS Pathogens, 3, e13.
  • Beiter, K., Wartha, F., Albiger, B., Normark, S., Zychlinsky, A. & Henriques-Normark, B. (2006) An endonuclease allows Streptococcus pneumoniae to escape from neutrophil extracellular traps. Current Biology, 16, 401407.
  • Bennett, C.L., Djulbegovic, B., Norris, L.B. & Armitage, J.O. (2013) Colony-stimulating factors for febrile neutropenia during cancer therapy. New England Journal of Medicine, 368, 11311139.
  • Bianchi, M., Hakkim, R.A., Brinkmann, V., Siler, U., Seger, R.A., Zychlinsky, A. & Reichenbach, J. (2009) Restoration of NET formation by gene therapy in CGD controls aspergillosis. Blood, 114, 26192622.
  • Bonnett, C.R., Cornish, E.J., Harmsen, A.G. & Burritt, J.B. (2006) Early neutrophil recruitment and aggregation in the murine lung inhibit germination of Aspergillus fumigatus conidia. Infection and Immunity, 74, 65286539.
  • Brandau, S., Trellakis, S., Bruderek, K., Schmaltz, D., Steller, G., Elian, M., Suttmann, H., Schenck, M., Welling, J., Zabel, P. & Lang, S. (2011) Myeloid-derived suppressor cells in the peripheral blood of cancer patients contain a subset of immature neutrophils with impaired migratory properties. Journal of Leukocyte Biology, 89, 311317.
  • Brandau, S., Dumitru, C.A. & Lang, S. (2013) Protumor and antitumor functions of neutrophil granulocytes. Seminars in Immunopathology, 35, 163176.
  • Brandes, M., Klauschen, F., Kuchen, S. & Germain, R.N. (2013) A systems analysis identifies a feedforward inflammatory circuit leading to lethal influenza infection. Cell, 154, 197212.
  • Brinkmann, V. & Zychlinsky, A. (2012) Neutrophil extracellular traps: is immunity the second function of chromatin? Journal of Cell Biology, 198, 773783.
  • Brinkmann, V., Reichard, U., Goosmann, C., Fauler, B., Uhlemann, Y., Weiss, D.S., Weinrauch, Y. & Zychlinsky, A. (2004) Neutrophil extracellular traps kill bacteria. Science, 303, 15321535.
  • von Brühl, M.L., Stark, K., Steinhart, A., Chandraratne, S., Konrad, I., Lorenz, M., Khandoga, A., Tirniceriu, A., Coletti, R., Kollnberger, M., Byrne, R.A., Laitinen, I., Walch, A., Brill, A., Pfeiler, S., Manukyan, D., Braun, S., Lange, P., Riegger, J., Ware, J., Eckart, A., Haidari, S., Rudelius, M., Schulz, C., Echtler, K., Brinkmann, V., Schwaiger, M., Preissner, K.T., Wagner, D.D., Mackman, N., Engelmann, B. & Massberg, S. (2012) Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo. Journal of Experimental Medicine, 209, 819835.
  • Bruns, S., Kniemeyer, O., Hasenberg, M., Aimanianda, V., Nietzsche, S., Thywißen, A., Jeron, A., Latge, J.P., Brakhage, A.A. & Gunzer, M. (2010) Production of extracellular traps against Aspergillus fumigatus in vitro and in infected lung tissue is dependent on invading neutrophils and influenced by hydrophobin RodA. PLoS Pathogens, 6, e1000873.
  • Campbell, A.M., Kashgarian, M. & Shlomchik, M.J. (2012) NADPH oxidase inhibits the pathogenesis of systemic lupus erythematosus. Science Translational Medicine, 4, 157ra141.
  • Cashen, A., Lopez, S., Gao, F., Calandra, G., MacFarland, R., Badel, K. & DiPersio, J. (2008) A phase II study of plerixafor (AMD3100) plus G-CSF for autologous hematopoietic progenitor cell mobilization in patients with Hodgkin lymphoma. Biology of Blood and Marrow Transplantation, 14, 12531261.
