Gastrin-releasing peptide receptor (GRPR) mediates chemotaxis in neutrophils

Neutrophil migration to inflamed sites is crucial for both the initiation of inflammation and resolution of infection, yet these cells are involved in perpetuation of different chronic inflammatory diseases. Gastrin-releasing peptide (GRP) is a neuropeptide that acts through G protein coupled receptors (GPCRs) involved in signal transmission in both central and peripheral nervous systems. Its receptor, gastrin-releasing peptide receptor (GRPR), is expressed by various cell types, and it is overexpressed in cancer cells. RC-3095 is a selective GRPR antagonist, recently found to have antiinflammatory properties in arthritis and sepsis models. Here we demonstrate that i.p. injection of GRP attracts neutrophils in 4 h, and attraction is blocked by RC-3095. Macrophage depletion or neutralization of TNF abrogates GRP-induced neutrophil recruitment to the peritoneum. In vitro, GRP-induced neutrophil migration was dependent on PLC-beta 2, PI3K, ERK, p38 and independent of G alpha i protein, and neutrophil migration toward synovial fluid of arthritis patients was inhibited by treatment with RC-3095. We propose that GRPR is an alternative chemotactic receptor that may play a role in the pathogenesis of inflammatory disorders.

Intracellular mechanisms of hydroquinone toxicity on endotoxin-activated neutrophils

Circulating neutrophils promptly react to different substances in the blood and orchestrate the beginning of the innate inflammatory response. We have shown that in vivo exposure to hydroquinone (HQ), the most oxidative compound of cigarette smoke and a toxic benzene metabolite, affects circulating neutrophils, making them unresponsive to a subsequent bacterial infection. In order to understand the action of toxic molecular mechanisms on neutrophil functions, in vitro HQ actions on pro-inflammatory mediator secretions evoked by Escherichia coli lipopolysaccharide (LPS) were investigated. Neutrophils from male Wistar rats were cultured with vehicle or HQ (5 or 10 mu M; 2 h) and subsequently incubated with LPS (5 mu g/ml; 18 h). Hydroquinone treatment impaired LPS-induced nitric oxide (NO), tumour necrosis factor alpha (TNF-alpha), interleukin (IL)-1 beta and IL-6 secretions by neutrophils. The toxic effect was not dependent on cell death, reduced expression of the LPS receptor or toll-like receptor-4 (TLR-4) or cell priming, as HQ did not induce reactive oxygen species generation or beta(2)integrin membrane expression. The action of toxic mechanisms on cytokine secretion was dependent on reduced gene synthesis, which may be due to decreased nuclear factor kappa B (NF-kappa B) nuclear translocation. Conversely, this intracellular pathway was not involved in impaired NO production because HQ treatments only affected inducible nitric oxide synthase protein expression and activity, suggesting posttranscriptional and/or posttranslational mechanisms of action. Altogether, our data show that HQ alters the action of different LPS-activated pathways on neutrophils, which may contribute to the impaired triggering of the host innate immune reaction detected during in vivo HQ exposure.

Ageing exacerbates damage of systemic and salivary neutrophils from patients presenting Candida-related denture stomatitis

Abstract Background Ageing leads to a decline in the function of the immune system, increasing the body's susceptibility to infections through the impairment of T-cells, macrophages, neutrophils and dendritic cells Denture stomatitis is a primary oral disease affecting elderly denture wearers. The major etiologic factor involved in this pathology is the infection by Candida albicans, an opportunistic pathogen that causes local and disseminated diseases in immunosuppressed humans. Neutrophils play a critical role in the immune response against C. albicans and are continually present in the salivary fluid and in the blood. The aim of this study was to determine ageing-related changes in salivary and blood neutrophils and their potential implications in Candida-related denture stomatitis. Results Our results showed a lower number of neutrophils in the saliva from patients presenting Candida-related denture stomatitis in comparison to their matched controls. Furthermore, fewer neutrophils were isolated from the saliva of aged control individuals in comparison to matched younger subjects. CXCR1, CD62L and CD11b expression were significantly greater on systemic neutrophils from younger control individuals. Elderly individuals showed more apoptotic salivary neutrophils and lower GM-CSF levels than younger ones, regardless of the occurrence of Candida infection. On the other hand, CXCL-8 concentrations were higher in the saliva from elderly individuals. Besides, TNF-α was detected at elevated levels in the saliva from infected elderly subjects. Salivary neutrophils from elderly and young patients presented impaired phagocytic activity against C. albicans. However, just systemic neutrophils from elderly showed decreased phagocytosis when compared to the younger ones, regardless of the occurrence of infection. In addition, neutrophils from aged individuals and young patients presented low fungicidal activity. Conclusion The data suggests that the Candida related-denture stomatitis is associated to neutrophils function deficiency, and ageing drastically appears to alter important characteristics of such cells, facilitating the establishment of this infection.

