Produced in the bone marrow at a rate of 1011 per day , neutrophils are the most abundant leukocyte in the human circulation and a critical component of the innate immune system. They provide front-line protection against infection with rapidly dividing bacteria, yeast and fungi, able to unleash an array of antimicrobial mechanisms aimed at containing and eliminating the invading pathogen. These strategies include the generation of reactive oxygen species (ROS), degranulation to release bactericidal granule contents such as myeloperoxidase, and the recently described release of extracellular traps enabling extracellular killing of bacteria [see earlier review in this series by Klaus T. Preissner et al. (pp. 3–9)].
In the absence of inflammation, neutrophils have a short blood half-life , after which they home to the spleen, liver or bone marrow to be destroyed . However, at sites of inflammation neutrophil lifespan is extended as a result of exposure to pro-inflammatory mediators such as granulocyte macrophage colony-stimulating factor (GM-CSF)  and type 1 interferons , or by environmental cues such as hypoxia , thus allowing them to retain their functional capacity.
Whilst toxic to the pathogen, neutrophil-mediated defence strategies are damaging to the surrounding tissue. If not tightly regulated, inappropriate survival of neutrophils leads to a chronic inflammatory state which can contribute to diseases such as rheumatoid arthritis (RA) [7,8]. Thus, it is critical that after performing their function, neutrophils are cleared in order to limit bystander tissue damage. This is achieved by induction of apoptosis, a form of programmed cell death. Upon entering apoptosis, neutrophils show characteristic morphological changes, including a reduction in cell size and nuclear condensation. Following apoptosis, neutrophils also display ‘eat me’ signals in order to aid their clearance by macrophages . Despite the importance of neutrophil apoptosis in the resolution of inflammation, the precise mechanisms that control this process are still incompletely understood, though recent research has shed light upon key mechanisms. Improved understanding of this process may therefore provide potential novel targets for the treatment of inflammatory diseases.
In the absence of pro-survival signals, neutrophils have a short blood half-life . In culture, neutrophils will survive for around 18–24 h before undergoing constitutive apoptosis [10,11]. The spontaneous entry of neutrophils into apoptosis is fundamental in maintaining the homeostasis of circulating neutrophil numbers, as well as ensuring the efficient resolution of the inflammatory response.
Neutrophil apoptosis was originally thought to occur by one of two pathways, the extrinsic and intrinsic pathways. The extrinsic pathway involves the ligation of death receptors, such as Fas, on the surface of neutrophils. Ligation of death receptors by their ligands promotes the recruitment of adaptor molecules such as Fas-associated protein with death domain and promotes the aggregation and activation of caspase-8, leading to apoptosis [12,13]. The intrinsic pathway is triggered by mitochondrial membrane depolarization resulting in the release of cytochrome-c. Once released from the mitochondria, cytochrome-c can bind to apoptosis-promoting activating factor-1 (Apaf-1) resulting in its oligomerization, leading to caspase-9 activation and apoptosis . However, our own studies have shown that neutrophils use a novel variant in that they can activate the extrinsic pathway in the absence of death receptor ligation. We showed that neutrophils are unable to maintain redox status which leads to the ROS-mediated activation of sphingomyelinase in the cell membrane and subsequent generation of ceramide . We propose that this leads to ligand-independent aggregation of death receptors into lipid rafts, explaining the activation of caspase 8 during neutrophil apoptosis which is not inhibited by blockade of Fas.
Central to these processes is the balance between pro and antiapoptotic Bcl-2 family members. In particular, it has been demonstrated that levels of the antiapoptotic protein Mcl-1 decrease during neutrophil lifespan , and that levels of this protein can be maintained by factors that extend neutrophil lifespan such as GM-CSF . Given its apparent central role in neutrophil survival, understanding the precise mechanism by which Mcl-1 levels are controlled in neutrophils is the subject of much research.