  • Caudrillier, A., Kessenbrock, K., Gilliss, B.M., Nguyen, J.X., Marques, M.B., Monestier, M., Toy, P., Werb, Z. & Looney, M.R. (2012) Platelets induce neutrophil extracellular traps in transfusion-related acute lung injury. The Journal of Clinical Investigation, 122, 26612671.
  • Christin, L., Wysong, D.R., Meshulam, T., Hastey, R., Simons, E.R. & Diamond, R.D. (1998) Human platelets damage Aspergillus fumigatus hyphae and may supplement killing by neutrophils. Infection and Immunity, 66, 11811189.
  • Chtanova, T., Schaeffer, M., Han, S.J., van Dooren, G.G., Nollmann, M., Herzmark, P., Chan, S.W., Satija, H., Camfield, K., Aaron, H., Striepen, B. & Robey, E.A. (2008) Dynamics of neutrophil migration in lymph nodes during infection. Immunity, 29, 487496.
  • Clark, S.R., Ma, A.C., Tavener, S.A., McDonald, B., Goodarzi, Z., Kelly, M.M., Patel, K.D., Chakrabarti, S., McAvoy, E., Sinclair, G.D., Keys, E.M., Allen-Vercoe, E., Devinney, R., Doig, C.J., Green, F.H. & Kubes, P. (2007) Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood. Nature Medicine, 13, 463469.
  • Conti, H.R., Shen, F., Nayyar, N., Stocum, E., Sun, J.N., Lindemann, M.J., Ho, A.W., Hai, J.H., Yu, J.J., Jung, J.W., Filler, S.G., Masso-Welch, P., Edgerton, M. & Gaffen, S.L. (2009) Th17 cells and IL-17 receptor signaling are essential for mucosal host defense against oral candidiasis. Journal of Experimental Medicine, 206, 299311.
  • Dancey, J.T., Deubelbeiss, K.A., Harker, L.A. & Finch, C.A. (1976) Neutrophil kinetics in man. The Journal of Clinical Investigation, 58, 705715.
  • De Filippo, K., Dudeck, A., Hasenberg, M., Nye, E., von Rooijen, N., Hartmann, K., Gunzer, M., Roers, A. & Hogg, N. (2013) Mast cell and macrophage chemokines CXCL1/CXCL2 control the early stage of neutrophil recruitment during tissue inflammation. Blood, 121, 49304937.
  • Demers, M., Krause, D.S., Schatzberg, D., Martinod, K., Voorhees, J.R., Fuchs, T.A., Scadden, D.T. & Wagner, D.D. (2012) Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis. Proceedings of the National Academy of Sciences of the United States of America, 109, 1307613081.
  • Dengler, V., Downey, G.P., Tuder, R.M., Eltzschig, H.K. & Schmidt, E.P. (2013) Neutrophil intercellular communication in acute lung injury. Emerging roles of microparticles and gap junctions. American Journal of Respiratory Cell and Molecular Biology, 49, 15.
  • Eash, K.J., Means, J.M., White, D.W. & Link, D.C. (2009) CXCR4 is a key regulator of neutrophil release from the bone marrow under basal and stress granulopoiesis conditions. Blood, 113, 47114719.
  • Essin, K., Salanova, B., Kettritz, R., Sausbier, M., Luft, F.C., Kraus, D., Bohn, E., Autenrieth, I.B., Peschel, A., Ruth, P. & Gollasch, M. (2007) Large-conductance calcium-activated potassium channel activity is absent in human and mouse neutrophils and is not required for innate immunity. American Journal of Physiology-Cell Physiology, 293, C45C54.
