Search

Gout is an inflammatory disease triggered by deposition of monosodium urate (MSU) crystals in the joints, resulting in high neutrophil influx and pain. Here, we studied the role of the inhibitory receptor CD300a in the resolution process in a murine model of gout. We found increased CD300a expression on neutrophils emigrated to the joint. When compared to WT mice, CD300a(−/−) mice had persistent neutrophil influx till 24 hr after MSU injection. This was associated with increased concentration of IL‐1β and greater tissue damage in the joints of CD300a(−/−) mice. There was an increase in the percentage of apoptotic neutrophils in the synovial lavage of WT mice, as compared to CD300a(−/−) mice. This difference was reflected in the decline of efferocytic events in the synovial cavity of CD300a(−/−) mice 24 hr after MSU injection. A CD300a agonistic antibody was shown, for the first time, to increase apoptosis of human neutrophils, and this was associated with cleavage of caspase‐8. In conclusion, our results reveal an important role of CD300a in the control of leucocyte infiltration, IL‐1β production and caspase‐8 cleavage in neutrophils, contributing to the resolution of inflammation triggered by MSU injection.

Background Neuromyelitis optica spectrum disorder (NMOSD), an autoimmune astrocytopathic disease associated with the anti-aquaporin-4 (AQP4) antibody, is characterized by extensive necrotic lesions primarily located on the optic nerves and spinal cord. Tanshinone IIA (TSA), an active natural compound extracted from Salvia miltiorrhiza Bunge, has profound immunosuppressive effects on neutrophils. Objective The present study aimed to evaluate the effect of TSA on NMOSD mice and explore the underlying mechanisms. Mice were initially administered TSA (pre-TSA group, n = 20) or vehicle (vehicle group, n = 20) every 8 h for 3 days, and then NMOSD model was induced by intracerebral injection of NMOSD-immunoglobulin G (NMO-IgG) and human complement (hC). In addition, post-TSA mice (n = 10) were administered equal dose of TSA at 8 h and 16 h after model induction. At 24 h after intracerebral injection, histological analysis was performed to assess the inhibitory effects of TSA on astrocyte damage, demyelination, and neuroinflammation in NMOSD mice, and western blotting was conducted to clarify the effect of TSA on the NF-κB and MAPK signaling pathways. Furthermore, flow cytometry and western blotting were conducted to verify the proapoptotic effects of TSA on neutrophils in vitro. Results There was a profound reduction in astrocyte damage and demyelination in the pre-TSA group and post-TSA group. However, prophylactic administration of TSA induced a better effect than therapeutic treatment. The number of infiltrated neutrophils was also decreased in the lesions of NMOSD mice that were pretreated with TSA. We confirmed that prophylactic administration of TSA significantly promoted neutrophil apoptosis in NMOSD lesions in vivo, and this proapoptotic effect was mediated by modulating the caspase pathway in the presence of inflammatory stimuli in vitro. In addition, TSA restricted activation of the NF-κB signaling pathway in vivo. Conclusion Our data provide evidence that TSA can act as a prophylactic agent that reduces NMO-IgG-induced damage in the mouse brain by enhancing the resolution of inflammation by inducing neutrophil apoptosis, and TSA may serve as a promising therapeutic agent for neutrophil-associated inflammatory disorders, such as NMOSD.

Aspergillus fumigatusairway infections are associated with increased rates of hospitalizations and declining lung function in patients with chronic lung disease. While the pathogenesis of invasiveA. fumigatusinfections is well studied, little is known about the development and progression of airway infections. Previous studies have demonstrated a critical role for the IL-1 cytokines, IL-1α and IL-1β in enhancing pulmonary neutrophil recruitment during invasive aspergillosis. Here we use a mouse model ofA. fumigatusairway infection to study the role of these IL-1 cytokines in immunocompetent mice. In the absence of IL-1 receptor signaling, mice exhibited reduced numbers of viable pulmonary neutrophils and increased levels of neutrophil apoptosis during fungal airway infection. Impaired neutrophil viability in these mice was associated with reduced pulmonary and systemic levels of G-CSF, and treatment with G-CSF restored both neutrophil viability and resistance toA. fumigatusairway infection. Taken together, these data demonstrate that IL-1 dependent G-CSF production plays a key role for host resistance toA. fumigatusairway infection through suppressing neutrophil apoptosis at the site of infection.

Neutrophils play a critical role in the eradication of pathogenic organisms, particularly bacteria. However, in the septic patient the prolonged activation and accumulation of neutrophils may augment tissue and organ injury. This review discusses the different activation states and chemotaxis of neutrophils in septic patients. Neutrophil killing of bacteria and the formation of neutrophil extracellular traps represent important components of the innate immune response and they become dysregulated during sepsis, possibly through changes in their metabolism. Delayed neutrophil apoptosis may contribute to organ injury, or allow better clearance of pathogens. Neutrophils provide a friendly immune response to clear infections, but excessive activation and recruitment has the potential to turn them into potent foes.

Discussion on targeting LXR and PPAR agonists as therapeutic alternatives to ustekinumab therapy in LAD-1.

LPS delays neutrophil apoptosis by a process generally assumed to involve cell-intrinsic TLR4 signaling. However, neutrophil survival responses to LPS have been reported to be monocyte-dependent, which would indicate more complexity than is currently appreciated. We compared the survival responses of conventionally purified vs highly purified neutrophils to confirm or refute the need for secondary cell-types and to identify the cellular or molecular mechanisms involved. Direct stimulation of TLR4 failed to extend the survival of highly purified neutrophils, but survival activity was retained in less pure neutrophil preparations containing low numbers of eosinophils, monocytes, platelets and CD3+ lymphocytes. Sequential depletions identified monocytes as the only cell type required. Transfer of culture supernatants after lipid A-conditioning revealed that purified monocytes were sufficient for production of nearly all of the survival activity observed in mixed populations. The survival factors secreted upon TLR4 stimulation remain unidentified, but were not correlated with IL-1β, IL-6 or TNF-α nor could survival activity be inhibited by Ab blockade of IL-8 or of several other candidate factors other than endogenously produced GM-CSF, which was responsible for about one-tenth of the survival activity present in conditioned supernatants. These observations confirm that ex vivo neutrophil survival responses to TLR4 agonists are not cell intrinsic and involve potentially novel factors secreted by TLR4-stimulated monocytes.