Recent work has suggested an unexpected role for cyclin-dependent kinases (CDKs) in the regulation of neutrophil apoptosis. CDKs are a family of proteins that were originally characterized by their ability to regulate cell cycle progression. It is perhaps surprising therefore that terminally differentiated cells would express CDKs. Indeed, in humans, promyeloid progenitor cells have been shown to lose their expression of cell cycle-dependent CDKs (CDK 1, 2, 4 and 6) as they differentiate towards neutrophils . The study by Rossi et al., however, reported that human neutrophils express CDK1, CDK2 and CDK5 at the protein level. Moreover, while levels of these proteins did not change during neutrophil lifespan, CDK1 activity was decreased rapidly during apoptosis in response to the activating Fas antibody CH11, thus suggesting a role for CDK activity in neutrophil apoptosis . In addition, this was shown to correlate with the down-regulation of Mcl-1 , suggestive of a role for CDKs in the regulation of Mcl-1 levels. More recent data from our laboratory have demonstrated that the cell cycle-independent CDK, CDK9, is a key regulator of spontaneous apoptosis in neutrophils isolated from human peripheral blood [19▪▪]. We found freshly isolated neutrophils expressed the cell cycle-independent CDKs, CDK5, CDK7 and CDK9, at the protein level, but did not express the cell cycle-dependent CDKs, CDK1, CDK2, CDK4 or CDK6 [19▪▪]. Importantly, CDK9 activity levels decreased during the neutrophil lifespan and were found to correlate with decreased expression of Mcl-1 and spontaneous neutrophil apoptosis [19▪▪]. In addition, neutrophil apoptosis and Mcl-1 down-regulation could be accelerated in the presence of flavopiridol, a specific CDK9 inhibitor, further supporting a role for CDK9 in regulating neutrophil lifespan [19▪▪]. Leitch et al.[20▪▪] have also found that human neutrophils express high levels of CDK7 and CDK9 and that the broad-range CDK inhibitor R-roscovitine, as well as the CDK7 and CDK9-specific inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), could reduce CDK7 and CDK9 activity and accelerate neutrophil apoptosis, which correlated with Mcl-1 down-regulation [20▪▪].
Taken together, these recent data demonstrate that CDK9 activity has an important role in neutrophil survival and that its activity may be responsible for alterations in Mcl-1 levels (summarized in Fig. 1a). The mechanisms by which CDKs regulate Mcl-1 levels, however, are yet to be elucidated, although a role for activation of RNA polymerase II leading to effects on transcriptional activity has been proposed [19▪▪,20▪▪]. The use of CDK inhibitors such as flavopiridol and R-roscovitine may prove a useful tool in the treatment of chronic inflammatory diseases such as RA (see later section).
INDUCTION OF NEUTROPHIL APOPTOSIS BY IMMUNE CELLS
As outlined above, neutrophil lifespan is extended at sites of infection as a result of exposure to pro-inflammatory cytokines and bacterial products. Regulating neutrophil lifespan at times of infection is therefore critical to prevent excessive and inappropriate neutrophil activity that would otherwise damage surrounding tissue.
In a mouse model of acute lung injury, D’Alessio et al. recovered significantly more live neutrophils from the bronchoalveolar lavage (BAL) of both Rag1−/− and T-regulatory cell (Treg)-depleted mice when compared to wild-type mice, suggesting a role for this T-cell subset in influencing neutrophil survival in vivo. Indeed, the adoptive transfer of Tregs into Rag1−/− mice was found to significantly increase neutrophil apoptosis, an effect the group also observed when Tregs were added directly to neutrophil cultures in vitro. These data confirmed previous work in humans that had shown neutrophil death to be accelerated in the presence of Tregs . Importantly, in both these studies, Tregs were shown to induce the apoptosis of neutrophils that had been previously exposed to lipopolysaccharide (LPS), suggesting that at a site of infection, Tregs would be able to override neutrophil pro-survival signals [21,22]. The mechanism by which Tregs induce neutrophil apoptosis is currently unknown, although a role for perforin/granzyme cytotoxicity has been proposed (Fig. 1b).
In addition to Tregs, natural killer (NK) cells are also capable of mediating neutrophil apoptosis. Requiring direct contact and mediated through the Fas pathway, human NK cells have been shown to induce apoptosis of both resting and GM-CSF-treated neutrophils, suggesting that as with Tregs, NK-mediated apoptosis would occur at an inflammatory site [23▪▪]. Expression of an as of yet unidentified ligand for the NK cell activatory receptor Nkp46 on the surface of neutrophils is thought to trigger NK activation since blocking antibodies against this receptor significantly protected neutrophils from NK cell-induced apoptosis [23▪▪] (Fig. 1b).