  • Femling, J.K., Cherny, V.V., Morgan, D., Rada, B., Davis, A.P., Czirjak, G., Enyedi, P., England, S.K., Moreland, J.G., Ligeti, E., Nauseef, W.M. & DeCoursey, T.E. (2006) The antibacterial activity of human neutrophils and eosinophils requires proton channels but not BK channels. Journal of General Physiology, 127, 659672.
  • Fiore, S. & Serhan, C.N. (1990) Formation of lipoxins and leukotrienes during receptor-mediated interactions of human platelets and recombinant human granulocyte/macrophage colony-stimulating factor-primed neutrophils. Journal of Experimental Medicine, 172, 14511457.
  • Flomenberg, N., Devine, S.M., Dipersio, J.F., Liesveld, J.L., McCarty, J.M., Rowley, S.D., Vesole, D.H., Badel, K. & Calandra, G. (2005) The use of AMD3100 plus G-CSF for autologous hematopoietic progenitor cell mobilization is superior to G-CSF alone. Blood, 106, 18671874.
  • Fuchs, T.A., Abed, U., Goosmann, C., Hurwitz, R., Schulze, I., Wahn, V., Weinrauch, Y., Brinkmann, V. & Zychlinsky, A. (2007) Novel cell death program leads to neutrophil extracellular traps. Journal of Cell Biology, 176, 231241.
  • Fuchs, T.A., Kremer Hovinga, J.A., Schatzberg, D., Wagner, D.D. & Lammle, B. (2012) Circulating DNA and myeloperoxidase indicate disease activity in patients with thrombotic microangiopathies. Blood, 120, 11571164.
  • Gabrilovich, D.I. & Nagaraj, S. (2009) Myeloid-derived suppressor cells as regulators of the immune system. Nature Reviews Immunology, 9, 162174.
  • Garcia-Romo, G.S., Caielli, S., Vega, B., Connolly, J., Allantaz, F., Xu, Z., Punaro, M., Baisch, J., Guiducci, C., Coffman, R.L., Barrat, F.J., Banchereau, J. & Pascual, V. (2011) Netting neutrophils are major inducers of type I IFN production in pediatric systemic lupus erythematosus. Science Translational Medicine, 3, 73ra20.
  • Gasser, O. & Schifferli, J.A. (2004) Activated polymorphonuclear neutrophils disseminate anti-inflammatory microparticles by ectocytosis. Blood, 104, 25432548.
  • Goodridge, H.S., Reyes, C.N., Becker, C.A., Katsumoto, T.R., Ma, J., Wolf, A.J., Bose, N., Chan, A.S., Magee, A.S., Danielson, M.E., Weiss, A., Vasilakos, J.P. & Underhill, D.M. (2011) Activation of the innate immune receptor Dectin-1 upon formation of a ‘phagocytic synapse’. Nature, 472, 471475.
  • Guimaraes-Costa, A.B., Nascimento, M.T., Froment, G.S., Soares, R.P., Morgado, F.N., Conceicao-Silva, F. & Saraiva, E.M. (2009) Leishmania amazonensis promastigotes induce and are killed by neutrophil extracellular traps. Proceedings of the National Academy of Sciences of the United States of America, 106, 67486753.
  • Haak, S., Croxford, A.L., Kreymborg, K., Heppner, F.L., Pouly, S., Becher, B. & Waisman, A. (2009) IL-17A and IL-17F do not contribute vitally to autoimmune neuro-inflammation in mice. The Journal of Clinical Investigation, 119, 6169.
  • Hakkim, A., Furnrohr, B.G., Amann, K., Laube, B., Abed, U.A., Brinkmann, V., Herrmann, M., Voll, R.E. & Zychlinsky, A. (2010) Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis. Proceedings of the National Academy of Sciences of the United States of America, 107, 98139818.
  • Hasenberg, M., Köhler, A., Bonifatius, S., Borucki, K., Riek-Burchardt, M., Achilles, J., Männ, L., Baumgart, K., Schraven, B. & Gunzer, M. (2011a) Rapid immunomagnetic negative enrichment of neutrophil granulocytes from murine bone marrow for functional studies in vitro and in vivo. PLoS ONE, 6, e17314.