Neutrophils are potent effector cells and contain a battery of harmful substances and degrading enzymes. A silent neutrophil death, i.e., apoptosis, is therefore of importance to avoid damage to the surrounding tissue and to enable termination of the acute inflammatory process. There is a pile of evidence supporting the role for pro-inflammatory cytokines in extending the life-span of neutrophils, but relatively few studies have been devoted to mechanisms actively driving apoptosis induction in neutrophils. We have previously demonstrated that natural killer (NK) cells can promote apoptosis in healthy neutrophils. In this study, we set out to investigate how neutrophil sensitivity to NK cell-mediated cytotoxicity is regulated under inflammatory conditions. Using in vitro-activated neutrophils and a human skin chamber model that allowed collection of in vivo-transmigrated neutrophils, we performed a comprehensive characterization of neutrophil expression of ligands to NK cell receptors. These studies revealed a dramatic downregulation of HLA class I molecules in inflammatory neutrophils, which was associated with an enhanced susceptibility to NK cell cytotoxicity. Collectively, our data shed light on the complex regulation of interactions between NK cells and neutrophils during an inflammatory response and provide further support for a role of NK cells in the resolution phase of inflammation.

Background Abnormal neutrophils are involved in many chronic endocrine diseases, including type 2 diabetes mellitus (T2DM), and in periodontitis (PD), which is a chronic inflammatory disease in which neutrophils play a vital role. The p38 mitogen-activated protein kinase (MAPK) signaling pathway participates in the apoptosis of many inflammatory cells. Additionally, 1,25-dihydroxyvitamin-D3 (1,25VitD3) as a regulator can induce responses to infection and tumor cell apoptosis. However, the effect of 1,25VitD3 in the pathogenic relationship between T2DM and PD remains unclear. The aim of this study was to assess the effect of 1,25VitD3 on neutrophil apoptosis in patients with T2DM and PD and the p38-MAPK-relevant signaling pathway mechanism in this process in vitro. Methods Neutrophils were stained with Wright's stain, and apoptosis was detected by flow cytometry and Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. Apoptosis- and p38-related mRNAs and proteins were examined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blotting and ELISA. The internal relationships were analyzed using a linear regression equation and Pearson's correlation coefficient. Results The highest rate of neutrophil apoptosis occurred in cultures treated with 10–8 mol/L 1,25VitD3 in the T2DM-PD group. The apoptosis rate in the T2DM-PD-p38 inhibitor group was higher than that in the healthy control group. Western blot, ELISA and qRT-PCR results showed that the mRNA and protein expression profiles of Caspase-3 and Bax were highly up-regulated and that Bcl-2 was down-regulated in the T2DM-PD-p38 inhibitor group. The expression levels of apoptotic mRNAs and proteins in the T2DM and T2DM-PD groups were significantly higher than those in the T2DM-p38 and T2DM-PD-p38 inhibitor groups. 1,25VitD3-induced neutrophil apoptosis and phosphorylated p38 (p-p38) expression were partially inhibited by the p38 inhibitor. Expression levels of apoptosis-related genes and p-p38 in neutrophils were positively associated with increasing concentrations of 1,25VitD3. p-p38 protein expression was positively associated with the level of serum 1,25VitD3. Conclusion 1,25VitD3 could promote peripheral blood neutrophil apoptosis in patients with T2DM and PD through activation of the p38-MAPK signaling pathway in vitro.

INTRODUCTION Altered neutrophil apoptosis might be responsible for recurrent bacterial infections encountered in hemodialysis (HD) and chronic kidney disease (CKD) patients. This work was designed to assess the neutrophil apoptotic activity and the impact of implementation of granulocyte macrophage colony stimulating factor (GM-CSF), as a survival factor, on neutrophil apoptosis among these patients. MATERIAL AND METHODS Twenty-five patients on regular HD along with 34 CKD patients on conservative treatment, as well as 15 healthy controls, were investigated for apoptotic rate via assessment of neutrophil expression of Annexin-V by flow cytometry, before and after 20 h culture in absence and presence of GM-CSF. Neutrophil viability was determined using light microscopy. The preservation of neutrophil activation in these patients was analyzed by flow cytometric CD18 neutrophil expression. Chronic inflammatory state was evaluated by estimating C-reactive protein (CRP) and soluble intercellular adhesion molecule-1 (sICAM-1). Obtained data were statistically analyzed. RESULTS Compared to controls, both HD and CKD groups had a significant increase of Annexin-V and CD18 expression and significant decrease in neutrophil viability. Culture of their neutrophils with GM-CSF showed significant decrease of apoptosis accompanied by improvement of neutrophil viability compared to their cultured cells without GM-CSF. These patients also showed significant elevation of CRP and sICAM-1. CONCLUSIONS Granulocyte macrophage colony stimulating factor demonstrated an evident impact on improving in vitro neutrophil survival and viability in HD and CKD patients. Therefore, this may represent promising preventive and/or therapeutic strategies against infection frequently observed in these patients and causing morbidity and mortality.

Vitamin C, also known as ascorbic acid (AA), is involved in all phases of wound healing. In the inflammatory phase it is required for neutrophil apoptosis and clearance. During the proliferative phase, AA contributes towards synthesis, maturation, secretion and degradation of collagen. Deficiencies affect the maturation phase by altering collagen production and scar formation. The body strives to maintain homeostasis of AA, thereby ensuring availability for collagen synthesis. After wounding, plasma and tissue levels of AA diminish and, as a consequence, supplements may be useful for healing, although levels beyond saturation are excreted. Clinicians need to be aware of both the nutritional status of patients with either acute or chronic wounds and the possibility of any AA deficiency which may hinder healing.

PMF of infancy is a recently described autosomal recessive disorder presenting with severe bone marrow failure, accelerated neutrophil apoptosis, and significant platelet dysfunction, caused by a mutation in the VPS45 gene. In this study, we update our group of patients with PMF, highlighting different aspects of this disease, and evaluating the effectiveness of HSCT for the treatment of this disorder. Update of clinical data, hematological features, molecular studies, treatment and final outcome of four children diagnosed with VPS 45-associated PMF of infancy. The patients described had clinical and hematological findings consistent with MF. Molecular studies showed that all patients were homozygous for the Thr224Asn mutation in the VPS 45 gene. HSCT was carried out in three patients and was successful in two. VPS 45-associated MF is a novel primary immune deficiency that can be successfully corrected by HSCT if applied early in the course of disease using appropriate conditioning. The diagnosis of VPS 45-associated PMF should be considered in all children presenting with SCN with subsequent development of pancytopenia. Long-term follow-up of these patients is necessary to identify extra-hematological manifestations of VPS45 deficiency.

