Neutrophil cell death in response to infection and its relation to coagulation
© Iba et al.; licensee BioMed Central Ltd. 2013
Received: 27 September 2013
Accepted: 13 November 2013
Published: 4 December 2013
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© Iba et al.; licensee BioMed Central Ltd. 2013
Received: 27 September 2013
Accepted: 13 November 2013
Published: 4 December 2013
Neutrophil is a major player in the pathophysiology of severe sepsis. Recent studies have revealed that the cell death mechanism of neutrophils directly relates to the development of organ dysfunction during sepsis. Here we discuss about the different types of neutrophil cell death such as necrosis, apoptosis, autophagy, and the unique cell death style dubbed NETosis. NETosis cells release neutrophil extracellular traps (NETs), which are composed of chromatin bound to granular and nucleic proteins. The primary purpose of NET release is thought to be the control of microbial infections; however, it acts as a danger signal for the host as well. The harmful substances such as DNA, histones, and high-mobility group box 1 (HMGB1) and many other danger-associated molecular patterns (DAMPs) released along with NETosis or from necrotic neutrophils also contribute to the pathogenesis of sepsis. At the same time, the coagulation system, which is closely tied to these neutrophil cell death mechanisms, is often over-activated. It is well known that individual bacterial pathogens express virulence factors that modulate cell death pathways and influence the coagulation disorder during sepsis. Moreover, extensive cross talk exists between these two phenomena, whereby inflammation leads to activation of coagulation and coagulation considerably affects inflammatory activity. A greater knowledge of cell death pathways in sepsis informs the potential for future therapies designed to ameliorate excessive immune responses during sepsis.
Neutrophils play a central role in the innate immune system . The well-known functions of neutrophils are the migration to the infection site where they engulf and inactivate microorganisms through the fusion of phagosomes with granules and the formation of phagolysosomes, in which antimicrobial enzymes and reactive oxygen species (ROS) act synergistically for the clearance of pathogens . Excessive neutrophil activation results in degranulation and release of ROS into the extracellular medium, which leads to host tissue injury , while neutrophil apoptosis contributes to the resolution of inflammation .
Neutrophil cell death is divided into necrosis, apoptosis (type 1 programmed cell death), autophagy (type 2 programmed cell death), the newly recognized NETosis, and some other types. Necrosis is characterized as cell death exhibiting cytoplasmic swelling, disorganized organelles, ruptured plasma membrane, and lytic nucleus . In contrast, chromatin condensation is the notable feature of apoptosis, which later proceeds to the fragmentation of the nucleus and formation of apoptotic bodies consisting of intact plasma membrane and various organelles. Autophagy is known as a process of degradation of the self-components with a purpose of recycling cytoplasmic components. It is characterized by the formation of autophagosomes, which are large vesicles containing cytosol and organelle, which then fuse with lysosomes and are degraded without cell damage . As a matter of fact, these forms of cell death appear to be transformable. For example, autophagy is thought to be a reversible cell death; however, once autophagic capacity is overwhelmed by certain stimulations, apoptosis is triggered. Apoptotic cells often turn to necrosis if the stimulus is too strong, and this type of cell death is known as aponecrosis.
The fourth style of neutrophil cell death NETosis is quite unique. NETosis is a type of programmed cell death. Neutrophils can kill pathogens extracellularly by releasing neutrophil extracellular traps (NETs) . The impact of NETs derives from the combined antimicrobial activities of chromatin, histone, elastase, and other cytoplasmic proteins. In this review, we introduce the mechanism and the morphological findings of major types of neutrophil deaths in the following part.
Neutrophils are inherently short-lived, approximately 5–6 days, and undergo spontaneous apoptosis . In infected tissues, their apoptosis can be delayed both by microbial constituents and by pro-inflammatory stimuli [10, 11]. Generally, the tissue neutrophils die in apoptosis; however, if the infection is serious enough, some undergo necrosis or other styles of cell death. Except for apoptotic and autophagic cell deaths, uncontrolled release of toxic substances from the dead neutrophils can propagate the inflammatory response leading to tissue damage. Therefore, avoidance of unprogrammed death and scavenging of the dying neutrophils is crucial for the maintenance of homeostasis [12, 13]. To terminate the inflammation, it is necessary not only to attenuate the generation of anti-inflammatory mediators but also to remove the inflammatory cells along with the microbes they have ingested .