  • Hasenberg, M., Behnsen, J., Krappmann, S., Brakhage, A. & Gunzer, M. (2011b) Phagocyte responses towards Aspergillus fumigatus. International Journal of Medical Microbiology, 301, 436444.
  • Hasenberg, M., Köhler, A., Bonifatius, S., Jeron, A. & Gunzer, M. (2011c) Direct observation of phagocytosis and NET-formation by neutrophils in infected lungs using 2-photon microscopy. Journal of Visualized Experiments, 52, p ii, 2659.
  • Hasenberg, M., Stegemann-Koniszewski, S. & Gunzer, M. (2013) Cellular immune reactions in the lung. Immunological Reviews, 251, 189214.
  • Hirano, Y., Hizume, K., Kimura, H., Takeyasu, K., Haraguchi, T. & Hiraoka, Y. (2012) Lamin B receptor recognizes specific modifications of histone H4 in heterochromatin formation. Journal of Biological Chemistry, 287, 4265442663.
  • Hirsch, J.G. (1959) Immunity to infectious diseases: review of some concepts of Metchnikoff. Bacteriological Reviews, 23, 4860.
  • Hoffmann, K., Dreger, C.K., Olins, A.L., Olins, D.E., Shultz, L.D., Lucke, B., Karl, H., Kaps, R., Muller, D., Vaya, A., Aznar, J., Ware, R.E., Sotelo, C.N., Lindner, T.H., Herrmann, H., Reis, A. & Sperling, K. (2002) Mutations in the gene encoding the lamin B receptor produce an altered nuclear morphology in granulocytes (Pelger-Huet anomaly). Nature Genetics, 31, 410414.
  • Hosseinzadeh, A., Messer, P.K. & Urban, C.F. (2012) Stable redox-cycling nitroxide Tempol inhibits NET formation. Frontiers in Immunology, 3, 391.
  • Hurst, J.K. (2012) What really happens in the neutrophil phagosome? Free Radical Biology and Medicine, 53, 508520.
  • Jenne, C.N., Wong, C.H., Zemp, F.J., McDonald, B., Rahman, M.M., Forsyth, P.A., McFadden, G. & Kubes, P. (2013) Neutrophils recruited to sites of infection protect from virus challenge by releasing neutrophil extracellular traps. Cell Host & Microbe, 13, 169180.
  • Kash, J.C., Tumpey, T.M., Proll, S.C., Carter, V., Perwitasari, O., Thomas, M.J., Basler, C.F., Palese, P., Taubenberger, J.K., Garcia-Sastre, A., Swayne, D.E. & Katze, M.G. (2006) Genomic analysis of increased host immune and cell death responses induced by 1918 influenza virus. Nature, 443, 578581.
  • Khandpur, R., Carmona-Rivera, C., Vivekanandan-Giri, A., Gizinski, A., Yalavarthi, S., Knight, J.S., Friday, S., Li, S., Patel, R.M., Subramanian, V., Thompson, P., Chen, P., Fox, D.A., Pennathur, S. & Kaplan, M.J. (2013) NETs are a source of citrullinated autoantigens and stimulate inflammatory responses in rheumatoid arthritis. Science Translational Medicine, 5, 178ra40.
  • Kim, J.V., Kang, S.S., Dustin, M.L. & McGavern, D.B. (2009) Myelomonocytic cell recruitment causes fatal CNS vascular injury during acute viral meningitis. Nature, 457, 191195.
  • Klebanoff, S.J., Kettle, A.J., Rosen, H., Winterbourn, C.C. & Nauseef, W.M. (2013) Myeloperoxidase: a front-line defender against phagocytosed microorganisms. Journal of Leukocyte Biology, 93, 185198.
