Increased Neutrophil Survival Research Articles

A culture model to analyze the acute biomaterial-dependent reaction of human primary neutrophils in vitro.

Neutrophils play a pivotal role in orchestrating the immune system response to biomaterials, the onset and resolution of chronic inflammation, and macrophage polarization. However, the neutrophil response to biomaterials and the consequent impact on tissue engineering approaches is still scarcely understood. Here, we report an in vitro culture model that comprehensively describes the most important neutrophil functions in the light of tissue repair. We isolated human primary neutrophils from peripheral blood and exposed them to a panel of hard, soft, naturally- and synthetically-derived materials. The overall trend showed increased neutrophil survival on naturally derived constructs, together with higher oxidative burst, decreased myeloperoxidase and neutrophil elastase and decreased cytokine secretion compared to neutrophils on synthetic materials. The culture model is a step to better understand the immune modulation elicited by biomaterials. Further studies are needed to correlate the neutrophil response to tissue healing and to elucidate the mechanism triggering the cell response and their consequences in determining inflammation onset and resolution.

Differential effects of antibiotics on neutrophils exposed to lipoteichoic acid derived from Staphylococcus aureus

BackgroundPersistent bacteremia occurs in at least 30% of patients with Staphylococcus aureus bloodstream infection (SAB) and may be attributable to a dysregulated host immune response. Neutrophils interact with a variety of S. aureus microbial factors, including lipoteichoic acid (LTA), to activate phagocytic function in a concentration-dependent manner. Antibiotics have been shown to exert both direct antimicrobial action as well as immunomodulatory effects. In this study, we compared the effects of different anti-staphylococcal antibiotics on LTA-mediated immune activation of neutrophils.MethodsNeutrophils obtained from healthy volunteers were exposed to two levels of LTA (1 and 10 μg/ml) with or without addition of antibiotics from different pharmacologic classes (vancomycin, daptomycin, ceftaroline). Neutrophil function was assessed by examining phagocytic response, activation (CD11b, CD62L expression), Toll-like receptor-2 expression, cell survival and apoptosis, and CXCL8 release.ResultsDifferential LTA-mediated antibiotic effects on neutrophil function were observed primarily at the high LTA exposure level. Ceftaroline in the presence of 10 μg/ml LTA had the most prominent effects on phagocytosis and CD11b and CD62L expression, with trends towards increased neutrophil survival and preservation of CXCL8 release when compared to daptomycin and vancomycin with the latter significantly dampening PMN CXCL8 release.ConclusionsSelect antimicrobial agents, such as ceftaroline, exert immunostimulatory effects on neutrophils exposed to S. aureus LTA, which when confirmed in vivo, could be leveraged for its dual immunomodulatory and antibacterial actions for the treatment of persistent SAB mediated by a dysregulated host response.

Increased mucosal neutrophil survival is associated with altered microbiota in HIV infection.

Gastrointestinal (GI) mucosal dysfunction predicts and likely contributes to non-infectious comorbidities and mortality in HIV infection and persists despite antiretroviral therapy. However, the mechanisms underlying this dysfunction remain incompletely understood. Neutrophils are important for containment of pathogens but can also contribute to tissue damage due to their release of reactive oxygen species and other potentially harmful effector molecules. Here we used a flow cytometry approach to investigate increased neutrophil lifespan as a mechanism for GI neutrophil accumulation in chronic, treated HIV infection and a potential role for gastrointestinal dysbiosis. We report that increased neutrophil survival contributes to neutrophil accumulation in colorectal biopsy tissue, thus implicating neutrophil lifespan as a new therapeutic target for mucosal inflammation in HIV infection. Additionally, we characterized the intestinal microbiome of colorectal biopsies using 16S rRNA sequencing. We found that a reduced Lactobacillus: Prevotella ratio associated with neutrophil survival, suggesting that intestinal bacteria may contribute to GI neutrophil accumulation in treated HIV infection. Finally, we provide evidence that Lactobacillus species uniquely decrease neutrophil survival and neutrophil frequency in vitro, which could have important therapeutic implications for reducing neutrophil-driven inflammation in HIV and other chronic inflammatory conditions.