Most neutrophils undergo apoptosis after they leave the peripheral circulation without infection . When apoptosis proceeds in an orderly fashion, tissue macrophages and other phagocytes ingest the apoptotic bodies which include potentially injurious granular enzymes. In contrast, necrosis is a turbulent cell death. If this accidental cell death is triggered by unexpected events, toxic constituents including proteolytic enzymes and oxidant-generating enzymes are released from the necrotic cells in an unregulated manner. Neutrophil necrosis is probably one of the major causes of tissue damage during infection , but little is known as to how they undergo necrosis, and there is no simple method that can detect the neutrophils undergoing necrosis.
Necrotic cells release a variety of danger signals known as damage-associated molecular patterns (DAMPs) such as high-mobility group box 1 (HMGB1), uric acid, heat shock proteins, DNA-chromatin complexes, and antimicrobial peptides. Many of these substances are recognized by specific receptors named pattern-recognizing receptors (PRRs) and stimulate the synthesis of pro-inflammatory mediators. For example, HMGB1, a nuclear protein binding to DNA and regulating gene transcription, is released from the necrotic cells and has been shown to stimulate inflammatory cytokine secretion by monocytes . Uric acid and its active form, monosodium urate (MSU), are released to the cytosolic compartment under inflammatory stimulation. MSU has recently attracted attention as a strong inducer of inflammatory reaction . DNA-chromatin complexes  and heat shock proteins [20, 21] have also been shown to stimulate pro-inflammatory cytokine production . Since PRRs have been known to recognize the molecular patterns of microorganisms and their related products, the intra- and extrainflammatory stimulus known as pathogen-associated molecular patterns (PAMPs) in sepsis is mediated through similar receptors. PAMPs are common components to many microbes, for example, lipopolysaccharide, peptidoglycan, and flagellin, which are of bacterial origin, as well as RNA and DNA, which can be of viral or bacterial origin. As for PRRs, Toll-like receptor (TLR) is the best known, and more than ten subtypes have been identified in humans. Among them, TLR-3 is a receptor for viral double-stranded RNA, which allows macrophages to recognize by-products of necrotic neutrophils, thereby stimulating the generation of pro-inflammatory cytokines .
In contrast to necrosis, which is a passive sequence of events leading to disintegration of the nuclear envelope and cytoplasmic membranes, apoptosis represents a highly organized programmed cell death . The ability to eliminate cells by apoptosis rather than necrosis is favorable to the host because the cell elimination in an orderly manner can limit the extent of cell death and inflammation caused by the uncontrolled release of toxic neutrophil products during necrosis. Apoptosis is initiated by either an intrinsic or an extrinsic pathway. The intrinsic pathway is activated by different noxious stimuli and involves the release of cytochrome C from mitochondria into the cytosol . This release triggers the activation of intracellular caspases responsible for the cleavage of DNA and structural cytoplasmic proteins . The second, extrinsic pathway of apoptosis is triggered by the binding of extracellular ligands such as tumor necrosis factor (TNF)-α or Fas ligand (FasL) to specific TNF receptors on the cell surface . The binding of these ligands then generates a transmembrane signal to activate the caspase sequence. In the case of neutrophil, the timing of apoptosis is strictly regulated. Neutrophil granulocytes in particular are primed to undergo apoptosis within 24–48 h after having left the systemic circulation, but the exact time at which this occurs is influenced by several factors [30, 31].
Autophagy is a homeostatic mechanism involved in the clearance of damaged organelles and in cellular survival under certain stresses or nutrient depletion to provide essential nutrients and proteins through recycling of the cytosolic organelles . The implication of autophagy in innate immunity is yet to be clarified, but one possible explanation for activated autophagy during sepsis is that the autophagic process results in the removal of intracellular pathogens (xenophagy) during sepsis [36, 37]. The regulation of autophagy by activation of ROS, TLR, and inflammatory cytokines such as TNF-α and interferons has been reported [38–40]. TLR activation does not result in excess oxidative burst, but the inhibition of apoptosis leads to the induction of autophagy [41, 42]. Considering the regulatory role of apoptosis in the inflammatory process, the pro-survival induction of autophagy in neutrophils enhances the inflammatory responses by delaying cell death and could be involved in the pathogenesis of sepsis related to suppression of apoptosis, leading to tissue injury. As mentioned previously, autophagy is basically the survival mechanism of cells; however, when the insult is excessive, autophagy results in autophagic cell death. Morphological features of this cell death include vacuolization, degradation of cytoplasmic content, and lack of chromatin condensation. Cells undergoing autophagic cell death may be internalized by neighboring cells. Therefore, this type of cell death is considered to be a noninflammatory form .