  • von Köckritz-Blickwede, M., Goldmann, O., Thulin, P., Heinemann, K., Norrby-Teglund, A., Rohde, M. & Medina, E. (2008) Phagocytosis-independent antimicrobial activity of mast cells by means of extracellular trap formation. Blood, 111, 30703080.
  • Köhler, A., De Filippo, K., Hasenberg, M., van den Brandt, C., Nye, E., Hosking, M.P., Lane, T.E., Männ, L., Ransohoff, R.M., Hauser, A.E., Winter, O., Schraven, B., Geiger, H., Hogg, N. & Gunzer, M. (2011) G-CSF mediated thrombopoietin release triggers neutrophil motility and mobilization from bone marrow via induction of Cxcr2 ligands. Blood, 117, 43494357.
  • Kolaczkowska, E. & Kubes, P. (2013) Neutrophil recruitment and function in health and inflammation. Nature Reviews Immunology, 13, 159175.
  • Kolls, J.K. & Linden, A. (2004) Interleukin-17 family members and inflammation. Immunity, 21, 467476.
  • Lämmermann, T., Afonso, P.V., Angermann, B.R., Wang, J.M., Kastenmüller, W., Parent, C.A. & Germain, R.N. (2013) Neutrophil swarms require LTB4 and integrins at sites of cell death in vivo. Nature, 498, 371375.
  • Lande, R., Ganguly, D., Facchinetti, V., Frasca, L., Conrad, C., Gregorio, J., Meller, S., Chamilos, G., Sebasigari, R., Riccieri, V., Bassett, R., Amuro, H., Fukuhara, S., Ito, T., Liu, Y.J. & Gilliet, M. (2011) Neutrophils activate plasmacytoid dendritic cells by releasing self-DNA-peptide complexes in systemic lupus erythematosus. Science Translational Medicine, 3, 73ra19.
  • Latimer, K.S., Kircher, I.M., Lindl, P.A., Dawe, D.L. & Brown, J. (1989) Leukocyte function in Pelger-Huet anomaly of dogs. Journal of Leukocyte Biology, 45, 301310.
  • Leonardi, C., Matheson, R., Zachariae, C., Cameron, G., Li, L., Edson-Heredia, E., Braun, D. & Banerjee, S. (2012) Anti-interleukin-17 monoclonal antibody ixekizumab in chronic plaque psoriasis. New England Journal of Medicine, 366, 11901199.
  • Li, P., Li, M., Lindberg, M.R., Kennett, M.J., Xiong, N. & Wang, Y. (2010) PAD4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps. Journal of Experimental Medicine, 207, 18531862.
  • Li, K.W., Turner, S.M., Emson, C.L., Hellerstein, M.K. & Dale, D.C. (2011) Deuterium and neutrophil kinetics. Blood, 117, 60526053.
  • Manitz, M.P., Horst, B., Seeliger, S., Strey, A., Skryabin, B.V., Gunzer, M., Frings, W., Schonlau, F., Roth, J., Sorg, C. & Nacken, W. (2003) Loss of S100A9 (MRP14) results in reduced interleukin-8-induced CD11b surface expression, a polarized microfilament system, and diminished responsiveness to chemoattractants in vitro. Molecular and Cellular Biology, 23, 10341043.
  • Martinod, K., Demers, M., Fuchs, T.A., Wong, S.L., Brill, A., Gallant, M., Hu, J., Wang, Y. & Wagner, D.D. (2013) Neutrophil histone modification by peptidylarginine deiminase 4 is critical for deep vein thrombosis in mice. Proceedings of the National Academy of Sciences of the United States of America, 110, 86748679.
  • Massberg, S., Grahl, L., von Bruehl, M.L., Manukyan, D., Pfeiler, S., Goosmann, C., Brinkmann, V., Lorenz, M., Bidzhekov, K., Khandagale, A.B., Konrad, I., Kennerknecht, E., Reges, K., Holdenrieder, S., Braun, S., Reinhardt, C., Spannagl, M., Preissner, K.T. & Engelmann, B. (2010) Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases. Nature Medicine, 16, 887896.