The cytokine environment influence on human skin-derived T cells.

Skin resident T cells provide immediate immunologic responses at their specific location and play a role in the pathogenesis of skin diseases such as psoriasis. Recently, IL-9–producing T cells were described as a major T-cell subtype present in the skin, but knowledge on the biology and in situ regulation of this T-cell subtype is scarce. Here, we investigated the cytokine influence on skin T cells with focus on IL-9–producing T cells because a better understanding of their biology may identify novel therapeutic approaches. Healthy human skin biopsies were cultured either in the presence of IL-2, IL-4, and TGF-β [T helper (Th)9–promoting condition (Th9-PC)] or IL-2 and IL-15 [standard condition (SC)]. Paired analysis of enzymatically isolated skin T cells and emigrated T cells after 4 wk of skin culture showed significant alterations of T-cell phenotypes, cytokine production, and IL-9–producing T-cell frequency. RNA sequencing analysis revealed differentially regulated pathways and identified CXCL8 and CXCL13 as top up-regulated genes in Th9-PC compared with SC. Functionally supernatant of stimulated skin-derived T cells, CXCL8 and CXCL13 increased neutrophil survival. We report that the cytokine environment alters skin-derived T-cell phenotype and functional properties.—Kienzl, P., Polacek, R., Reithofer, M., Reitermaier, R., Hagenbach, P., Tajpara, P., Vierhapper, M., Gschwandtner, M., Mildner, M. Jahn-Schmid, B., Elbe-Bürger, A. The cytokine environment influence on human skin–derived T cells.

Targets of Neutrophil Influx and Weaponry: Therapeutic Opportunities for Chronic Obstructive Airway Disease.

Neutrophils are important effector cells of antimicrobial immunity in an acute inflammatory response, with a primary role in the clearance of extracellular pathogens. However, in respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD), there is excessive infiltration and activation of neutrophils, subsequent production of reactive oxygen species, and release of serine proteases, matrix metalloproteinases, and myeloperoxidase—resulting in collateral damage as the cells infiltrate into the tissue. Increased neutrophil survival through dysregulated apoptosis facilitates continued release of neutrophil-derived mediators to perpetuate airway inflammation and tissue injury. Several target mechanisms have been investigated to address pathologic neutrophil biology and thereby provide a novel therapy for respiratory disease. These include neutrophil influx through inhibition of chemokine receptors CXCR2, CXCR1, and PI3Kγ signaling and neutrophil weaponry by protease inhibitors, targeting matrix metalloproteinases and neutrophil serine proteases. In addition, neutrophil function can be modulated using selective PI3Kδ inhibitors. This review highlights the latest advances in targeting neutrophils and their function, discusses the opportunities and risks of neutrophil inhibition, and explores how we might better develop future strategies to regulate neutrophil influx and function for respiratory diseases in dire need of novel effective therapies.

Fever-range hyperthermia improves the anti-apoptotic effect induced by low pH on human neutrophils promoting a proangiogenic profile.

