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Here we elaborate on the origin of low(er)‐density neutrophils (LDNs) to better understand the variation found in literature. Supplemented with original data, we test the hypothesis that buoyant density of neutrophils is characterized by a spectrum that as a whole shifts to a lower density after activation. Both the 20% highest density (HDNs) and 20% lowest density (LDNs) neutrophils from healthy donors were isolated by Percoll of different densities. Using this method we found that LDNs were significantly better in T‐cell suppression and bacterial containment than their 20% highest density counterparts. We found no statistically relevant differences in neutrophil survival or bacterial phagocytosis. Stimulation of healthy donor neutrophils with N‐formyl‐methionyl‐leucyl‐phenylalanine induced LDNs co‐segregating with peripheral blood mononuclear cells after Ficoll separation. These in vitro induced LDNs showed increased activation markers compared to HDNs and were comparable to the activation markers found on the LDN fraction seen in patients with chronic inflammatory conditions such as present in cancer patients. This all fits with the hypothesis that the density of neutrophils is distributed in a spectrum partially coupled to maturation. Additionally a shift in this spectrum can be induced by in vitro stimulation or by activation in disease., Nowadays the term low(er) density neutrophils (LDN) is used to describe a specific subset of neutrophils with a certain buoyant density, but these LDNs are characterized by a wide variety in both functional and morphologic properties, especially in disease.

Contains fulltext : 229398.pdf (Publisher’s version ) (Open Access) Acute infection is characterized by eosinopenia. However, the underlying mechanism(s) are poorly understood and it is unclear whether decreased mobilization/production of eosinophils in the bone marrow (BM) and/or increased homing to the tissues play a role. The objective of this study was to investigate the differentiation and activation status of eosinophils in the human BM and blood upon experimental human endotoxemia, a standardized, controlled, and reproducible model of acute systemic inflammation. A BM aspirate and venous blood was obtained from seven healthy volunteers before, 4 h after, and 1 week after intravenous challenge with 2 ng/kg endotoxin. Early progenitors (CD34+/IL-5Rα+), eosinophil promyelocytes, myelocytes, metamyelocytes, and mature eosinophils were identified and quantified in the bone marrow and blood samples using flowcytometry based on specific eosinophil markers (CD193 and IL-5Rα). Activation status was assessed using antibodies against known markers on eosinophils: Alpha-4 (CD49d), CCR3 (CD193), CR1 (CD35), CEACAM-8 (CD66b), CBRM 1/5 (activation epitope of MAC-1), and by plasma cytokine analysis. Four hours after endotoxin administration, numbers of mature eosinophils in the blood and in the BM markedly declined compared with baseline, whereas numbers of all eosinophil progenitors did not change. The remaining eosinophils did not show signs of activation or degranulation despite significantly increased circulating levels of eotaxin-1. Furthermore, the expression of CD49d and CD193 on eosinophils was lower compared to baseline, but normalized after 7 days. Together these data imply that circulatory eosinopenia after an innate immune challenge is mediated by CD49d-mediated homing of eosinophils to the tissues.

Neutrophils are the most abundant white blood cells and are indispensable for host defense. Recently, they have also been implicated in immune regulation and suppression. The latter functions seem hard to reconcile with the widely held view that neutrophils are very short-lived, with a circulatory half-life of