  • McDonald, B., Pittman, K., Menezes, G.B., Hirota, A.A., Slaba, I., Waterhouse, C.C.M., Beck, P.L., Muruve, D.A. & Kubes, P. (2010) Intravascular danger signals guide neutrophils to sites of sterile inflammation. Science, 330, 362366.
  • McDonald, B., Urrutia, R., Yipp, B.G., Jenne, C.N. & Kubes, P. (2012) Intravascular neutrophil extracellular traps capture bacteria from the bloodstream during sepsis. Cell Host & Microbe, 12, 324333.
  • Menegazzi, R., Decleva, E. & Dri, P. (2012) Killing by neutrophil extracellular traps: fact or folklore? Blood, 119, 12141216.
  • Mocsai, A. (2013) Diverse novel functions of neutrophils in immunity, inflammation, and beyond. Journal of Experimental Medicine, 210, 12831299.
  • Nauseef, W.M. (2007) How human neutrophils kill and degrade microbes: an integrated view. Immunological Reviews, 219, 88102.
  • Nauseef, W.M. (2012) Editorial: Nyet to NETs? A pause for healthy skepticism. Journal of Leukocyte Biology, 91, 353355.
  • Niesner, R.A., Narang, P., Spiecker, H., Andresen, V., Gericke, K.H. & Gunzer, M. (2008) Selective detection of NADPH oxidase in polymorphonuclear cells by means of NAD(P)H-based fluorescence lifetime imaging. Journal of Biophysics, 2008, Article ID 602639.
  • Norris, B.A., Uebelhoer, L.S., Nakaya, H.I., Price, A.A., Grakoui, A. & Pulendran, B. (2013) Chronic but not acute virus infection induces sustained expansion of myeloid suppressor cell numbers that inhibit viral-specific T cell immunity. Immunity, 38, 309321.
  • Nunes, P., Cornut, D., Bochet, V., Hasler, U., Oh-Hora, M., Waldburger, J.M. & Demaurex, N. (2012) STIM1 juxtaposes ER to phagosomes, generating Ca(2)(+) hotspots that boost phagocytosis. Current Biology, 22, 19901997.
  • Ostrand-Rosenberg, S., Sinha, P., Beury, D.W. & Clements, V.K. (2012) Cross-talk between myeloid-derived suppressor cells (MDSC), macrophages, and dendritic cells enhances tumor-induced immune suppression. Seminars in Cancer Biology, 22, 275281.
  • Papp, K.A., Leonardi, C., Menter, A., Ortonne, J.P., Krueger, J.G., Kricorian, G., Aras, G., Li, J., Russell, C.B., Thompson, E.H. & Baumgartner, S. (2012) Brodalumab, an anti-interleukin-17-receptor antibody for psoriasis. New England Journal of Medicine, 366, 11811189.
  • Patel, D.D., Lee, D.M., Kolbinger, F. & Antoni, C. (2013) Effect of IL-17A blockade with secukinumab in autoimmune diseases. Annals of the Rheumatic Diseases, 72(Suppl. 2), ii116ii123.
  • Peters, N.C., Egen, J.G., Secundino, N., Debrabant, A., Kimblin, N., Kamhawi, S., Lawyer, P., Fay, M.P., Germain, R.N. & Sacks, D. (2008) In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies. Science, 321, 970974.
  • Pillay, J., den Braber I., Vrisekoop, N., Kwast, L.M., de Boer, R.J., Borghans, J.A., Tesselaar, K. & Koenderman, L. (2010) In vivo labeling with 2H2O reveals a human neutrophil lifespan of 5.4 days. Blood, 116, 625627.