SummaryInflammation is a double-edged sword in the outcome of pneumonia. On the one hand, an effective and timely inflammatory response is required to eliminate the invading respiratory pathogen. On the other, a toxic and prolonged inflammatory response may result in lung injury and poor outcomes, even in those receiving advanced medical care. This review focuses on recent understanding of the dynamics of the cytokine response, neutrophil activity, and responsiveness to cytokines and neutrophil lifespan as major elements of lung inflammation resulting in favorable or poor outcomes in lung infection primarily due to pneumococcus and influenza virus. Although some progress has been made in our understanding of the molecular mechanisms of the pneumonia inflammation axis composed of cytokines modulating neutrophil activation and neutrophil apoptosis, important questions remain to be answered. The degree of neutrophil activation, generation of reactive oxygen species, and the release of granule antimicrobial peptides play a key role in microbial pathogen clearance; however, prolonged neutrophil activation may contribute to lung injury and poor outcomes in pneumonia. Molecular markers of the mechanisms regulating neutrophil survival and apoptosis may help in the identification of novel therapeutic targets to modulate inflammation by inducing timely neutrophil apoptosis. A major task is to identify the mechanisms of dysregulation in inflammation leading to toxic responses, thereby targeting a biomarker and enabling timely therapies to modulate inflammation.

Delayed neutrophil apoptosis has been demonstrated in sepsis and may contribute to organ damage. It has recently been proposed that apolipoprotein L (ApoL) may be involved in programmed cell death, but the expression and functions of ApoLs in leukocytes (especially neutrophils) during sepsis and other inflammatory conditions are currently unknown. In this prospective observational study in a 36-bed university hospital medico-surgical intensive care unit (ICU), we included 78 adult ICU patients with (n = 41) or without (n = 37) sepsis and 47 healthy volunteers. We analyzed ApoL mRNA expression using quantitative polymerase chain reaction in whole blood leukocytes and protein expression in CD15 isolated neutrophils using western blotting. Neutrophil apoptosis was assessed using the APO-BRDU method. ApoL mRNA was downregulated in whole blood leukocytes and neutrophils in ICU patients compared to healthy volunteers and this effect translated at the protein level as indicated by western blot analysis of neutrophils. There was a negative correlation between ApoL expression in neutrophils and C-reactive protein (CRP) levels and a positive correlation between the number of apoptotic neutrophils and mRNA levels of ApoL1 and 2. The degree of neutrophil apoptosis in critically ill patients is therefore correlated with modified expression profiles of ApoLs., SCOPUS: ar.j, info:eu-repo/semantics/published

Neutrophils are the first responders to sites of acute tissue damage and infection. Recent studies suggest that in addition to neutrophil apoptosis, resolution of neutrophil inflammation at wounds can be mediated by reverse migration from tissues and transmigration back into the vasculature. In settings of chronic inflammation, neutrophils persist in tissues, and this persistence has been associated with cancer progression. However, the role of neutrophils in the tumor microenvironment remains controversial, with evidence for both pro- and anti-tumor roles. Here we review the mechanisms that regulate neutrophil recruitment and resolution at sites of tissue damage, with a specific focus on the tumor microenvironment. We discuss the current understanding as to how neutrophils alter the tumor microenvironment to support or hinder cancer progression, and in this context outline gaps in understanding and important areas of inquiry.

Recently, unexpected biological features of polymorphonuclear neutrophils have been revealed. In addition to their pivotal role in the defence against pathogens, neutrophils display a high degree of plasticity and contribute to control of adaptive immune responses. An emerging aspect of neutrophils is their ability to modulate their survival in response to both intrinsic and extrinsic factors. This review focuses on recent advances that have uncovered proliferating cell nuclear antigen (PCNA) and other cell cycle regulatory proteins as novel players regulating neutrophil survival. A better understanding of the mechanisms involved in neutrophil fate might pave the way for the identification of new anti-inflammatory molecules.

Nussbaum G, Shapira L: How has neutrophil research improved our understanding of periodontal pathogenesis? J Clin Periodontol 2011; 38 (Suppl. 11): 49–59. doi: 10.1111/j.1600-051X.2010.01678.x. Abstract Background: Neutrophils are the predominant cells responsible for host defence against bacterial infection. Loss of neutrophil defence, due either to deficient number or function, strongly predisposes to bacterial infections such as periodontitis. Yet, the neutrophil oxidative and proteolytic arsenal has also been implicated in perpetrating periodontal tissue damage in periodontitis. Aim: In this review, we focus on recent developments that shed light on these two aspects of neutrophil function in periodontitis. Methods: Primary search: using PubMed search for “neutophil”, “periodontal”, and “periodontitis”. Secondary search: using references from the articles found in the first stage. Results: Early histological studies showed that infiltrating neutrophils form a wall of cells abutting the junctional epithelium in periodontal inflammatory lesions. The chronic standoff between these neutrophils and the bacterial community suggests that bacterial evasion of neutrophil clearance is a major characteristic of periodontitis. Indeed, not all functional neutrophil deficiencies increase the risk of periodontitis, an observation that points the way towards identification of particular anti-bacterial pathways essential for protection against periodontal pathogens. The net result in the majority of periodontitis patients who exhibit normal neutrophil number and function, is that neutrophils accumulate in the periodontal tissue where they are available to participate in tissue destruction. Diminished neutrophil clearance further contributes to the persistence of activated neutrophils in the periodontal tissue. Conclusions: Data on the role of neutrophils in the pathogenesis of periodontitis are mixed. Neutrophils are a critical arm of the defence against periodontitis, but bacterial evasion of the neutrophil microbicidal machinery coupled with delayed neutrophil apoptosis may transform the neutrophil from defender to perpetrator. At this stage of knowledge, attempts to induce host modulation through neutrophil suppression or activation are premature.