Interestingly, in the absence of direct contact, human NK cells appear to promote the survival of neutrophils. In two studies, it has been shown that exposing neutrophils to supernatants derived from cytokine-activated NK cells  or culturing neutrophils with NK cells in a transwell system  markedly reduces the rate of neutrophil apoptosis, which in both instances was attributed to the production of interferon-γ and GM-CSF by activated NK cells [24,25]. In the study by Costantini et al., the pro-survival effect of NK cells on neutrophil survival was reduced when activated NK cells and neutrophils were cultured together in the absence of a transwell, providing further evidence that direct NK–neutrophil interaction promotes neutrophil cell death. Indeed, when the cells were co-cultured in the presence of neutralizing antibodies against CD18 to prevent their interaction, neutrophil survival rates were comparable to those observed in the transwell system .
TARGETING NEUTROPHIL APOPTOSIS FOR THE TREATMENT OF INFLAMMATORY DISEASE
Inhibition of neutrophil apoptosis is associated with numerous chronic inflammatory diseases including RA  and acute respiratory distress syndrome (ARDS) . Because of this, targeting the induction of neutrophil apoptosis has emerged as a potential treatment for chronic inflammatory diseases. Indeed, it has been demonstrated that the injection of apoptotic neutrophils was able to protect mice from LPS-induced death . This was due in part to the fact that apoptotic neutrophils cause macrophages to switch from a tumour necrosis factor (TNF)-α-producing inflammatory phenotype to an interleukin-10-producing regulatory phenotype . Thus, inducing neutrophil apoptosis may aid the resolution of inflammation not only by the removal of the neutrophils themselves, but also by altering macrophage phenotype.
DEATH RECEPTOR LIGANDS
Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane receptor and a member of the TNF-receptor family. TRAIL can bind to two receptors expressed on the surface of neutrophils, namely TRAIL-R1  and TRAIL-R2 . Once bound, these receptors can recruit adaptor proteins which contain cytoplasmic death domains and are able to activate caspase-8, leading to apoptosis .
Recently McGrath et al.[31▪] demonstrated that targeting TRAIL in order to induce neutrophil apoptosis may hold promise as a potential treatment for inflammatory disease. In two murine models of inflammation, zymosan-induced peritonitis and LPS-induced lung injury, the authors were able to demonstrate that mice deficient in TRAIL showed an enhanced inflammatory response and increased neutrophil numbers concomitant with reduced neutrophil apoptosis [31▪]. Importantly they went on to show that exogenous administration of TRAIL resulted in increased neutrophil apoptosis and accelerated resolution of inflammation in both wild-type and TRAIL-deficient mice [31▪]. In addition, they found no effects of TRAIL administration on other cell types suggestive of a neutrophil-dependent mechanism [31▪]. This use of TRAIL to induce inflammatory resolution is particularly exciting as studies using other death receptor ligands have not proved as promising. For example, administration of Fas-L to the lungs of mice leads to neutrophil infiltration and pulmonary inflammation .
CYCLIN-DEPENDENT KINASE INHIBITORS
As eluded to earlier, CDKs, via their ability to regulate expression of the antiapoptotic protein Mcl-1, are important regulators of neutrophil lifespan [18,19▪▪,20▪▪]. Therefore, inhibition of CDK activity represents a potential therapeutic strategy through which to drive inflammatory resolution. Indeed, in the first study of its kind, Rossi et al. demonstrated in three murine models of inflammation (carrageenan-induced pleural inflammation, bleomycin-induced lung injury and passively induced arthritis) that administration of the broad-acting CDK inhibitor R-roscovitine could accelerate resolution. In all three models, treatment was associated with a reduction in tissue damage, which in the case of carrageenan-induced pleural inflammation was attributed in part to the induction of inflammatory cell apoptosis . The group have since confirmed in a separate study the ability of R-roscovitine and a second CDK inhibitor, DRB, to drive resolution in the bleomycin-induced lung injury model [20▪▪]. However, in contrast to their previous work, the group delivered the CDK inhibitors via the intra-tracheal route in an effort to mimic the delivery of standard inhalation therapy [20▪▪]. In addition, earlier studies demonstrated that the CDK9 inhibitor flavopiridol was effective at reducing joint inflammation in the collagen-induced arthritis model, although the cellular target was not found in this study . The success of these therapies in animal models has exciting implications for the possible translation of these findings into human clinical trials.