  • Pillay, J., van, H.E., Visser, T., Tak, T., Lammers, J.W., Ulfman, L.H., Leenen, L.P., Pickkers, P. & Koenderman, L. (2012) A subset of neutrophils in human systemic inflammation inhibits T cell responses through Mac-1. The Journal of Clinical Investigation, 122, 327336.
  • Proebstl, D., Voisin, M.B., Woodfin, A., Whiteford, J., D'Acquisto, F., Jones, G.E., Rowe, D. & Nourshargh, S. (2012) Pericytes support neutrophil subendothelial cell crawling and breaching of venular walls in vivo. Journal of Experimental Medicine, 209, 12191234.
  • Puel, A., Cypowyj, S., Bustamante, J., Wright, J.F., Liu, L., Lim, H.K., Migaud, M., Israel, L., Chrabieh, M., Audry, M., Gumbleton, M., Toulon, A., Bodemer, C., El-Baghdadi, J., Whitters, M., Paradis, T., Brooks, J., Collins, M., Wolfman, N.M., Al-Muhsen, S., Galicchio, M., Abel, L., Picard, C. & Casanova, J.L. (2011) Chronic mucocutaneous candidiasis in humans with inborn errors of interleukin-17 immunity. Science, 332, 6568.
  • Puellmann, K., Kaminski, W.E., Vogel, M., Nebe, C.T., Schroeder, J., Wolf, H. & Beham, A.W. (2006) A variable immunoreceptor in a subpopulation of human neutrophils. Proceedings of the National Academy of Sciences of the United States of America, 103, 1444114446.
  • Reeves, E.P., Lu, H., Jacobs, H.L., Messina, C.G., Bolsover, S., Gabella, G., Potma, E.O., Warley, A., Roes, J. & Segal, A.W. (2002) Killing activity of neutrophils is mediated through activation of proteases by K+ flux. Nature, 416, 291297.
  • Remijsen, Q., Kuijpers, T.W., Wirawan, E., Lippens, S., Vandenabeele, P. & Vanden Berghe, T. (2011) Dying for a cause: NETosis, mechanisms behind an antimicrobial cell death modality. Cell Death and Differentiation, 18, 581588.
  • Rowat, A.C., Jaalouk, D.E., Zwerger, M., Ung, W.L., Eydelnant, I.A., Olins, D.E., Olins, A.L., Herrmann, H., Weitz, D.A. & Lammerding, J. (2013) Nuclear envelope composition determines the ability of neutrophil-type cells to passage through micron-scale constrictions. Journal of Biological Chemistry, 288, 86108618.
  • Sasse, C., Hasenberg, M., Weyler, M., Gunzer, M. & Morschhäuser, J. (2013) White-opaque switching of Candida albicans allows immune evasion in an environment-dependent fashion. Eukaryotic Cell, 12, 5058.
  • Segal, A.W. (2005) How neutrophils kill microbes. Annual Review of Immunology, 23, 197223.
  • Springer, T.A. (1994) Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell, 76, 301314.
  • Stein, S., Scholz, S., Schwaeble, J., Sadat, M.A., Modlich, U., Schultze-Strasser, S., Diaz, M., Chen-Wichmann, L., Mueller-Kuller, U., Brendel, C., Fronza, R., Kaufmann, K.B., Naundorf, S., Pech, N.K., Travers, J.B., Matute, J.D., Presson, R.G. Jr, Sandusky, G.E., Kunkel, H., Rudolf, E., Dillmann, A., von, K.C., Kuehlcke, K., Baum, C., Schambach, A., Dinauer, M.C., Schmidt, M. & Grez, M. (2013) From bench to bedside: preclinical evaluation of a SIN gammaretroviral vector for the gene therapy of X-linked chronic granulomatous disease. Human Gene Therapy Clinical Development, 24, 8698.
  • Steinberg, B.E. & Grinstein, S. (2007) Unconventional roles of the NADPH oxidase: signaling, ion homeostasis, and cell death. Science Signaling, 2007, e11.