ARDS pathophysiology is characterized by complex mechanisms that involve cells of inflammation, lung tissue cells, cytokines, chemokines, as well as apoptosis activators and inhibitors. There are two important theories that link apoptosis with ARDS and suggest that epithelial cell apoptosis, as well as the accumulation of neutrophils in the lung, may contribute to a cascade of events and, finally, ARDS. The activation of the Fas/FasL pathway is an important mechanism of alveolar epithelial injury in the lungs of patients with ALI. In addition, neutrophilic inflammation in the alveolar spaces is characteristic of ALI in humans and in most animal models of ALI. The enhanced phagocytosis of apoptotic neutrophils could lead to resolution of inflammation and repair during ARDS. In this review, we will focus on elucidating the role of apoptosis in the pathophysiology of ARDS and the contribution of Fas-mediated inflammation in ARDS. Furthermore, we will give evidence that TNF-alpha, IL-1beta and IL-13 attenuate the pro-cell death effects of Fas/CD95 on A549 epithelial cells, at least partially, by the NF-kB and PI3-K pathways, suggesting that induction of the expression of antiapoptotic genes protects the epithelial cells from cell death. (c) 2009 Elsevier GmbH. All rights reserved. Pathology Research and Practice

Apoptosis, or programmed cell death, is a highly conserved cellular suicide mechanism. Apoptosis is critical to the effective resolution of inflammation, particularly in regulating the lifespan of neutrophils. Neutrophils are key components of the first line of defense against microorganisms. Thus, subversion of this critical host defense mechanism by pathogens can contribute to susceptibility to severe and recurrent infections. In this review, we describe the molecular mechanisms involved in PMN death in relationship with viral infections.

Polymorphonuclear leukocytes or neutrophils are the main executors of cellular death, both in septic inflammation during bacterial infection and in sterile inflammation during trauma or surgery. Whereas in septic inflammation neutrophils perform a useful function to fortify the host's defense against infection, in sterile inflammation, by contrast, they contribute to unwelcome tissue damage. Regardless of the situation, activated neutrophils exhibit a prolonged lifespan and delayed apoptotic death which, under normal conditions, is a prerequisite for their natural renewal. Traditionally, delayed neutrophil apoptosis was considered to promote trauma or surgical injury. According to the results of recent studies, however surprising they may appear, the reverse might be in keeping with what happens IN VIVO. Apoptotic signaling in neutrophils could, by contrast, contribute to intrinsic protection of the host's tissues. This review article, aimed preferentially but not exclusively at the cardiac surgeon, presents some new information in support of this viewpoint, which fits in with our own observations.

Apoptosis, or programmed cell death, is a highly conserved cellular suicide mechanism. Apoptosis is critical to the effective resolution of inflammation, particularly in regulating the lifespan of the inflammatory neutrophil. Pathological dysregulation of neutrophil apoptosis prevents resolution of inflammation and is implicated in numerous inflammatory conditions. Similarly, subversion of this critical host defense mechanism by pathogens can prevent resolution of infection. Below, we describe the effects of different bacterial pathogens on regulation of neutrophil apoptosis and consider how re-engagement of these subverted mechanisms may facilitate the development of new strategies to combat infectious diseases.

Anne-Sophie Guedj,1 Arnold J Kell,2 Michael Barnes,2 Sandra Stals,1 David Gonçalves,3 Denis Girard,3 Carole Lavigne11National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB, 2National Research Council of Canada, Ottawa, ON, 3Laboratoire de recherche en inflammation et physiologie des granulocytes, Université du Québec, INRS-Institut Armand-Frappier, Laval,QC, CanadaAbstract: Following infection, HIV establishes reservoirs within tissues that are inaccessible to optimal levels of antiviral drugs or within cells where HIV lies latent, thus escaping the action of anti-HIV drugs. Macrophages are a persistent reservoir for HIV and may contribute to the rebound viremia observed after antiretroviral treatment is stopped. In this study, we further investigate the potential of poly(lactic-co-glycolic) acid (PLGA)-based nanocarriers as a new strategy to enhance penetration of therapeutic molecules into macrophages. We have prepared stable PLGA nanoparticles (NPs) and evaluated their capacity to transport an active molecule into the human monocyte/macrophage cell line THP-1 using bovine serum albumin (BSA) as a proof-of-concept compound. Intracellular localization of fluorescent BSA molecules encapsulated into PLGA NPs was monitored in live cells using confocal microscopy, and cellular uptake was quantified by flow cytometry. In vitro and in vivo toxicological studies were performed to further determine the safety profile of PLGA NPs including inflammatory effects. The size of the PLGA NPs carrying BSA (PLGA-BSA) in culture medium containing 10% serum was ~126nm in diameter, and they were negatively charged at their surface (zeta potential =-5.6mV). Our confocal microscopy studies and flow cytometry data showed that these PLGA-BSA NPs are rapidly and efficiently taken up by THP-1 monocyte-derived macrophages (MDMs) at low doses. We found that PLGA-BSA NPs increased cellular uptake and internalization of the protein in vitro. PLGA NPs were not cytotoxic for THP-1 MDM cells, did not modulate neutrophil apoptosis in vitro, and did not show inflammatory effect in vivo in the murine air pouch model of acute inflammation. In contrast to BSA alone, BSA encapsulated into PLGA NPs increased leukocyte infiltration in vivo, suggesting the in vivo enhanced delivery and protection of the protein by the polymer nanocarrier. We demonstrated that PLGA-based nanopolymer carriers are good candidates to efficiently and safely enhance the transport of active molecules into human MDMs. In addition, we further investigated their inflammatory profile and showed that PLGA NPs have low inflammatory effects in vitro and in vivo. Thus, PLGA nanocarriers are promising as a drug delivery strategy in macrophages for prevention and eradication of intracellular pathogens such as HIV and Mycobacterium tuberculosis.Keywords: PLGA nanoparticles, BSA delivery, inflammatory profile, neutrophil apoptosis, murine air pouch, HIV reservoir

Neutrophils are short-lived leukocytes that play a vital role in immune responses to bacteria, yeast, and fungi. This study was performed to investigate the effect of 4-O-methyl-ascochlorin (MAC), an anti-tumor, antibiotic, and anti-fungal prenyl-phenol compound on the spontaneous apoptosis of human neutrophils. MAC time- and dose-dependently accelerated the spontaneous apoptosis of human neutrophils. The effect of MAC on neutrophil apoptosis was blocked by pre-treatment of the neutrophils with specific inhibitors of pancaspase (zVAD-fmk), caspase-8 (zIETD-fmk), or caspase-3 (zDEVD-fmk). The cleavage of procaspase-8 and procaspase-3 was increased by MAC. Mitochondrial permeability, which was measured by the retention of DiOC_6(3), was dose-dependently increased by MAC but the change of mitochondrial permeability was not blocked by pretreatment of neutrophils with zIETD-fmk. These results suggest that MAC induces neutrophil apoptosis by caspase-8-dependent but mitochondria-independent manner.