Recent advances in the field of neutrophil apoptosis have uncovered an emerging role for CDKs in the regulation of neutrophil lifespan, as well as a newly identified role for Tregs and NK cells in the induction of neutrophil apoptosis. The importance of these findings is likely to extend beyond merely increasing our understanding of neutrophil biology and may present novel avenues for the treatment of chronic inflammatory diseases.
Conflicts of interest
There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
- ▪ of special interest
- ▪▪ of outstanding interest
Additional references related to this topic can also be found in the Current World Literature section in this issue (p. 67).
1. Athens JW, Haab OP, Raab SO, et al. Leukokinetic studies. IV. The total blood, circulating and marginal granulocyte pools and the granulocyte turnover rate in normal subjects. J Clin Invest 1961; 40:989–995.
2. Dancey JT, Deubelbeiss KA, Harker LA, Finch CA. Neutrophil kinetics in man. J Clin Invest 1976; 58:705–715.
3. Martin C, Burdon PC, Bridger G, et al. Chemokines acting via CXCR2 and CXCR4 control the release of neutrophils from the bone marrow and their return following senescence. Immunity 2003; 19:583–593.
4. Brach MA, deVos S, Gruss HJ, Herrmann F. Prolongation of survival of human polymorphonuclear neutrophils by granulocyte-macrophage colony-stimulating factor is caused by inhibition of programmed cell death. Blood 1992; 80:2920–2924.
5. Wang K, Scheel-Toellner D, Wong SH, et al. Inhibition of neutrophil apoptosis by type 1 IFN depends on cross-talk between phosphoinositol 3-kinase, protein kinase C-delta, and NF-kappa B signaling pathways. J Immunol 2003; 171:1035–1041.
6. Cross A, Barnes T, Bucknall RC, et al. Neutrophil apoptosis in rheumatoid arthritis is regulated by local oxygen tensions within joints. J Leukoc Biol 2006; 80:521–528.
7. Edwards SW, Hallett MB. Seeing the wood for the trees: the forgotten role of neutrophils in rheumatoid arthritis. Immunol Today 1997; 18:320–324.
8. Raza K, Scheel-Toellner D, Lee CY, et al. Synovial fluid leukocyte apoptosis is inhibited in patients with very early rheumatoid arthritis. Arthritis Res Ther 2006; 8:R120.
9. Savill J, Dransfield I, Hogg N, Haslett C. Vitronectin receptor-mediated phagocytosis of cells undergoing apoptosis. Nature 1990; 343:170–173.
10. Savill JS, Wyllie AH, Henson JE, et al. Macrophage phagocytosis of aging neutrophils in inflammation. Programmed cell death in the neutrophil leads to its recognition by macrophages. J Clin Invest 1989; 83:865–875.
11. Scheel-Toellner D, Wang K, Craddock R, et al. Reactive oxygen species limit neutrophil life span by activating death receptor signaling. Blood 2004; 104:2557–2564.
12. Scaffidi C, Fulda S, Srinivasan A, et al. Two CD95 (APO-1/Fas) signaling pathways. EMBO J 1998; 17:1675–1687.
13. Schulze-Osthoff K, Ferrari D, Los M, et al. Apoptosis signaling by death receptors. Eur J Biochem 1998; 254:439–459.
14. Li P, Nijhawan D, Budihardjo I, et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 1997; 91:479–489.
15. Leuenroth SJ, Grutkoski PS, Ayala A, Simms HH. The loss of Mcl-1 expression in human polymorphonuclear leukocytes promotes apoptosis. J Leukoc Biol 2000; 68:158–166.
16. Moulding DA, Quayle JA, Hart CA, Edwards SW. Mcl-1 expression in human neutrophils: regulation by cytokines and correlation with cell survival. Blood 1998; 92:2495–2502.