Neutrophils have the shortest lifespan among leukocytes and usually die via apoptosis, limiting their deleterious potential. However, this tightly regulated cell death program can be modulated by pathogen-associated molecular patterns (PAMPs), danger-associated molecular pattern (DAMPs), and inflammatory cytokines. We have previously reported that low pH, a hallmark of inflammatory processes and solid tumors, moderately delays neutrophil apoptosis. Here we show that fever-range hyperthermia accelerates the rate of neutrophil apoptosis at neutral pH but markedly increases neutrophil survival induced by low pH. Interestingly, an opposite effect was observed in lymphocytes; hyperthermia plus low pH prevents lymphocyte activation and promotes the death of lymphocytes and lymphoid cell lines. Analysis of the mechanisms through which hyperthermia plus low pH increased neutrophil survival revealed that hyperthermia further decreases cytosolic pH induced by extracellular acidosis. The fact that two Na+/H+ exchanger inhibitors, 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and amiloride, reproduced the effects induced by hyperthermia suggested that it prolongs neutrophil survival by inhibiting the Na+/H+ antiporter. The neutrophil anti-apoptotic effect induced by PAMPs, DAMPs, and inflammatory cytokines usually leads to the preservation of the major neutrophil effector functions such as phagocytosis and reactive oxygen species (ROS) production. In contrast, our data revealed that the anti-apoptotic effect induced by low pH and hyperthermia induced a functional profile characterized by a low phagocytic activity, an impairment in ROS production and a high ability to suppress T-cell activation and to produce the angiogenic factors VEGF, IL-8, and the matrix metallopeptidase 9 (MMP-9). These results suggest that acting together fever and local acidosis might drive the differentiation of neutrophils into a profile able to promote both cancer progression and tissue repair during the late phase of inflammation, two processes that are strongly dependent on the local production of angiogenic factors by infiltrating immune cells.

Myeloid conditional deletion and transgenic models reveal a threshold for the neutrophil survival factor Serpinb1.

Serpinb1 is an inhibitor of neutrophil granule serine proteases cathepsin G, proteinase-3 and elastase. One of its core physiological functions is to protect neutrophils from granule protease-mediated cell death. Mice lacking Serpinb1a (Sb1a-/-), its mouse ortholog, have reduced bone marrow neutrophil numbers due to cell death mediated by cathepsin G and the mice show increased susceptibility to lung infections. Here, we show that conditional deletion of Serpinb1a using the Lyz2-cre and Cebpa-cre knock-in mice effectively leads to recombination-mediated deletion in neutrophils but protein-null neutrophils were only obtained using the latter recombinase-expressing strain. Absence of Serpinb1a protein in neutrophils caused neutropenia and increased granule permeabilization-induced cell death. We then generated transgenic mice expressing human Serpinb1 in neutrophils under the human MRP8 (S100A8) promoter. Serpinb1a expression levels in founder lines correlated positively with increased neutrophil survival when crossed with Sb1a-/- mice, which had their defective neutrophil phenotype rescued in the higher expressing transgenic line. Using new conditional and transgenic mouse models, our study demonstrates the presence of a relatively low Serpinb1a protein threshold in neutrophils that is required for sustained survival. These models will also be helpful in delineating recently described functions of Serpinb1 in metabolism and cancer.

Oxygen levels determine the ability of glucocorticoids to influence neutrophil survival in inflammatory environments

GCs are highly effective in treating a wide range of inflammatory diseases but are limited in their ability to control neutrophilic lung inflammation in conditions such as COPD. Neutrophil apoptosis, a central feature of inflammation resolution, is delayed in response to microenvironmental cues, such as hypoxia and inflammatory cytokines, present at inflamed sites. GCs delay neutrophil apoptosis in vitro, and this may therefore limit the ability of GCs to control neutrophilic inflammation. This study assesses the effect GCs have on hypoxia- and inflammatory cytokine-induced neutrophil survival. Human neutrophils were treated with GCs in the presence or absence of GM-CSF or inflammatory macrophage-CM at a range of oxygen concentrations (21-1% oxygen). Neutrophil apoptosis and survival were assessed by flow cytometry and morphological analysis and neutrophil function, by stimulus-induced shape change and respiratory burst. Dexamethasone promoted neutrophil survival at 21%, 10%, and 5% oxygen but not at 1% oxygen. Interestingly, GM-CSF and inflammatory CM increased neutrophil survival significantly, even at 1% oxygen, with cells remaining functionally active at 96 h. Dexamethasone was able to reduce the prosurvival effect of GM-CSF and inflammatory CM in a hypoxic environment. In conclusion, we found that GCs do not augment neutrophil survival in the presence of severe hypoxia or proinflammatory mediators. This suggests that GCs would not promote neutrophil survival at sites of inflammation under these conditions.