Systemic inflammation results in release of refractory neutrophils which can cause susceptibility to infections and activates differentiated neutrophils, which can mediate tissue damage. Neutrophils play an important role in host defense. However, deregulation of neutrophils contributes to tissue damage in severe systemic inflammation. In contrast to complications mediated by an overactive neutrophil compartment, severe systemic inflammation is a risk factor for development of immune suppression and as a result, infectious complications. The role of neutrophils in this clinical paradox is poorly understood, and in this study, we tested whether this paradox could be explained by distinct neutrophil subsets and their functionality. We studied the circulating neutrophil compartment immediately after induction of systemic inflammation by administering 2 ng/kg Escherichia coli LPS i.v. to healthy volunteers. Neutrophils were phenotyped by expression of membrane receptors visualized by flow cytometry, capacity to interact with fluorescently labeled microbes, and activation of the NADPH-oxidase by oxidation of Amplex Red and dihydrorhodamine. After induction of systemic inflammation, expression of membrane receptors on neutrophils, such as CXCR1 and -2 (IL-8Rs), C5aR, FcγRII, and TLR4, was decreased. Neutrophils were also refractory to fMLF-induced up-regulation of membrane receptors, and suppression of antimicrobial function was shown by decreased interaction with Staphylococcus epidermis. Simultaneously, activation of circulating neutrophils was demonstrated by a threefold increase in release of ROS. The paradoxical phenotype can be explained by the selective priming of the respiratory burst. In contrast, newly released, CD16dim banded neutrophils display decreased antimicrobial function. We conclude that systemic inflammation leads to a functionally heterogeneous neutrophil compartment, in which newly released refractory neutrophils can cause susceptibility to infections, and activated, differentiated neutrophils can mediate tissue damage.

Interleukin (IL)-5 is a hematopoietic cytokine able to regulate differentiation, survival, and effector functions of eosinophils. It binds specifically to its receptor, which is composed of a cytokine-specific α-chain and a β-chain shared with the receptors for IL-3 and the granulocyte macrophage-colony stimulating factor. The molecular mechanisms by which IL-5 modulates eosinophil survival remain unclear. In this study, we demonstrate that IL-5 withdrawal induces eosinophil apoptosis through a mitochondria-dependent pathway, independently of Fas receptor activation. The lipid kinase phosphatidylinositol-3 kinase plays a crucial role in the maintenance of eosinophil survival, as inhibition of its activity results in apoptosis. IL-5 induces phosphorylation and thus, inhibition of the Forkhead transcription factor FOXO3a and glycogen synthase kinase 3 (GSK-3). We analyzed expression of FOXO3a-dependent transcriptional targets: Fas ligand or Bim (a proapoptotic Bcl-2 family member), but neither was detected in apoptotic eosinophils. We further show that GSK-3 is activated after IL-5 withdrawal, and inhibition of its activity rescues eosinophils from apoptosis. β-catenin, a direct GSK-3 substrate, is present in the nucleus of IL-5-stimulated eosinophils, but it is translocated to the plasma membrane in the absence of cytokine in a GSK-3-dependent manner. This is the first report describing a potential role for GSK-3 and β-catenin in regulating eosinophil survival and suggests a novel mechanism by which IL-5 inhibits the constitutive apoptotic program in these cells.

SummaryRespiratory syncytial virus (RSV) is a common cause of lower respiratory tract disease (LRTD) in infants. Eosinophils have been suggested to play a role in the disease pathogenesis of LRTD. Inflammation can induce functional and morphological alterations of peripheral blood granulocytes. In patients with RSV LRTD, we aimed to investigate the eosinophil activation status by analysing surface markers. In vitro stimulation of eosinophils with cytokines leads to up-regulation of CD11b and priming markers recognized by the recently developed priming markers A17 and A27, whereas interleukin (IL)-5Rα is being down-regulated. In 51 patients and 10 controls we examined the expression of these surface markers on eosinophils in moderate to severe RSV-induced LRTD patients at the time of admission and 6 weeks later during the convalescence phase. RSV-patients were characterized by a higher eosinophil CD11b expression compared to controls. Although basal A17 and A27 expression was not increased, we observed a significantly higher expression of these priming epitopes on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated cells of RSV patients compared with cells of controls, indicative of prior in vivo priming. Furthermore, IL-5Rα expression was down-regulated on peripheral blood eosinophils of these patients. Follow-up blood samples showed normalization of all markers but CD11b, which was persistently increased. Utilizing cellular markers, we observed that peripheral blood eosinophils from infants with RSV LRTD are in a more activated state compared to eosinophils of controls, which normalizes only partially during convalescence.