This study evaluated the expression of neutrophil apoptosis and the effects of melatonin at different concentrations on delayed neutrophil apoptosis in different severities of acute pancreatitis in patients.The study population was comprised of 10 patients with severe acute pancreatitis (SAP) and 10 with mild acute pancreatitis (MAP). A total of 10 mL of blood was drawn 24 hours after the onset of the clinical disease for isolation and incubation of the human neutrophils with 4 different concentrations of melatonin. Neutrophil apoptosis activity, CD18 expression, and respiratory burst activity were assessed with flow cytometry 12 hours after incubation. Another group of neutrophils from a healthy control group was used (n = 6) for comparison.Neutrophil apoptosis in patients with SAP is delayed compared with that of patients with MAP. Neutrophils from patients with SAP or MAP are functionally activated. Melatonin at concentrations of 10(-8), 10(-7), or 10(-6) M reverses the delayed process and enhances apoptosis activity in neutrophils in patients with MAP. Melatonin at concentrations of 10(-7) and 10(-6) M reverses the delayed process and increases apoptosis activity in neutrophils in patients with SAP. Neutrophils from patients with SAP and MAP showed significantly increased CD18 expression and respiratory burst activity. Melatonin at concentrations of 10(-7) or 10(-6) M reverses CD18 expression and respiratory burst activity in neutrophils in patients with SAP.This study highlights the importance of neutrophil apoptosis in patients with SAP and raises the possibility of a therapeutic strategy. Study data show that melatonin promotes neutrophil apoptosis in human acute pancreatitis.

The accumulation of neutrophils during inflammation is essential for the destruction and removal of invading microorganisms. However, for resolution of inflammation to occur, neutrophils must also be removed from the inflammatory site since these cells are capable of releasing tissue toxic molecules. Neutrophil removal has been shown to occur via apoptosis and phagocyte clearance of apoptotic cells. Therefore, manipulation of these processes is likely to be a key therapeutic strategy in the management of inflammatory disease. In this review, we examine mediators of neutrophil survival and apoptosis and the signalling pathways that regulate the balance between life and death in these cells.

The primitive roles of neutrophils are known as phagocyte to engulf and kill microorganisms which invade human bodies. Recently, it is well established that neutrophils play important roles to modulate inflammation. Clearance of apoptotic neutrophils by cells such as macrophages can lead to the resolution of inflammation. It is also important to clarify the mechanisms of neutrophilic inflammation and to develop therapeutic strategies based on the induction of neutrophil apoptosis in order to reverse or attenuate an inflammatory response. Several modulators of neutrophil apoptosis and activators of neutrophil priming have been discovered, they have been discussed in the pathogenesis of several diseases. However, we have no potential therapeutic strategy on the property so far. New clinical perspectives in future are expected.

The action of antibacterial cathelicidin CAP11 (cationic antibacterial polypeptide of 11 kDa) on the lipopolysaccharide (LPS)-induced suppression of neutrophil apoptosis was evaluated in vitro.Human neutrophils (10(6) cells/ml) were incubated alone or with mononuclear cells (6 x 10(5) cells/ml) in the presence of LPS (10 ng/ml) and CAP 11 (0.1 approximately 10 microg/ml), and neutrophil apoptosis was determined.LPS suppressed neutrophil apoptosis, accompanied with the activation of NF-kappaB, phosphorylation of extracellular signal-related protein kinase (ERK), expression of Bcl-XL (an anti-apoptotic protein) and inhibition of caspase 3 activity. Interestingly, CAP11 (1 microg/ml) reversed the actions of LPS to trigger these changes, and induced neutrophil apoptosis (p0.0001). Moreover, neutralizing antibodies against Mac-1 (CD11b/CD18) and Toll-like receptor (TLR) 4 completely blocked the LPS-induced suppression of neutrophil apoptosis (p0.0001), suggesting a major role of Mac-1 and TLR4 in the LPS-mediated neutrophil activation. In addition, LPS activated monocytes to produce proinflammatory cytokines (IL-1beta, TNF-alpha and IL-8) and inhibited neutrophil apoptosis. Importantly, CAP11 (1 microg/ml) reduced the cytokine production, thereby inducing neutrophil apoptosis (p0.0001). Finally, CAP11 (1 microg/ml) strongly suppressed the LPS-binding to neutrophils and monocytes (p0.01).CAP11 is able to block the LPS-induced survival of neutrophils via the suppression of anti-apoptotic signaling in neutrophils and cytokine production from monocytes by inhibiting the binding of LPS to target cells.

Neutrophil apoptosis is a constitutive process that can be enhanced or delayed by signals triggered by various stimuli. In this work, we investigated the action mechanism of phospholipase D (PLD) and its expression in the inhibition of spontaneous and Fas-mediated apoptosis. Anti-Fas antibody-stimulated apoptosis of neutrophils was significantly blocked by the exogenous addition of bacterial PLD from Streptomyces chromofuscus (scPLD), and neutrophils cultured for 24 h in the presence of anti-Fas antibody showed lower agonist-stimulated PLD activity compared to untreated cells. The amount of PLD1a protein reduced time-dependently in cultured neutrophils, but was recovered by treating with LPS or GM-CSF. The reduction in PLD1a protein level was blocked by caspase inhibitors. The exogenous addition of scPLD blocked the up-regulation of Fas-associated death domain expression, mitochondrial permeability, and the cleavages of procaspase-8, procaspase-3, and protein kinase C-delta. We also found that the protein level of apoptosis-inducing factor was increased in cultured neutrophils but its expression was reduced by scPLD. However, sulfasalazine-induced apoptosis and change of protein expression were not blocked by scPLD. Taken together, the activity and protein levels of PLD play a role as an anti-apoptotic factor by acting at multiple levels of the apoptotic cascade in neutrophils.

Neutrophils rapidly undergo spontaneous apoptosis, but this process can be considerably delayed by exposure to a variety of agents such as pro-inflammatory cytokines. The anti-apoptotic protein of the Bcl-2 family, Mcl-1, plays a key role in the regulation of neutrophil apoptosis. The protein has some unusual properties compared with other family members, including an extremely high turnover rate. Many factors, such as cytokines and local oxygen concentrations, can regulate cellular levels of Mcl-1 via transcription and post-transcriptional modification, control the survival time of neutrophils within tissues and thereby influence the inflammatory response.

Neutrophils and macrophages are involved in inflammation. Neutrophils debride the area of local contamination and then die (apoptosis) after being engulfed by macrophages. This is vital before the wound can move to the next stage of healing.