Activation profile of CXCL8-stimulated neutrophils and aging

Neutrophils are pivotal effector cells of innate immunity representing the first line of defense against aggression. They are the first cells to arrive at the site of the aggression, where they can directly eliminate the invading microorganisms. Their activation and recruitment into peripheral tissues is indispensable for host defense. With aging, there are alterations of the receptor by driven functions of human neutrophils as a decrease in the functional changes in signaling elicited by specific receptors, as CXCR1. We investigated the activation of neutrophils from elderly after the cells were cultivated with CXCL8. Although, CXCL8 induced elastase (ELA) secretion, data showed neither myeloperoxidase (MPO) activity nor production of IL-6, IL-10, GM-CSF by neutrophils from elderly compared with young individuals. On the other hand, in the presence of only LPS or LPS associated with CXCL8 neutrophils from elderly individuals, there were significant levels of IL-6, IL-10, GM-CSF but not MPO. These results indicate that neutrophils from elderly do not respond to CXCL8 stimulus, but they are activated by LPS to produce cytokines. However, MPO activity from elderly individuals was not different in the presence or absence of LPS and CXCL8

Antimicrobial peptides and nitric oxide production by neutrophils from periodontitis subjects

Neutrophils play an important role in periodontitis by producing nitric oxide (NO) and antimicrobial peptides, molecules with microbicidal activity via oxygen-dependent and -independent mechanisms, respectively. It is unknown whether variation in the production of antimicrobial peptides such as LL-37, human neutrophil peptides (HNP) 1-3, and NO by neutrophils influences the pathogenesis of periodontal diseases. We compared the production of these peptides and NO by lipopolysaccharide (LPS)-stimulated neutrophils isolated from healthy subjects and from patients with periodontitis. Peripheral blood neutrophils were cultured with or without Aggregatibacter actinomycetemcomitans-LPS (Aa-LPS), Porphyromonas gingivalis-LPS (Pg-LPS) and Escherichia coli-LPS (Ec-LPS). qRT-PCR was used to determine quantities of HNP 1-3 and LL-37 mRNA in neutrophils. Amounts of HNP 1-3 and LL-37 proteins in the cell culture supernatants were also determined by ELISA. In addition, NO levels in neutrophil culture supernatants were quantitated by the Griess reaction. Neutrophils from periodontitis patients cultured with Aa-LPS, Pg-LPS and Ec-LPS expressed higher HNP 1-3 mRNA than neutrophils from healthy subjects. LL-37 mRNA expression was higher in neutrophils from patients stimulated with Aa-LPS. Neutrophils from periodontitis patients produced significantly higher LL-37 protein levels than neutrophils from healthy subjects when stimulated with Pg-LPS and Ec-LPS, but no difference was observed in HNP 1-3 production. Neutrophils from periodontitis patients cultured or not with Pg-LPS and Ec-LPS produced significantly lower NO levels than neutrophils from healthy subjects. The significant differences in the production of LL-37 and NO between neutrophils from healthy and periodontitis subjects indicate that production of these molecules might influence individual susceptibility to important periodontal pathogens.

The silent survival of Staphylococcus aureus phagocyted by HL-60 derived neutrophils

Staphylococcus aureus is a Gram positive pathogen that causes various human infections and represents one of the most common causes of bacteremia. S. aureus is able to invade a variety of non-professional phagocytes and that can survive engulfment by neutrophils, producing both secreted and surface components that compromise innate immune responses. In the contest of our study we evaluated the functional activity of vaccine specific antibodies by opsonophagocytosis killing assay (OPKA). Interestingly a low level of killing of the staphylococcal cells has been observed. In the meanwhile intracellular survival studies showed that S. aureus persisted inside phagocytes for several hours until a burst of growth after 5 hours in the supernatant. These data suggest that the strong ability of S. aureus to survive in the phagocytes could be the cause of the low killing measured by OPKA. Moreover parallel studies on HL-60 cells infected with S. aureus done by using transmission electron microscopy (TEM) interestingly showed that staphylococcal cells have an intracellular localization (endosomal vacuoles) and that they are able not only to maintain the integrity of their membrane but also to replicate inside vacuolar compartments. Finally in order to generate 3D volume of whole bacteria when present inside neutrophilic vacuoles, we collected a series of tomographic two-dimensional (2D) images by using a transmission electron microscope, generating 5 different tomograms. The three-dimensional reconstruction reveals the presence of intact bacteria within neutrophil vacuoles. The S. aureus membrane appears completely undamaged and integral in contrast with the physiological process of phagosytosis through vacuoles progression. S. aureus bacteria show a homogenous distribution of the density in all the three dimensions (X, Y, Z). All these evidences definitely explain the ability of the pathogen to survive inside the endosomal vacuoles and should be the cause of the low killing level.

