Peculiarities of cell death mechanisms in neutrophils

Analyses of neutrophil death mechanisms have revealed many similarities with other cell types; however, a few important molecular features make these cells unique executors of cell death mechanisms. For instance, in order to fight invading pathogens, neutrophils possess a potent machinery to produce reactive oxygen species (ROS), the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Evidence is emerging that these ROS are crucial in the execution of most neutrophil cell death mechanisms. Likewise, neutrophils exhibit many diverse granules that are packed with cytotoxic mediators. Of those, cathepsins were recently shown to activate pro-apoptotic B-cell lymphoma-2 (Bcl-2) family members and caspases, thus acting on apoptosis regulators. Moreover, neutrophils have few mitochondria, which hardly participate in ATP synthesis, as neutrophils gain energy from glycolysis. In spite of relatively low levels of cytochrome c in these cells, the mitochondrial death pathway is functional. In addition to these pecularities defining neutrophil death pathways, neutrophils are terminally differentiated cells, hence they do not divide but undergo apoptosis shortly after maturation. The initial trigger of this spontaneous apoptosis remains to be determined, but may result from low transcription and translation activities in mature neutrophils. Due to the unique biological characteristics of neutrophils, pharmacological intervention of inflammation has revealed unexpected and sometimes disappointing results when neutrophils were among the prime target cells during therapy. In this study, we review the current and emerging models of neutrophil cell death mechanisms with a focus on neutrophil peculiarities.

Inflammation-associated autophagy-related programmed necrotic death of human neutrophils characterized by organelle fusion events

The most common form of neutrophil death, under both physiological and inflammatory conditions, is apoptosis. In this study, we report a novel form of programmed necrotic cell death, associated with cytoplasmic organelle fusion events, that occurs in neutrophils exposed to GM-CSF and other inflammatory cytokines upon ligation of CD44. Strikingly, this type of neutrophil death requires PI3K activation, a signaling event usually involved in cellular survival pathways. In the death pathway reported in this study, PI3K is required for the generation of reactive oxygen species, which somehow trigger the generation of large cytoplasmic vacuoles, generated by the fusion of CD44-containing endosomes with autophagosomes and secondary, but not primary, granules. Neutrophils demonstrating vacuolization undergo rapid cell death that depends on receptor-interacting protein 1 kinase activity and papain family protease(s), but not caspases, that are most likely activated and released, respectively, during or as a consequence of organelle fusion. Vacuolized neutrophils are present in infectious and autoimmune diseases under in vivo conditions. Moreover, isolated neutrophils from such patients are highly sensitive toward CD44-mediated PI3K activation, reactive oxygen species production, and cell death, suggesting that the newly described autophagy-related form of programmed neutrophil necrosis plays an important role in inflammatory responses.

Autophagy regulation in macrophages and neutrophils

Autophagy is a conserved proteolytic mechanism that degrades cytoplasmic material including cell organelles. Accumulating evidence exists that autophagy also plays a major role in immunity and inflammation. Specifically, it appears that autophagy protects against infections and inflammation. Here, we review recent work performed in macrophages and neutrophils, which both represent critical phagocytes in mammalians.

Apoptotic neutrophils release macrophage migration inhibitory factor upon stimulation with tumor necrosis factor-alpha

Macrophage migration inhibitory factor (MIF) is an important cytokine involved in the regulation of innate immunity and present at increased levels during inflammatory responses. Here we demonstrate that mature blood and tissue neutrophils constitutively express MIF as a cytosolic protein not associated with azurophil granules. Functionally active MIF, but not proteases stored in azurophil granules, was released from apoptotic neutrophils following short term tumor necrosis factor (TNF)-alpha stimulation in a caspase-dependent manner and prior to any detectable phagocytosis by monocyte-derived macrophages. Moreover, TNF-alpha-mediated MIF release was blocked by glyburide and propenicide, both inhibitors of ATP-binding cassette-type transporters, suggesting that this transporter system is activated during neutrophil apoptosis. Taken together, apoptotic mature neutrophils release MIF upon short term TNF-alpha stimulation. Therefore, apoptosis may not always occur without the induction of pro-inflammatory mechanisms.