Following inflammatory-cell recruitment in the lung, neutrophil apoptosis and subsequent engulfment by macrophages are regarded as important components in the resolution of pulmonary inflammation. The goal of this study was to further investigate the role of apoptosis and its influence, if any, on the pulmonary inflammatory process following exposures to the following particulate types: amorphous (AMO) or crystalline silica (Si), lipopolysaccharide (LPS), or pigment-grade titanium dioxide (TiO2). Rats were intratracheally instilled either with TiO2, AMO, or Si particles at doses of 1 or 5 mg/kg or 6 microg LPS. Following exposures, bronchoalveolar lavage fluids and lung tissues were collected and evaluated at 12, 24, 48, or 168 h (i.e., 1 wk). At the 1 mg/kg dose, AMO instillation produced the highest pulmonary inflammatory response, concomitant with a rise in apoptotic cells that mirrored temporally the transient nature of the inflammatory response. At 5 mg/kg, amorphous silica and crystalline silica particles induced substantial pulmonary inflammation [approximately 50-60% neutrophils (PMNs)] at 12 h postexposure (pe). A fundamental difference between the two inflammatory patterns, however, was the subsequent reversibility of inflammation in the AMO-exposed rats at 168 h postexposure and the sustained inflammatory effect in the Si-exposed animals measured through 168 h pe (approximately 40% PMNs). Pulmonary apoptotic responses in AMO-exposed rats mirrored temporally and correlated with the time-course reduction of inflammatory responses, leading to resolution. In the Si-exposed rats, apoptotic levels remained elevated, concomitant with sustained inflammation measured through 168 h pe. High doses of TiO2 particles produced transient lung inflammation, but with low levels of apoptosis. In addition, instillation of LPS produced a transient inflammatory response which mirrored the time course of apoptosis levels and was resolved by 168 h pe. cDNA microarray methods demonstrated that gene expression was altered for several apoptosis-related genes in AMO-, Si-, and LPS-exposed animals at 24 h pe. The results of these studies demonstrate that, following exposures, the resolution of lung inflammation correlated temporally with apoptotic levels of neutrophils in AMO- and LPS-exposed rats. Alternatively, instillation of crystalline silica resulted in sustained pulmonary inflammation and measurable apoptosis at 1 wk postexposure, but the apoptotic cell processes were not effective in resolving the inflammatory response. The findings suggest that the coordination between the resolution of inflammation and inflammatory cell apoptosis in the lung is dependent on the particle-type and that other factors, such as particle cytotoxicity, may also be important.

Neutrophil apoptosis is delayed in medical conditions associated to anoxia or hypoxia, prolonging tissue destruction and fostering the inflammation. Hypoxia Inducible Factor-1α (HIF-1α), is a main regulator of delayed neutrophil apoptosis but the mechanism of action is poorly characterized. Neutrophil gelatinase-associated lipocalin (24p3) participates actively in iron metabolism and the regulation of iron-responsive genes. Recently, a connection has been described between HIF-1α and 24p3. The purpose of the present study was to determine whether constitutive apoptosis in neutrophils requires 24p3 and whether HIF-1α represses 24p3 affecting cell death iron intracellular levels. To this end we used in vivo ischemic models and anoxic approaches based on the reactivation of the delayed apoptosis. We found that the stabilization of HIF-α during anoxic periods provoked a delay in neutrophil apoptosis through decrease of 24p3 expression and intracellular iron content. The ischemia drastically inhibited the synthesis of 24p3 in circulating neutrophils, increasing the tissue damage. Reactivation of neutrophil apoptosis with opsonized E.coli induced increases in intracellular levels of iron and 24p3. In conclusion, contrary to other cell types, constitutive apoptosis in neutrophils requires 24p3. During hypoxia or ischemia, HIF-1α stabilization represses 24p3 expression, consequently iron levels are depleted and neutrophil apoptosis is delayed. Copyright © by BIOLIFE, s.a.s.

Delayed apoptosis of primed neutrophils (PMNs) may facilitate PMN-mediated tissue injury leading to multiple organ failure (MOF). We previously reported delayed apoptosis and priming of PMNs in severely injured patients at risk for MOF. Our in vitro and in vivo data have implicated phospholipids in PMN cytotoxicity following trauma and shock. The phospholipid signaling pathway remains to be elucidated, but may involve protein kinase C (PKC). We hypothesized that circulating platelet-activating factor (PAF) and PAF-like proinflammatory phospholipids mediate delayed postinjury PMN apoptosis and that PKC is integral to the signaling pathway.Blood was drawn from severely injured patients (n = 6; mean injury severity score = 21 and transfusion = 10 units) at 6 h postinjury. The plasma fraction was isolated and incubated (5% CO(2), 37 degrees C, 24 h) with PMNs harvested from healthy volunteers. Some PMNs were preincubated with a PAF receptor antagonist (WEB 2170, 400 microM) or a PKC inhibitor (Bis I, 1 microM). Apoptotic index (% PMNs undergoing apoptosis) was assessed morphologically.Trauma patients' plasma delayed PMN apoptosis compared with plasma from controls. The PMN apoptotic index was not altered by WEB 2170 or Bis I alone; however, WEB 2170 or Bis I pretreatment abrogated delayed PMN apoptosis in response to trauma patients' plasma.Trauma patients' plasma delays apoptosis of PMNs. Our data implicate PAF-like phospholipids in this effect, and PKC appears to be integral in the signaling process. Further elucidation of specific lipids and signaling pathways may reveal clinically accessible therapeutic targets to prevent PMN-mediated hyperinflammation.