Acidosis downregulates platelet haemostatic functions and promotes neutrophil proinflammatory responses mediated by platelets

Acidosis is one of the hallmarks of tissue injury such as trauma, infection, inflammation, and tumour growth. Although platelets participate in the pathophysiology of all these processes, the impact of acidosis on platelet biology has not been studied outside of the quality control of laboratory aggregation assays or platelet transfusion optimization. Herein, we evaluate the effect of physiologically relevant changes in extracellular acidosis on the biological function of platelets, placing particular emphasis on haemostatic and secretory functions. Platelet haemostatic responses such as adhesion, spreading, activation of αIIbβ3 integrin, ATP release, aggregation, thromboxane B2 generation, clot retraction and procoagulant activity including phosphatidilserine exposure and microparticle formation, showed a statistically significant inhibition of thrombin-induced changes at pH of 7.0 and 6.5 compared to the physiological pH (7.4). The release of alpha granule content was differentially regulated by acidosis. At low pH, thrombin or collagen-induced secretion of vascular endothelial growth factor and endostatin were dramatically reduced. The release of von Willebrand factor and stromal derived factor-1α followed a similar, albeit less dramatic pattern. In contrast, the induction of CD40L was not changed by low pH, and P-selectin exposure was significantly increased. While the generation of mixed platelet-leukocyte aggregates and the increased chemotaxis of neutrophils mediated by platelets were further augmented under acidic conditions in a P-selectin dependent manner, the increased neutrophil survival was independent of P-selectin expression. In conclusion, our results indicate that extracellular acidosis downregulates most of the haemostatic platelet functions, and promotes those involved in amplifying the neutrophil-mediated inflammatory response.

HIV-1–Infected Peripheral Blood Mononuclear Cells Enhance Neutrophil Survival and HLA-DR Expression Via Increased Production of GM-CSF: Implications for HIV-1 Infection

HIV-1 bound to intact neutrophils efficiently infects activated peripheral blood mononuclear cells (PBMC). Here, we evaluated the effect of the local milieu created by activated PBMC before and after HIV-1 infection on neutrophil survival and HLA-DR expression, with emphasis placed on a role for GM-CSF. PBMC of healthy adult individuals were activated by phytohemagglutinin (PHA) or anti-CD3/anti-CD28 and were subsequently cultured without (HIV-1⁻) or with HIV-1 (HIV-1+). The effects of the culture supernatants or recombinant GM-CSF on survival and HLA-DR expression by neutrophils of healthy adult individuals and of HIV-1-infected individuals were evaluated using flow cytometry. Conditioned medium from PHA-activated PBMC (HIV-1⁻ and HIV-1+) increased neutrophil survival and induced HLA-DR expression by neutrophils of healthy individuals in a GM-CSF dependent fashion. HIV-1 infection variably, but consistently, increased GM-CSF production by PHA-activated PBMC but not GM-CSF production by anti-CD3/anti-CD28-activated PBMC. The latter was correlated with a loss of CD3+GMCSF+ cells after infection. Neutrophils of elite controllers exhibited a diminished HLADR response to GM-CSF in culture, whereas neutrophils of HIV-1+ subjects having a low viral load on anti-retroviral therapy or subjects with a high viral load exhibited a range of HLA-DR responses. GM-CSF production within the mucosa or draining lymph nodes may promote HIV-1 infection by facilitating sustained contact between viable neutrophils with bound HIV-1 and CD4 lymphocytes. The minimal effect of GM-CSF on HLA-DR expression by neutrophils of elite controllers provides indirect support for this conclusion.