Targeting survivin via PI3K but not c-akt/PKB by anticancer drugs in immature neutrophils

Myelosuppression is the most common unwanted side effect associated with the administration of anticancer drugs, and infections remain a common cause of death in chemotherapy-treated patients. Several mechanisms of the cytotoxicity of these drugs have been proposed and may synergistically operate in a given cell. Survivin expression has been associated with cancer, but recent reports suggest that this molecule is also expressed in several immature and mature hematopoietic cells. Here, we provide evidence that treatment of immature neutrophils with anticancer drugs reduced endogenous survivin levels causing apoptosis. The anticancer drugs did not directly target survivin, instead they blocked the activity of phosphatidylinositol-3-OH kinase, which regulated survivin expression and apoptosis in these cells. Strikingly, and in contrast to other cells, this pathway did not involve the serine/threonine kinase c-akt/PKB. Moreover, in combination with anticancer drug therapy, rapamycin did not induce increased myelosuppression in an experimental lymphoma mouse model. These data suggest that drugs that block either c-akt/PKB or signaling molecules located distal to c-akt/PKB may preferentially induce apoptosis of cancer cells as they exhibit no cytotoxicity for immature neutrophils.

Autophagic-like cell death in neutrophils induced by autoantibodies

Human neutrophils undergo autophagic-like cell death following Sialic acid binding immunoglobulin-like lectin-9 (Siglec-9) ligation and concurrent stimulation with certain, but not all, neutrophil survival cytokines. Caspase inhibition by these cytokines is required, but is not sufficient, to trigger this particular form of cell death. Additional mechanisms may involve reactive oxygen species (ROS), and blocking of ROS or prevention of ROS production prevents autophagic-like neutrophil death. Interestingly, human intravenous immunoglobulin (IVIg) preparations contain natural anti-Siglec-9 autoantibodies, which are able to ligate Siglec-9 on neutrophils and induce autophagic-like cell death in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and some other survival cytokines. Here, we discuss the pathophysiological and therapeutic implications of these recent findings.

Cleavage of CXCR1 on neutrophils disables bacterial killing in cystic fibrosis lung disease

Interleukin-8 (IL-8) activates neutrophils via the chemokine receptors CXCR1 and CXCR2. However, the airways of individuals with cystic fibrosis are frequently colonized by bacterial pathogens, despite the presence of large numbers of neutrophils and IL-8. Here we show that IL-8 promotes bacterial killing by neutrophils through CXCR1 but not CXCR2. Unopposed proteolytic activity in the airways of individuals with cystic fibrosis cleaved CXCR1 on neutrophils and disabled their bacterial-killing capacity. These effects were protease concentration-dependent and also occurred to a lesser extent in individuals with chronic obstructive pulmonary disease. Receptor cleavage induced the release of glycosylated CXCR1 fragments that were capable of stimulating IL-8 production in bronchial epithelial cells via Toll-like receptor 2. In vivo inhibition of proteases by inhalation of alpha1-antitrypsin restored CXCR1 expression and improved bacterial killing in individuals with cystic fibrosis. The cleavage of CXCR1, the functional consequences of its cleavage, and the identification of soluble CXCR1 fragments that behave as bioactive components represent a new pathophysiologic mechanism in cystic fibrosis and other chronic lung diseases.

TLR expression on neutrophils at the pulmonary site of infection: TLR1/TLR2-mediated up-regulation of TLR5 expression in cystic fibrosis lung disease

Cystic fibrosis (CF) lung disease is characterized by infection with Pseudomonas aeruginosa and a sustained accumulation of neutrophils. In this study, we analyzed 1) the expression of MyD88-dependent TLRs on circulating and airway neutrophils in P. aeruginosa-infected CF patients, P. aeruginosa-infected non-CF bronchiectasis patients, and noninfected healthy control subjects and 2) studied the regulation of TLR expression and functionality on neutrophils in vitro. TLR2, TLR4, TLR5, and TLR9 expression was increased on airway neutrophils compared with circulating neutrophils in CF and bronchiectasis patients. On airway neutrophils, TLR5 was the only TLR that was significantly higher expressed in CF patients compared with bronchiectasis patients and healthy controls. Studies using confocal microscopy and flow cytometry revealed that TLR5 was stored intracellularly in neutrophils and was mobilized to the cell surface in a protein synthesis-independent manner through protein kinase C activation or after stimulation with TLR ligands and cytokines characteristic of the CF airway microenvironment. The most potent stimulator of TLR5 expression was the bacterial lipoprotein Pam(3)CSK(4). Ab-blocking experiments revealed that the effect of Pam(3)CSK(4) was mediated through cooperation of TLR1 and TLR2 signaling. TLR5 activation enhanced the phagocytic capacity and the respiratory burst activity of neutrophils, which was mediated, at least partially, via a stimulation of IL-8 production and CXCR1 signaling. This study demonstrates a novel mechanism of TLR regulation in neutrophils and suggests a critical role for TLR5 in neutrophil-P. aeruginosa interactions in CF lung disease.