  • Stewart, C.L., Roux, K.J. & Burke, B. (2007) Blurring the boundary: the nuclear envelope extends its reach. Science, 318, 14081412.
  • Sundd, P., Gutierrez, E., Koltsova, E.K., Kuwano, Y., Fukuda, S., Pospieszalska, M.K., Groisman, A. & Ley, K. (2012) ‘Slings’ enable neutrophil rolling at high shear. Nature, 488, 399403.
  • Tak, T., Tesselaar, K., Pillay, J., Borghans, J.A. & Koenderman, L. (2013) What's your age again? Determination of human neutrophil half-lives revisited. Journal of Leukocyte Biology, 94, 595601.
  • Tittel, A.P., Heuser, C., Ohliger, C., Llanto, C., Yona, S., Hammerling, G.J., Engel, D.R., Garbi, N. & Kurts, C. (2012) Functionally relevant neutrophilia in CD11c diphtheria toxin receptor transgenic mice. Nature Methods, 9, 385390.
  • Tofts, P.S., Chevassut, T., Cutajar, M., Dowell, N.G. & Peters, A.M. (2011) Doubts concerning the recently reported human neutrophil lifespan of 5.4 days. Blood, 117, 60506052.
  • Underhill, D.M. & Goodridge, H.S. (2012) Information processing during phagocytosis. Nature Reviews Immunology, 12, 492502.
  • Urban, C.F., Reichard, U., Brinkmann, V. & Zychlinsky, A. (2006) Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms. Cellular Microbiology, 8, 668676.
  • Urban, C.F., Ermert, D., Schmid, M., Abu-Abed, U., Goosmann, C., Nacken, W., Brinkmann, V., Jungblut, P.R. & Zychlinsky, A. (2009) Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans. PLoS Pathogens, 5, e1000639.
  • Wang, S., Voisin, M.B., Larbi, K.Y., Dangerfield, J., Scheiermann, C., Tran, M., Maxwell, P.H., Sorokin, L. & Nourshargh, S. (2006) Venular basement membranes contain specific matrix protein low expression regions that act as exit points for emigrating neutrophils. Journal of Experimental Medicine, 203, 15191532.
  • Wang, K., Lin, B., Lin, J. & Lan, X. (2013) A novel mutation in the myeloperoxidase gene in a Chinese female with complete myeloperoxidase deficiency: the role of nonsense-mediated mRNA decay. Gene, 515, 205207.
  • Weaver, C.T., Hatton, R.D., Mangan, P.R. & Harrington, L.E. (2007) IL-17 family cytokines and the expanding diversity of effector T cell lineages. Annual Review of Immunology, 25, 821852.
  • Winterbourn, C.C. & Kettle, A.J. (2013) Redox reactions and microbial killing in the neutrophil phagosome. Antioxidants & Redox Signaling, 18, 642660.
  • Yousefi, S., Gold, J.A., Andina, N., Lee, J.J., Kelly, A.M., Kozlowski, E., Schmid, I., Straumann, A., Reichenbach, J., Gleich, G.J. & Simon, H.U. (2008) Catapult-like release of mitochondrial DNA by eosinophils contributes to antibacterial defense. Nature Medicine, 14, 949953.
  • Zhang, X., Kluger, Y., Nakayama, Y., Poddar, R., Whitney, C., DeTora, A., Weissman, S.M. & Newburger, P.E. (2004) Gene expression in mature neutrophils: early responses to inflammatory stimuli. Journal of Leukocyte Biology, 75, 358372.
  • Zhou, S.L., Dai, Z., Zhou, Z.J., Wang, X.Y., Yang, G.H., Wang, Z., Huang, X.W., Fan, J. & Zhou, J. (2012) Overexpression of CXCL5 mediates neutrophil infiltration and indicates poor prognosis for hepatocellular carcinoma. Hepatology, 56, 22422254.