NADPH oxidase activity: In the crossroad of neutrophil life and death

Neutrophils are terminally differentiated leukocytes, specialized in detecting and annihilating possible pathogens. For this function, neutrophils contain a number of cytotoxic systems, which can both kill the intruder or promote extensive tissue injury. The most stereotyped neutrophil cytotoxic mechanism is the extracellular and intra-phagosomal production of high amounts of superoxide (O2-) and other reactive oxygen species (ROS) via the activation of the complex NADPH oxidase (NADPHox). It has been proposed that the short neutrophil lifespan would be a mechanism of counter-regulating the indiscriminate release of its cytotoxic content, as well as aborting the excessive production of ROS. Studies performed in the last decades point out the role of NADPHox activity as one of the major systems involved in the up-regulation of neutrophil apoptosis. However, a growing number of evidence suggests that NADPHox-derived ROS are involved in the activation of signaling pathways that may lead to increased neutrophil survival. In this review, we evaluate the implication of NADPHox activity in the control of neutrophil's life and death, highlighting the signaling pathways modulated by NADPHox-derived ROS.

HIV‐1‐Infected Peripheral Blood Mononuclear Cells (PBMC) Promote Neutrophil Survival and Induce GM‐CSF‐Dependent Expression of HLA‐DR and IFN‐γ‐Dependent Expression of CD64

We examined the ability of HIV‐1 infected PBMC to influence neutrophil function. Incubating neutrophils with supernatant of PHA‐activated PBMC that had been cultured with HIV‐1 for six days (HIV‐1+ culture supernatant) significantly increased neutrophil survival at 48 hr. Depletion of the HIV‐1 virions by centrifugation did not alter the pro‐survival activity of the HIV‐1+ culture supernatant. The HIV‐1+ culture supernatant also induced expression of HLA‐DR and CD64 by neutrophils. The induction of HLA‐DR paralleled induction of HLA‐DR expression by GM‐CSF. The HIV‐1+ culture supernatant, however, did not induce neutrophil expression of CD80, CD86, or CD40. The HIV‐1+ culture supernatant stimulated concentration‐dependent increases in neutrophil HLA‐DR and CD64 at supernatant concentrations containing 5 to 50 pg GM‐CSF/ml and 3 to 30 pg IFN‐γ/ml, respectively. Neutralizing anti‐GM‐CSF antibody inhibited HLA‐DR expression and neutralizing anti‐IFN‐γ antibody inhibited CD64 expression induced by the HIV‐1+ culture supernatant. These results indicate that HIV‐1‐infected PBMC can prolong neutrophil survival and potentially influence neutrophil immune activity through the production of GM‐CSF and IFN‐γ. Supported by NIH Grant R21 DE070170

Inhibition of caspase‐dependent spontaneous apoptosis via a cAMP‐protein kinase A dependent pathway in neutrophils from sickle cell disease patients

Sickle cell disease (SCD) is a chronic inflammatory condition characterized by high leucocyte counts, altered cytokine levels and endothelial cell injury. As the removal of inflammatory cells by apoptosis is fundamental for the resolution of inflammation, we aimed to determine whether the leucocyte apoptotic process is altered in SCD. Neutrophils from SCD individuals showed an inhibition of spontaneous apoptosis when cultured in vitro, in the presence of autologous serum for 20 h. Intracellular cyclic adenosine monophosphate (cAMP) levels were approximately twofold increased in SCD neutrophils; possible cAMP-upregulating factors present in SCD serum include interleukin-8, granulocyte-macrophage colony-stimulating factor and prostaglandin. Accordingly, co-incubation of SCD neutrophils with KT5720, a cAMP-dependent protein kinase (PKA) inhibitor, abrogated increased SCD neutrophil survival. Caspase-3 activity was also significantly diminished in SCD neutrophils cultured for 16 h and this activity was restored when cells were co-incubated with KT5720. BIRC2 (encoding cellular inhibitor of apoptosis protein 1, cIAP(1)), MCL1 and BAX expression were unaltered in SCD neutrophils; however, BIRC3 (encoding the caspase inhibitor, cIAP(2)), was expressed at significantly higher levels. Thus, we report an inhibition of spontaneous SCD neutrophil apoptosis that appears to be mediated by upregulated cAMP-PKA signalling and decreased caspase activity. Increased neutrophil survival may have significant consequences in SCD; contributing to leucocytosis, tissue damage and exacerbation of the chronic inflammatory state.