Flotillins interact with PSGL-1 in neutrophils and, upon stimulation, rapidly organize into membrane domains subsequently accumulating in the uropod

BACKGROUND: Neutrophils polarize and migrate in response to chemokines. Different types of membrane microdomains (rafts) have been postulated to be present in rear and front of polarized leukocytes and disruption of rafts by cholesterol sequestration prevents leukocyte polarization. Reggie/flotillin-1 and -2 are two highly homologous proteins that are ubiquitously enriched in detergent resistant membranes and are thought to shape membrane microdomains by forming homo- and hetero-oligomers. It was the goal of this study to investigate dynamic membrane microdomain reorganization during neutrophil activation. METHODOLOGY/PRINCIPAL FINDINGS: We show now, using immunofluorescence staining and co-immunoprecipitation, that endogenous flotillin-1 and -2 colocalize and associate in resting spherical and polarized primary neutrophils. Flotillins redistribute very early after chemoattractant stimulation, and form distinct caps in more than 90% of the neutrophils. At later time points flotillins accumulate in the uropod of polarized cells. Chemotactic peptide-induced redistribution and capping of flotillins requires integrity and dynamics of the actin cytoskeleton, but does not involve Rho-kinase dependent signaling related to formation of the uropod. Both flotillin isoforms are involved in the formation of this membrane domain, as uropod location of exogenously expressed flotillins is dramatically enhanced by co-overexpression of tagged flotillin-1 and -2 in differentiated HL-60 cells as compared to cells expressing only one tagged isoform. Flotillin-1 and -2 associate with P-selectin glycoprotein ligand 1 (PSGL-1) in resting and in stimulated neutrophils as shown by colocalization and co-immunoprecipitation. Neutrophils isolated from PSGL-1-deficient mice exhibit flotillin caps to the same extent as cells isolated from wild type animals, implying that PSGL-1 is not required for the formation of the flotillin caps. Finally we show that stimulus-dependent redistribution of other uropod-located proteins, CD43 and ezrin/radixin/moesin, occurs much slower than that of flotillins and PSGL-1. CONCLUSIONS/SIGNIFICANCE: These results suggest that flotillin-rich actin-dependent membrane microdomains are importantly involved in neutrophil uropod formation and/or stabilization and organize uropod localization of PSGL-1.

Viable neutrophils release mitochondrial DNA to form neutrophil extracellular traps

Neutrophil extracellular traps (NETs) represent extracellular structures able to bind and kill microorganisms. It is believed that they are generated by neutrophils undergoing cell death, allowing these dying or dead cells to kill microbes. We show that, following priming with granulocyte/macrophage colony-stimulating factor (GM-CSF) and subsequent short-term toll-like receptor 4 (TLR4) or complement factor 5a (C5a) receptor stimulation, viable neutrophils are able to generate NETs. Strikingly, NETs formed by living cells contain mitochondrial, but no nuclear, DNA. Pharmacological or genetic approaches to block reactive oxygen species (ROS) production suggested that NET formation is ROS dependent. Moreover, neutrophil populations stimulated with GM-CSF and C5a showed increased survival compared with resting neutrophils, which did not generate NETs. In conclusion, mitochondrial DNA release by neutrophils and NET formation do not require neutrophil death and do also not limit the lifespan of these cells.

RhoH/TTF negatively regulates leukotriene production in neutrophils

Leukotriene B(4) (LTB(4)) is an important proinflammatory lipid mediator generated by neutrophils upon activation. GM-CSF stimulation is known to enhance agonist-mediated LTB(4) production of neutrophils within minutes, a process called "priming". In this study, we demonstrate that GM-CSF also limits the production of LTB(4) by neutrophils via a transcriptional mechanism at later time points. We identified hemopoietic-specific Ras homologous (RhoH)/translocation three four (TTF), which was induced following GM-CSF stimulation in neutrophils, as a key regulator in this process. Neutrophils derived from RhoH/TTF-deficient (Rhoh(-/-)) mice demonstrated increased LTB(4) production upon activation compared with normal mouse neutrophils. Moreover, neutrophils from cystic fibrosis patients expressed enhanced levels of RhoH/TTF and generated less LTB(4) upon activation compared with normal human neutrophils. Taken together, these data suggest that RhoH/TTF represents an inducible feedback inhibitor in neutrophils that is involved in the limitation of innate immune responses.