The term ‘NETosis’ for neutrophil cell death leads to the formation of NETs. NETosis is the third programmed neutrophil cell death, which is quite different from other types of cell death. Under the circumstances of bacterial, fungal, or parasitic infection, microbial components such as lipopolysaccharide and lipoteichoic acid, and ROS including hydrogen peroxide, can induce peculiar morphological changes in neutrophils . Rapid NET formation is also induced by platelets activated via TLR-4 . Similarly, alarmines such as heat shock proteins, HMGB1, as well as RNA and DNA of host origin are detected as the initiators of NETosis. With regard to PRRs, RNA and DNA are reported to be sensed by TLR-9 , while histones are reported to be sensed through TLR-2 and TLR-4 .
Under a certain stimulation, ROS is activated as the first step, then neutrophil elastase (NE) and myeloperoxidase (MPO) migrate from granules to the nucleus, and finally, the processing of histones leads to rupture of the cell. One of the most common inducers of NETosis is phorbol myristate acetate (PMA), which directly stimulates protein kinase C (PKC) and subsequently leads to the production of ROS. One of the distinctive appearances of NETosis is the homogenous nucleoplasm, and this change depends on the activity of NE and MPO. NE is initially stored in azurophilic granules in the cytosol. After the stimulation, NE is released from the granules and enters the nucleus, where it degrades the linker histone H1 and processes core histones . MPO also migrates to the nucleus and it enhances chromatin decondensation. Thus, NE and MPO cooperate to undergo further histone modifications to decondense the chromatin structure. Eventually, NETs are quickly removed once the infection is resolved. NETs are susceptible to DNase1 , and the debris left by DNase1 will be cleared by macrophages and neutrophils recruited to the inflammatory site .
One of the major roles of neutrophils is the elimination of microorganisms. For that purpose, NETs are expected to trap microbes and prevent their dissemination to the circulating blood. Inactivation of the virulence factors and the extermination of pathogens are also requested. Trapping microbes, most likely through charge interaction , prevents their dissemination and encloses them at the initial site of infection. Interestingly, Group A Streptococcus pyogenes, pneumococcus, and Staphylococcus aureus are capable of liberating themselves from NETs since they encode endonucleases. As a matter of fact, the expression of DNase is essential for these bacteria to be pathogenic . Other than DNA, NETs contain several proteins toxic to microbes. These include lysozyme, antimicrobial peptides, ion chelators (calgranulin), and histones. The antimicrobial activity of NETs is likely the result of the combination of these components. Their effects are enhanced by the combination work and the high local concentrations achieved on NETs. MPO on NETs is also essential to kill microbes. The antifungal activity of NETs has been assigned to calgranulin . Histones are the main toxic components of NETs; however, the mechanism of histone toxicity is poorly understood. In severe sepsis, extranuclear histones can be detected in circulating blood, which are released abundantly during NETosis . Since circulating histones are also harmful for the host cells , histones are the target of the new therapeutic strategy.
There is universal agreement that dysfunction in coagulation develops during sepsis and leads to inappropriate intravascular thrombus formation. In contrast, discussions are still going on whether coagulopathy has pathogenic roles in the progress of sepsis or is a mere response to the insult. This debate is still inconclusive since the results of the clinical trials using anticoagulants are inconsistent [56–59].
Thrombus formation causes obstruction in the microvasculature and induces tissue ischemia and injury, thereby contributing to multiple organ failure and death. Therefore, if microthrombus formation is prevented or shut off, the organ function should be preserved. But, in practice, there are multilayered confounders in such settings, including population heterogeneity, comorbidities, and contraindications to standard care therapeutics. Moreover, as stated in this review, since thrombus formation is an essential part of the host defense mechanism, neither unconditional application of anticoagulant therapy nor modulation of neutrophil death will be beneficial to the host.
Neutrophil cell death plays a pivotal role in the pathophysiology of sepsis. Although apoptosis has been the major focus of investigation, necrosis, autophagic cell death, and the new comer NETosis also play significant roles in this critical situation. Actually, a variety of styles of cell death coexist in sepsis, and the population of necrosis and NETosis are especially important to regulate the host condition. The specific virulence factors expressed by individual pathogens, the degree of insult, the duration of stimulation, and the response of the host immune system may determine the distribution of cell death phenotypes. It is apparent that cell disintegration and molecules such as histones, nucleosomes, proteases, and tissue factor released from the dead cells all play governing roles in the inflammatory and coagulatory responses, therefore host immune competence, during sepsis.
Bactericidal permeability-increasing protein
Damage-associated molecular patterns
High-mobility group box 1
Neutrophil extracellular traps
Pathogen-associated molecular patterns
Protein kinase C
Phorbol myristate acetate
Reactive oxygen species
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (#25462831).
A part of this review was presented in the 40th Annual Meeting of the Japanese Society of Intensive Care Medicine.
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