In vivo regulation of neutrophil apoptosis by C5a during sepsis

Delayed neutrophil apoptosis is characteristic of sepsis and may accentuate organ injury. It has been shown that PI-3K and MAPK pathways provide survival signaling in neutrophils. In this study, we demonstrate that neutrophils isolated from septic rats are resistant to apoptosis in comparison with the cells from normal animals. In contrast to normal serum, septic sera induced strong phosphorylation of AKT and p44/42 in neutrophils obtained from normal rats, resulting in marked resistance of these cells to apoptosis. Protection from apoptosis by septic sera was abrogated completely by inhibition of PI-3K and partially diminished by MEK inhibition. Increased neutrophil survival in septic rats was associated with increased levels of Bcl-xL in neutrophils and decreased levels of Bim expression. In vivo blockade of C5a in cecal ligation and puncture rats by anti-C5a antibody markedly restored the susceptibility of neutrophils to undergo apoptosis. C5a activated AKT and p44/42 and also enhanced X-linked inhibitor of apoptosis expression in neutrophils. LPS and C5a were able to induce Bcl-xL expression. Thus, neutrophil survival signals derived from effects of septic sera could be linked to activation of ERK1/2 and PI-3K, increased antiapoptotic protein expression, and ultimately, delayed neutrophil apoptosis.

Airway neutrophilia in COPD is not associated with increased neutrophil survival

Neutrophilic airway inflammation is a prominent feature of chronic obstructive pulmonary disease (COPD) and correlates with disease severity. The mechanisms that determine the extent of neutrophilia could involve increased influx or prolonged survival of neutrophils. The aim of the study was to assess whether neutrophil pro-survival mechanisms are increased in the airways of subjects with COPD owing to the presence of anti-apoptotic factors in the bronchial lining fluid. Induced sputum samples were collected from 20 subjects with stable COPD, 14 healthy smokers and 14 healthy controls. Quantification of apoptotic neutrophils was based on typical morphological cell changes. Anti-apoptotic, pro-survival activity in the sputum was studied by culturing peripheral blood neutrophils with the fluid phase of induced sputum. Apoptosis was assessed both by morphology and flow cytometry using Annexin V/7-aminoactinomycin D staining. COPD patients and healthy smokers had significantly higher percentages of sputum neutrophils than healthy controls. However, there were no significant differences between the three subject groups in either the proportion of apoptotic neutrophils in sputum or the in vitro anti-apoptotic activity detected in the sputum fluid phase. In conclusion, prolonged survival of neutrophils in sputum is not a feature of chronic obstructive pulmonary disease and cannot explain the increased numbers of airway neutrophils in this disease.

Regulation of C5a on Neutrophil Apoptosis During Sepsis

Delayed neutrophil apoptosis is characteristic of sepsis and may accentuate organ injury. It has been shown that phosphatidylinositol 3-kinase (PI3-K) and MAPK pathways provide survival signaling in neutrophils. In this study, we demonstrate that neutrophils isolated from septic rats are resistant to apoptosis in comparison to the cells from normal animals. In contrast to normal serum, septic sera induced strong phosphorylation of AKT and p44/42 in neutrophils obtained from normal rats, resulting in marked resistance of these cells to apoptosis. Protection from apoptosis by septic sera was completely abrogated by inhibition of PI3-K and partially diminished by MAP kinase (MEK) inhibition. Increased neutrophil survival in septic rats was associated with increased levels of Bcl-xL in neutrophils and decreased levels of Bim expression. In vivo blockade of C5a in CLP rats by anti-C5a antibody markedly restored the susceptibility of neutrophils to undergo apoptosis. C5a activated AKT and p44/42, and also enhanced XIAP expression in neutrophils. This enhancement was further augmented by LPS co-stimulation. Both LPS and C5a were able to induce Bcl-xL expression. Thus, neutrophil survival signals derived from effects of septic sera could be linked to activation of ERK1/2 and PI3-K, increased anti-apoptotic protein expression, and ultimately delayed neutrophil apoptosis. This work is supported by NIH grants GM-61656, GM-29507 and HL-31963.