Hyperglycaemia but not hyperlipidaemia decreases serum amylase and increases neutrophils in the exocrine pancreas of cats

The goal of the study was to determine whether hyperglycaemia or hyperlipidaemia causes pancreatitis in cats and to assess the effect of excess serum glucose and lipids on amylase and lipase activity. Ten-day hyperglycaemic and hyperlipidaemic clamps were carried out in five and six healthy cats, respectively. Ten healthy cats received saline and served as controls. The activity of amylase was below the normal range in 4 of 5 hyperglycaemic cats by day 10. The activity of lipase did not vary in any of the cats. Samples of exocrine pancreas were normal on histological examination, but the number of tissue neutrophils was increased in hyperglycaemic cats (P<0.05). In a retrospective study 14 of 40 (35%) cats with naturally occurring diabetes mellitus had amylase activities below the reference range at the time of admission. Amylase activities normalised within 1 week of insulin therapy and subsequent glycaemic control. Lipase activity was increased in 26 of 40 (65%) diabetic cats and remained elevated despite glycaemic control. In conclusion, hyperglycaemia, but not hyperlipidaemia, increases pancreatic neutrophils in cats. However, because the histological morphology of the exocrine pancreas was normal, hyperglycaemia may play only a minor role in the pathogenesis of pancreatitis. Low amylase activities in diabetic cats may reflect an imbalance in glucose metabolism rather than pancreatitis.

A novel TNFR1-triggered apoptosis pathway mediated by class IA PI3Ks in neutrophils

The most common form of neutrophil death is apoptosis. In the present study, we report surprising differences in the molecular mechanisms used for caspase activation between FAS/CD95-stimulated and TNF receptor 1 (TNFR1)-stimulated neutrophils. Whereas FAS-induced apoptosis was followed by caspase-8 activation and required Bid to initiate the mitochondrial amplification loop, TNF-?-induced apoptosis involved class IA PI3Ks, which were activated by MAPK p38. TNF-?-induced PI3K activation resulted in the generation of reactive oxygen species, which activated caspase-3, a mechanism that did not operate in neutrophils without active NADPH oxidase. We conclude that in neutrophils, proapoptotic pathways after TNFR1 stimulation are initiated by p38 and PI3K, but not by caspase-8, a finding that should be considered in anti-inflammatory drug-development strategies.

Protein overexpression following lentiviral infection of primary mature neutrophils is due to pseudotransduction

Neutrophils are terminally differentiated cells with a short life-span due to constitutive apoptosis. Because of these characteristics, genetic manipulation of neutrophils has been difficult, although it is highly desired given the importance of neutrophils in the immune system. Here we demonstrate that transduction of primary human mature neutrophils with enhanced green fluorescent protein (eGFP)-encoding lentiviral particles results in GFP-containing cells as previously reported. Yet, our data further show that GFP expression in neutrophils upon transduction is largely due to protein transfer, a process called lentiviral pseudotransduction, and not due to bona fide transduction. Thus, inhibition of viral genome integration by the reverse transcriptase inhibitor 3'-azido-3'-deoxythymidine (AZT) or of protein biosynthesis by cycloheximide (CHX) did not abolish GFP levels in transduced neutrophils. Importantly, lentiviral pseudotransduction of the enzyme death-associated protein kinase 2 (DAPK2) into primary human mature neutrophils resulted in increased protein levels, but not enzymatic functionality. Based on our data and previous reports of unspecific viral effects on immune cells following lentiviral transduction, we discourage scientists to use lentiviral transduction methods to manipulate primary mature neutrophils.

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.

The neurovascular unit as a selective barrier to polymorphonuclear granulocyte (PMN) infiltration into the brain after ischemic injury

The migration of polymorphonuclear granulocytes (PMN) into the brain parenchyma and release of their abundant proteases are considered the main causes of neuronal cell death and reperfusion injury following ischemia. Yet, therapies targeting PMN egress have been largely ineffective. To address this discrepancy we investigated the temporo-spatial localization of PMNs early after transient ischemia in a murine transient middle cerebral artery occlusion (tMCAO) model and human stroke specimens. Using specific markers that distinguish PMN (Ly6G) from monocytes/macrophages (Ly6C) and that define the cellular and basement membrane boundaries of the neurovascular unit (NVU), histology and confocal microscopy revealed that virtually no PMNs entered the infarcted CNS parenchyma. Regardless of tMCAO duration, PMNs were mainly restricted to luminal surfaces or perivascular spaces of cerebral vessels. Vascular PMN accumulation showed no spatial correlation with increased vessel permeability, enhanced expression of endothelial cell adhesion molecules, platelet aggregation or release of neutrophil extracellular traps. Live cell imaging studies confirmed that oxygen and glucose deprivation followed by reoxygenation fail to induce PMN migration across a brain endothelial monolayer under flow conditions in vitro. The absence of PMN infiltration in infarcted brain tissues was corroborated in 25 human stroke specimens collected at early time points after infarction. Our observations identify the NVU rather than the brain parenchyma as the site of PMN action after CNS ischemia and suggest reappraisal of targets for therapies to reduce reperfusion injury after stroke.

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.