Induction of Bim limits cytokine-mediated prolonged survival of neutrophils

Under inflammatory conditions, neutrophil apoptosis is delayed due to survival-factor exposure, a mechanism that prevents the resolution of inflammation. One important proinflammatory cytokine involved in the regulation of neutrophil survival/activation is granulocyte-macrophage colony-stimulating factor (GM-CSF). Although GM-CSF mediates antiapoptotic effects in neutrophils, it does not prevent apoptosis, and the survival effect is both time dependent and limited. Here, we identified the proapoptotic Bcl-2 family member Bim as an important lifespan limiting molecule in neutrophils, particularly under conditions of survival factor exposure. Strikingly, GM-CSF induced Bim expression in both human and mouse neutrophils that was blocked by pharmacological inhibition of phosphatidylinositol-3 kinase (PI3K). Increased Bim expression was also seen in human immature bone marrow neutrophils as well as in blood neutrophils from septic shock patients; both cell populations are known to be exposed to GM-CSF under in vivo conditions. The functional role of Bim was investigated using Bim-deficient mouse neutrophils in the presence and absence of the survival cytokines interleukin (IL)-3 and GM-CSF. Lack of Bim expression resulted in a much higher efficacy of the survival cytokines to block neutrophil apoptosis. Taken together, these data demonstrate a functional role for Bim in the regulation of neutrophil apoptosis and suggest that GM-CSF and other neutrophil hematopoietins initiate a proapoptotic counterregulation that involves upregulation of Bim.

Living and dying for inflammation: neutrophils, eosinophils, basophils

Neutrophils, eosinophils, and basophils play essential roles during microbe-induced and sterile inflammation. The severity of such inflammatory processes is controlled, at least in part, by factors that regulate cell death and survival of granulocytes. In recent years, major progress has been made in understanding the molecular mechanisms of granulocyte cell death and in identifying novel damage- and pathogen-associated molecular patterns as well as regulatory cytokines impacting granulocyte viability. Furthermore, an increased interest in innate immunity has boosted our overall understanding of granulocyte biology. In this review, we describe and compare factors and mechanisms regulating neutrophil, eosinophil, and basophil lifespan. Because dysregulation of death pathways in granulocytes can contribute to inflammation-associated immunopathology, targeting granulocyte lifespan could be therapeutically promising.

DAPK2 positively regulates motility of neutrophils and eosinophils in response to intermediary chemoattractants

The tight regulation of granulocyte chemotaxis is crucial for initiation and resolution of inflammation. Here, we show that DAPK2, a Ca(2+)/CaM-sensitive serine/threonine kinase known to modulate cell death in various cell types, is a novel regulator of migration in granulocytes. We demonstrate that human neutrophils and eosinophils express DAPK2 but unlike other leukocytes, no DAPK1 or DAPK3 protein. When DAPK activities were blocked by inhibitors, we found that neither granulocyte lifespan nor phagocytosis was affected. However, such pharmacological inactivation of DAPK activity abolished motility of granulocytes in response to intermediary but not end-target chemoattractants ex vivo. The defect in chemotaxis in DAPK2-inactive granulocytes is likely a result of reduced polarization of the cells, mediated by a lack of MLC phosphorylation, resulting in radial F-actin and pseudopod formation. As neutrophils treated with DAPKi also showed reduced recruitment to the site of inflammation in a mouse peritonitis model, DAPK2 may be a novel target for anti-inflammatory therapies.

Mesenchymal stromal cells improve survival during sepsis in the absence of heme oxygenase-1: the importance of neutrophils

The use of mesenchymal stromal cells (MSCs) for treatment of bacterial infections, including systemic processes like sepsis, is an evolving field of investigation. This study was designed to investigate the potential use of MSCs, harvested from compact bone, and their interactions with the innate immune system, during polymicrobial sepsis induced by cecal ligation and puncture (CLP). We also wanted to elucidate the role of endogenous heme oxygenase (HO)-1 in MSCs during a systemic bacterial infection. MSCs harvested from the bones of HO-1 deficient (-/-) and wild-type (+/+) mice improved the survival of HO-1(-/-) and HO-1(+/+) recipient mice when administered after the onset of polymicrobial sepsis induced by CLP, compared with the administration of fibroblast control cells. The MSCs, originating from compact bone in mice, enhanced the ability of neutrophils to phagocytize bacteria in vitro and in vivo and to promote bacterial clearance in the peritoneum and blood after CLP. Moreover, after depleting neutrophils in recipient mice, the beneficial effects of MSCs were entirely lost, demonstrating the importance of neutrophils for this MSC response. MSCs also decreased multiple organ injury in susceptible HO-1(-/-) mice, when administered after the onset of sepsis. Taken together, these data demonstrate that the beneficial effects of treatment with MSCs after the onset of polymicrobial sepsis is not dependent on endogenous HO-1 expression, and that neutrophils are crucial for this therapeutic response.