Anti-Apoptotic Effect, Mediated by Elevated Intracellular Cyclic AMP Levels, in Neutrophils of Sickle Cell Disease Patients.

Sickle cell disease (SCD) is now widely regarded as a chronic inflammatory condition, characterized by high base-line leukocyte counts, alterations in inflammatory cytokine levels, vascular endothelial injury and increased red and white cell adhesiveness. We hypothesized that this inflammatory state may be exacerbated by alterations in the apoptotic process in inflammatory cells, such as neutrophils. Neutrophils were isolated from the peripheral blood of healthy controls and individuals with SCD in steady state by separation over a ficoll-paque gradient. Following washing and lysis of contaminating red cells, neutrophils were cultured in DMEM supplemented with 10% (v/v) autologous serum and antibiotics for 20 h (4 x 106 cells/ml, 37oC, 5% CO2). Apoptosis was assessed by flow cytometry using a fluorescein isocyanate-labeled recombinant annexin V antibody and propidium iodide staining. Morphological analysis confirmed the apoptotic state of cells. After 20 h in culture, the percentage of non-apoptotic neutrophils from SCD patients (SCD neutrophils; 13.34 ± 1.03 %, n=8) was significantly higher than the percentage of non-apoptotic normal neutrophils (7.68 ± 1.59, n=7; P<0.01). Apoptosis is mediated by a number of signaling pathways, with the cAMP-dependent pathway being known to have an important anti-apoptotic role in neutrophils. Accordingly, measurement of cAMP in isolated normal and SCD neutrophils by ELISA demonstrated levels of cAMP to be significantly increased in the neutrophils of SCD individuals (4.55 ± 0.38 pMol/1x 106 cells compared to 2.18 ± 0.39 pMol/1x 106 cells in normal individuals, n>14, P<0.001). Co-incubation of SCD neutrophils during 20h-culture with a cAMP-dependent protein kinase (PKA) inhibitor, KT5720 (3μM), significantly decreased the percentage of non-apoptotic cells (12.88 ± 1.49 %, decreased to 6.18 ± 0.51 %, n=6; p=0.01) to levels similar to those seen in normal neutrophil cultures, indicating that the anti-apoptotic effect seen in SCD neutrophils is probably mediated by a cAMP-PKA dependent mechanism. Interestingly, when SCD neutrophils were cultured in medium containing fetal calf serum instead of autologous serum, the number of non-apoptotic cells at 20 h of culture was not different to the number of normal non-apoptotic neutrophils (8.91 ± 2.55 % compared to 9.08 ± 2.11 %, respectively, n= 7, P>0.05). This finding may indicate that this anti-apoptotic effect may be maintained by survival factors contained in serum; indeed incubation of normal neutrophils with SCD patient serum (10 % v/v, 30 min, 37oC), but not control serum, significantly augmented intracellular cAMP levels by 117.9 ± 10.5 % (n=8, p<0.01). In conclusion, we have observed an increased survival of neutrophils from SCD individuals under culture conditions, probably mediated by an up-regulated cAMP-PKA pathway and possibly dependent on cAMP-elevating survival factors contained in the serum. Since neutrophil apoptosis is imperative for the resolution of inflammation, increased neutrophil survival may be an important contributing factor to the chronic inflammatory state that characterizes SCD.