17. Klausen P, Bjerregaard MD, Borregaard N, Cowland JB. End-stage differentiation of neutrophil granulocytes in vivo is accompanied by up-regulation of p27kip1 and down-regulation of CDK2, CDK4, and CDK6. J Leukoc Biol 2004; 75:569–578.
18. Rossi AG, Sawatzky DA, Walker A, et al. Cyclin-dependent kinase inhibitors enhance the resolution of inflammation by promoting inflammatory cell apoptosis. Nat Med 2006; 12:1056–1064.
19▪▪. Wang K, Hampson P, Hazeldine J, et al. Cyclin-dependent kinase 9 activity regulates neutrophil spontaneous apoptosis. PLoS One 2012; 7:e30128.
This study was the first to describe a role for CDK9 activity in the regulation of neutrophil lifespan. In addition this study idenitifies flavopiridol as a potential novel treatment for chronic inflammatory disease.
20▪▪. Leitch AE, Lucas CD, Marwick JA, et al
. Cyclin-dependent kinases 7 and 9 specifically regulate neutrophil transcription and their inhibition drives apoptosis to promote resolution of inflammation. Cell Death Differ 2012. [Epub ahead of print]
This study described a role for both CDK9 and CDK7 activity in the regulation of neutrophil lifespan. The study is also the first to show that CDKs do this by driving neutrophil transcription.
21. D’Alessio FR, Tsushima K, Aggarwal NR, et al. CD4+CD25+Foxp3+ Tregs resolve experimental lung injury in mice and are present in humans with acute lung injury. J Clin Invest 2009; 119:2898–2913.
22. Lewkowicz P, Lewkowicz N, Sasiak A, Tchorzewski H. Lipopolysaccharide-activated CD4+CD25+ T regulatory cells inhibit neutrophil function and promote their apoptosis and death. J Immunol 2006; 177:7155–7163.
23▪▪. Thoren FB, Riise RE, Ousback J, et al. Human NK cells induce neutrophil apoptosis via an NKp46- and Fas-dependent mechanism. J Immunol 2012; 188:1668–1674.
This study was the first to show that NK cells could directly induce neutrophil apoptosis. This required cell to cell contact and was mediated by the activating NK cell receptor NKp46 and the Fas pathway.
24. Bhatnagar N, Hong HS, Krishnaswamy JK, et al. Cytokine-activated NK cells inhibit PMN apoptosis and preserve their functional capacity. Blood 2010; 116:1308–1316.
25. Costantini C, Micheletti A, Calzetti F, et al. Neutrophil activation and survival are modulated by interaction with NK cells. Int Immunol 2010; 22:827–838.
26. Aldridge AJ. Role of the neutrophil in septic shock and the adult respiratory distress syndrome. Eur J Surg 2002; 168:204–214.
27. Ren Y, Xie Y, Jiang G, et al. Apoptotic cells protect mice against lipopolysaccharide-induced shock. J Immunol 2008; 180:4978–4985.
28. Pan G, O’Rourke K, Chinnaiyan AM, et al. The receptor for the cytotoxic ligand TRAIL. Science 1997; 276:111–113.
29. Chaudhary PM, Eby M, Jasmin A, et al. Death receptor 5, a new member of the TNFR family, and DR4 induce FADD-dependent apoptosis and activate the NF-kappaB pathway. Immunity 1997; 7:821–830.
30. Gonzalvez F, Ashkenazi A. New insights into apoptosis signaling by Apo2L/TRAIL. Oncogene 2010; 29:4752–4765.
31▪. McGrath EE, Marriott HM, Lawrie A, et al. TNF-related apoptosis-inducing ligand (TRAIL) regulates inflammatory neutrophil apoptosis and enhances resolution of inflammation. J Leukoc Biol 2011; 90:855–865.
This study highlights the potential therapeutic benefit of TRAIL in neutrophilic inflammation.
32. Wortinger MA, Foley JW, Larocque P, et al. Fas ligand-induced murine pulmonary inflammation is reduced by a stable decoy receptor 3 analogue. Immunology 2003; 110:225–233.
33. Sekine C, Sugihara T, Miyake S, et al. Successful treatment of animal models of rheumatoid arthritis with small-molecule cyclin-dependent kinase inhibitors. J Immunol 2008; 180:1954–1961.