We examined the ability of TNF-α to modulate human neutrophil apoptosis. Neutrophils cultured with TNF-α alone undergo a low but significant increase in the number of apoptotic cells. More interestingly, when neutrophils were pretreated with TNF-α for 1–2 min at 37°C and then were exposed to a variety of agents such as immobilized IgG, IgG-coated erythrocytes, complement-treated erythrocytes, zymosan, PMA, zymosan-activated serum, fMLP, Escherichia coli, and GM-CSF for 3 h at 37°C, a marked stimulation of apoptosis was observed. Similar results were obtained in neutrophils pretreated with TNF-α for 30 min, 1 h, 3 h, and 18 h. Dose-dependent studies showed that TNF-α enhances neutrophil apoptosis at concentrations ranging from 1 to 100 ng/ml. In contrast to the observations made in neutrophils pretreated with TNF-α, there was no stimulation of apoptosis when TNF-α was added to neutrophils previously activated by conventional agonists. Experiments performed to establish the mechanism through which TNF-α promotes neutrophil apoptosis showed that neither reactive oxygen intermediates nor the Fas/Fas ligand system appear to be involved. Our results suggest that TNF-α plays a critical role in the control of neutrophil survival by virtue of its ability to induce an apoptotic death program which could be triggered by a variety of conventional agonists. Polymorphonuclear neutrophils are short-lived cells. In the absence of appropriate stimuli, they rapidly undergo characteristic changes indicative of apoptosis, including cell shrinkage, nuclear chromatin condensation, DNA fragmentation into nucleosome-length fragments, and cell surface exposure of phosphatidylserine (PS)3 (1, 2). Apoptosis, which represents an alternative fate to necrosis, not only determines neutrophil uptake by macrophages but also is associated with a loss of neutrophil functions, such as chemotaxis, phagocytosis, stimulated shape change, degranulation, and respiratory burst (3, 4, 5). For these reasons, neutrophil apoptosis may be considered a mechanism that contributes to the resolution of acute inflammation (1, 2, 3, 4, 5). As a first line of defense against host insult, neutrophils are rapidly recruited to inflammatory sites, where the expression of their apoptotic program can be modified by a number of agents. In vitro studies have identified a variety of agents that modulate neutrophil apoptosis. GM-CSF, IL-2, leukotriene B4, corticosteroids, and LPS inhibit neutrophil apoptosis (6, 7, 8, 9, 10, 11), whereas proteolytic enzymes, immune complexes, bacteria and virus induce neutrophil apoptosis (12, 13, 14, 15). Controversial results, on the other hand, have been reported regarding the effects of C5a, fMLP, G-CSF, and IL-6 (3, 5, 6, 7, 8, 9). During the course of inflammatory processes, macrophages, lymphocytes, and/or mast cells produce TNF-α, a powerful priming agonist of neutrophils (16, 17). Previous in vitro studies have shown that TNF-α enhances the expression of CD11b/CD18 on neutrophils, increases neutrophil adhesion to endothelium, triggers adherent neutrophils to release large amounts of reactive oxygen intermediates (ROI), and promotes neutrophil degranulation, phagocytosis, and Ab-dependent cell-mediated cytotoxicity (18, 19, 20, 21, 22). TNF-α has been variably reported either to induce, delay, or have no effect on neutrophil apoptosis (6, 23, 24, 25). These contrasting results could be explained, at least in part, by recent findings of Murray et al. (26), who showed that although prolonged incubation (>18 h) of human neutrophils with TNF-α indeed causes a decrease in the extent of apoptosis, TNF-α can induce apoptosis in a proportion of cells at earlier times (

In the present study, to investigate the apoptosis of the polymorphonuclear neutrophil (PMN) from healthy dogs, we carried out TUNEL assay and DNA analysis by electrophoresis on dog PMNs. The TUNEL assay indicated that apoptotic PMNs in dogs were 0.15+/-5% before incubation, 0.3+/-5% at 4h incubation, 1+/-6% at 8h, 9+/-4% at 12h and 28+/-5% at 24h, respectively. The ladder formation was much more clearly observed in DNA from PMNs after 24h incubation at 37 degrees C than that before incubation. The results in this study indicated that healthy dog PMNs undergo apoptosis spontaneously within hours to days, and that the apoptosis of PMNs might be related to the high turnover of these circulating cells in dogs.

To elucidate the role of A1, a new member of the Bcl-2 family of apoptosis regulators active in hematopoietic cell apoptosis, we established mice lacking A1-a, a subtype of the A1 gene in mice (A1-a−/− mice). Spontaneous apoptosis of peripheral blood neutrophils of A1-a−/− mice was enhanced compared with that of either wild-type mice or heterozygous mutants (A1-a+/− mice). Neutrophil apoptosis inhibition induced by lipopolysaccharide treatment in vitro or transendothelial migration in vivo observed in wild-type mice was abolished in both A1-a−/− and A1-a+/− animals. On the other hand, the extent of tumor necrosis factor α–induced acceleration of neutrophil apoptosis did not differ among A1-a−/−, A1-a+/−, and wild-type mice. The descending order of A1 mRNA expression was wild-type, A1-a+/−, and A1-a−/−. Taken together, these results suggest that A1 is involved in inhibition of certain types of neutrophil apoptosis.

Neutrophil apoptosis is an important mechanism that has implications for understanding the life span and toxic potentials of neutrophils at inflamed sights. In this study the authors examined the possibility that reactive oxygen species (ROS) released by neutrophils can regulate neutrophil survival. Cu,Zn-superoxide dismutase (Cu,Zn-SOD), Mn-SOD, and catalase in culture media were significantly effective in delaying the spontaneous apoptosis, suggesting that ROS play an important role in the resolution of inflammation by inducing neutrophil apoptosis. In this experiment, boiled Cu,Zn-SOD had no effect on inhibiting the apoptosis, but boiled Mn-SOD from Bacillus stearothermophilus was more effective in inhibiting the apoptosis than untreated Mn-SOD at the same dose. However, the boiled Mn-SOD showed only 80% of O2- inhibitable activity compared with the untreated Mn-SOD. This effect may be attributed to the partial liberation of manganese because MnCl2 inhibited the apoptosis effectively. Furthermore, Cu,Zn-SOD was effective in delaying apoptosis only when added to the culture within the first 3 h of incubation, suggesting that the isolation of neutrophils from peripheral blood enhances apoptosis of neutrophils.

There is growing interest in inflammation due to its involvement in many diverse medical conditions, including Alzheimer's disease, cancer, arthritis and asthma. The traditional view that resolution of inflammation is a passive process is now being superceded by an alternative hypothesis whereby its resolution is an active, anti-inflammatory process that can be manipulated therapeutically. This shift in mindset has stimulated a resurgence of interest in the biological mechanisms by which inflammation resolves. The anti-inflammatory processes central to the resolution of inflammation revolve around macrophages and are closely related to pro-inflammatory processes mediated by neutrophils and their ability to damage healthy tissue. We develop a spatially averaged model of inflammation centring on its resolution, accounting for populations of neutrophils and macrophages and incorporating both pro- and anti-inflammatory processes. Our ordinary differential equation model exhibits two outcomes that we relate to healthy and unhealthy states. We use bifurcation analysis to investigate how variation in the system parameters affects its outcome. We find that therapeutic manipulation of the rate of macrophage phagocytosis can aid in resolving inflammation but success is critically dependent on the rate of neutrophil apoptosis. Indeed our model predicts that an effective treatment protocol would take a dual approach, targeting macrophage phagocytosis alongside neutrophil apoptosis.