Protein Phosphatase 2A Regulates Apoptosis in Neutrophils by Dephosphorylating Both p38 MAPK and Its Substrate Caspase 3

The induction of apoptosis in neutrophils is an essential event in the resolution of an inflammatory process. We found recently that the reduction of the activity of the neutrophil survival factor p38 MAPK and dephosphorylation and thus activation of caspases must occur to initiate such cell death in these leukocytes. Here, we report a previously undetected early and transient activation of protein phosphatase 2A (PP2A) in neutrophils undergoing apoptosis. The pharmacological inhibition of this phosphatase during Fas-induced apoptosis augmented the levels of phosphorylation of both p38 MAPK and caspase 3, resulting in a decreased activity of caspase 3 and an increased neutrophil survival. The complementary finding of a time-dependent association among PP2A, p38 MAPK, and caspase 3 in intact neutrophils indicated that there is a direct regulatory link among these signaling enzymes during Fas-provoked apoptosis. Moreover, immunoprecipitated active p38 MAPK and recombinant phosphorylated caspase 3 were dephosphorylated by exposure to purified PP2A in vitro. Consequently, the early and temporary activation of PP2A in neutrophils impaired not only the p38 MAPK-mediated inhibition of caspase 3 but also restored the activity to caspase 3 that had already been phosphorylated and thereby inactivated. These findings indicate that PP2A plays a pivotal dual role in the induction of neutrophil apoptosis and therefore also in the resolution of inflammation.

SHP-1: a regulator of neutrophil apoptosis

The Src homology domain 2 (SH2)-containing tyrosine phosphatase-1 (SHP-1) has been implicated in the regulation of differentiation, proliferation, and activation of hematopoietic cells. In this review, we discuss the potential role of SHP-1 in modulating apoptosis pathways in neutrophils. Low enzymatic SHP-1 was associated with increased neutrophil survival in vitro and SHP-1-deficient mice were reported to develop severe neutrophilic inflammatory responses. In contrast, high expression of this phosphatase was observed in neutropenic patients. Moreover, when neutrophils were exposed to Fas ligand, TNF-α, or TRAIL, the anti-apoptotic effects of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), or IFN-γ were blocked, most likely by SHP-1-mediated inactivation of anti-apoptotic signaling molecules. In summary, the current available data point to an important role of SHP-1 in the regulation of neutrophil apoptosis.

Modulation of Leukotriene B4 Receptor-1 Expression by Dexamethasone: Potential Mechanism for Enhanced Neutrophil Survival

Glucocorticoids can down-regulate many inflammatory and immune responses and constitute a powerful therapeutic tool in a number of diseases. However, they have a somewhat paradoxical effect on neutrophils, in that they prolong their survival. Because leukotriene B(4) (LTB(4)) can also extend neutrophil survival, we proposed that glucocorticoids could prevent neutrophil apoptosis by up-regulating their expression of the high-affinity LTB(4) receptor (BLT1). Here we show that, indeed, dexamethasone (DEX) up-regulates the steady-state levels of BLT1 mRNA in human neutrophils. The effect was time and concentration dependent, being maximal at 4 h and at 10-100 nM DEX. The effect was also dependent on transcriptional activity, whereas BLT1 mRNA stability was not affected. DEX-induced up-regulation of BLT1 expression was prevented by pretreatment with the LTB(4) antagonist LY255283. Moreover, LTB(4) itself up-regulated the expression of BLT1 mRNA. BLT1 protein expression on neutrophils exposed to DEX for 24 h was also up-regulated 2- to 3-fold, and DEX-treated as well as LTB(4)-treated cells showed enhanced responsiveness to LTB(4) in terms of intracellular Ca(2+) mobilization and chemotaxis. Whereas DEX and LTB(4) alone decreased neutrophil apoptosis by approximately 50%, neutrophils treated with both LTB(4) and DEX showed >90% survival at 24 h. Moreover, BLT1 antagonists prevented the increased neutrophil survival induced by DEX as well as by LTB(4). Taken together, our results suggest that DEX-induced up-regulation of BLT1 expression in neutrophils may be one mechanism through which glucocorticoids can prolong neutrophil survival, namely by enhancing cell responses to the antiapoptotic effect of LTB(4).