This case report shows a striking correlation of remarkable brief high levels of pro- and anti-inflammatory cytokines coupled with increased neutrophil activation, followed by a sharp decrease in cytokine levels and increased neutrophil apoptosis associated with the favorable clinical outcomes of a patient with severe influenza infection. The host response examined in our case is not complete, given it did not assess the full spectrum of host response. The brief neutrophil and cytokine response seen in our case in the absence of antiviral therapy and in the presence of methotrexate immunosuppressive therapy rise the question as to whether the latter optimally modulated the macrophage function, resulting in a favorable outcome of severe influenza viral infection.

Background The death receptor ligand tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) shows considerable clinical promise as a therapeutic agent. TRAIL induces leukocyte apoptosis, reducing acute inflammatory responses in the lung. It is not known whether TRAIL modifies chronic lung injury or whether TRAIL has a role in human idiopathic pulmonary fibrosis (IPF). We therefore explored the capacity of TRAIL to modify chronic inflammatory lung injury and studied TRAIL expression in patients with IPF. Methods TRAIL � /� and wild-type mice were instilled with bleomycin and inflammation assessed at various time points by bronchoalveolar lavage and histology. Collagen deposition was measured by tissue hydroxyproline content. TRAIL expression in human IPF lung samples was assessed by immunohistochemistry and peripheral blood TRAIL measured by ELISA. Results TRAIL � /� mice had an exaggerated delayed inflammatory response to bleomycin, with increased neutrophil numbers (mean 3.1960.8 wild type vs 11.565.4310 4 TRAIL � /� ,p

Background: ANCA-Associated Systemic Vasculitis (AASV) is characterized by leukocytoclasis, accumulation of unscavenged apoptotic and necrotic neutrophils in perivascular tissues. Dysregulation of neutrophil cell death may contribute directly to the pathogenesis of AASV. less thanbrgreater than less thanbrgreater thanMethods: Neutrophils from Healthy Blood Donors (HBD), patients with AASV most in complete remission, Polycythemia Vera (PV), Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA) and renal transplant recipients (TP) were incubated in vitro, and the rate of spontaneous apoptosis was measured by FACS. Plasma levels of cytokines and sFAS were measured with cytometric bead array and ELISA. Expression of pro/anti-apoptotic factors, transcription factors C/EBP-alpha, C/EBP-beta and PU.1 and inhibitors of survival/JAK2-pathway were measured by real-time-PCR. less thanbrgreater than less thanbrgreater thanResults: AASV, PV and RA neutrophils had a significantly lower rate of apoptosis compared to HBD neutrophils (AASV 50 +/- 14% vs. HBD 64 +/- 11%, p andlt; 0.0001). In RA but not in AASV and PV, low apoptosis rate correlated with increased plasma levels of GM-CSF and high mRNA levels of anti-apoptotic factors Bcl-2A1 and Mcl-1. AASV patients had normal levels of G-CSF, GM-CSF and IL-3. Both C/EBP-alpha, C/EBP-beta were significantly higher in neutrophils from AASV patients than HBD. Levels of sFAS were significantly higher in AASV compared to HBD. less thanbrgreater than less thanbrgreater thanConclusion: Neutrophil apoptosis rates in vitro are decreased in AASV, RA and PV but mechanisms seem to differ. Increased mRNA levels of granulopoiesis-associated transcription factors and increased levels of sFAS in plasma were observed in AASV. Additional studies are required to define the mechanisms behind the decreased apoptosis rates, and possible connections with accumulation of dying neutrophils in regions of vascular lesions in AASV patients. Funding Agencies|Swedish Research Council|71X-15152|Crafoord Foundation

Leukocytes are known to exacerbate inflammatory and vaso-occlusive processes in sickle cell disease (SCD). The aim of this study was to determine whether alterations in neutrophil maturity and/or cell-death modulating factors in the circulation contribute to the increased leukocyte counts and leukocyte survival observed in SCD. The maturity of circulating neutrophils from healthy control individuals (CON), SCD and SCD patients on hydroxyurea therapy (SCDHU) was determined immunophenotypically. Serum factors affecting neutrophil apoptosis (determined by annexin V-binding) were analyzed by culturing control neutrophils (CON neutrophils) with pooled serum from CON, SCD and SCDHU individuals. Immunophenotypic characterization of neutrophils suggested a slight, but significant, increase in the circulation of immature neutrophils in SCD. While SCD neutrophils cultured in the presence of CON serum presented delayed apoptosis, unexpectedly, the culture of CON neutrophils with SCD serum significantly augmented apoptosis and caspase-9 activity. Inhibition of the activity of serum interleukin-8, a neutrophil-apoptosis-inhibiting cytokine, significantly increased SCD serum-induced CON neutrophil apoptosis, indicating that SCD serum may have both apoptotic and antiapoptotic properties. The decreased maturity of SCD neutrophils observed is suggestive of an accelerated immigration of leukocytes from the bone marrow to the circulating pool that may contribute to an increase in cell survival, subject to modulation by a complex balance of both anti- and proapoptotic factors contained in the circulation of SCD individuals.

In this issue of Blood , Elks et al describe a novel role for activated hypoxia inducible factor (HIF)–1α in sustaining inflammation by delaying neutrophilic retrograde emigration and preventing neutrophil apoptosis through the inhibition of prolyl hydroxylase (PHD) activity. This property is a novel function for HIFs, the master regulators of our body9s response to hypoxia.

Human tissue inflammation is terminated, at least in part, by the death of inflammatory neutrophils by apoptosis. The regulation of this process is therefore key to understanding and manipulating inflammation resolution. Previous data have suggested that the short-lived pro-survival Bcl-2 family protein, Mcl-1, is instrumental in determining neutrophil lifespan. However, Mcl-1 can be cleaved following caspase activity, and the possibility therefore remains that the observed fall in Mcl-1 levels is due to caspase activity downstream of caspase activation, rather than being a key event initiating apoptosis in human neutrophils. We demonstrate that apoptosis in highly purified neutrophils can be almost completely abrogated by caspase inhibition with the highly effective di-peptide caspase inhibitor, Q-VD. OPh, confirming the caspase dependence of neutrophil apoptosis. Effective caspase inhibition does not prevent the observed fall in Mcl-1 levels early in ultrapure neutrophil culture, suggesting that this fall in Mcl-1 levels is not a consequence of neutrophil apoptosis. However, at later timepoints, declines in Mcl-1 can be reversed with effective caspase inhibition, suggesting that Mcl-1 is both an upstream regulator and a downstream target of caspase activity in human neutrophils.