Tyrosine phosphorylation is thought to be critical in the regulation of neutrophil functioning, and members of the Src family of tyrosine kinases have recently been shown to be regulated in activated granulocytes. We have used a specific pharmacological inhibitor of Src kinases, pyrazolpyrimidine 1 (PP1), to evaluate the role of Src kinases in cytokine/chemoattractant-induced regulation of neutrophil function. PP1 inhibits PKB phosphorylation but not STAT5 phosphorylation or the activation of MAP kinases by fMLP or GM-CSF. Pretreatment of neutrophils with PP1 and with the PI3K inhibitor LY294002 resulted in a strong inhibition of fMLP-induced superoxide production and cytokine-mediated survival but not fMLP-induced migration. It is interesting that the kinetics of inhibition of actin polymerization and the respiratory burst are very similar. Although initiation of both processes was not affected, sustained activation was inhibited by PP1. Taken together, our results demonstrate a critical role for Src kinases in regulating neutrophil cytotoxic-effector functioning through PI3K-PKB.

Inflammation in allergic asthma is characterized by an influx of eosinophils and the presence of eosinophil products in the bronchial tissue. Orchestration of this inflammatory response is in part mediated by cytokines and chemoattractants, but final activation can require additional stimuli. IgA, the most abundant immunoglobulin at mucosal surfaces, is potentially a potent trigger for eosinophil activation. Previously, we have shown that binding IgA-coated targets is dependent on in vitro stimulation of cells with cytokines. Here, we demonstrate that eosinophils isolated from the blood of allergic asthmatic patients bind IgA beads independently of prior in vitro stimulation. Furthermore, we found that the proinflammatory cytokine, TNF-α, is a potent enhancer of IgA binding to eosinophils from allergic asthmatics, and it does not activate FcαR on eosinophils isolated from normal donors. The difference in IgA binding by FcαRs on normal and patient eosinophils might be explained by the activation of different signal transduction pathways. Studying intracellular signaling, we found an enhanced basal activity of phosphatidylinositol 3-kinase (PI3K) in eosinophils derived from allergic asthmatics. Moreover, inhibition of PI3K in these cells blocked the background and the TNF-α-induced IgA binding completely. In summary, these data demonstrate that the responsiveness of human eosinophils to TNF-α might be an important contribution for fine-tuning the allergic inflammatory reaction. Furthermore, the preactivation of PI3K results in a broader sensitivity to subsequent challenge with inflammatory cytokines.

The eosinophil-derived neurotoxin (EDN), a member of the mammalian ribonuclease family, is found in the large specific granules of human eosinophilic leukocytes. We have investigated the role of the C/EBP transcription factor family in the regulation of EDN promoter activity. Here we show that the C/EBP family is involved in intrinsic regulation of EDN promoter activity. We have identified a C/EBP binding site located at – 124 in the proximal promoter of the EDN gene. Mutation of this C/EBP site results in a decrease of promoter activity in HL-60-eos cells as well as in eosinophils differentiated in vitro from CD34+ cells. Different C/EBP proteins are able to bind to the C/EBP site as shown by gel shift assay. Our results indicate the importance of the C/EBP family in the regulation of the EDN gene in eosinophils. J. Leukoc. Biol. 66: 683–688; 1999.

We report that a transient incubation of human eosinophils with the protein kinase C (PKG) inhibitor CGP39-360 (staurosporine) or the more PKC-specific inhibitors CGP41-251 and CGP44-800 prior to activation of the respiratory burst with opsonized particles results in priming of this response. This priming effect was concentration dependent and occurred in the range in which the phorbol myristate acetate-induced respiratory burst was inhibited. CGP39-360 priming was minimally affected in Ca2+-depleted cells, indicating that an increase in [Ca2+]i is not important. Also, the binding of serum-treated zymosan (STZ) particles was strongly enhanced by the inhibitors. On the other hand, the release of plateletactivating factor (PAF) induced by opsonized particles was enhanced only by CGP39-360 and not by CGP41-251 and CGP44-800. Therefore, priming of the respiratory burst is not due to an aspecific enhancing effect of the inhibitors. These data indicate that different signal transduction routes are involved in priming of the STZ-induced respiratory burst and PAF release in human eosinophils.

We report that pretreatment of human eosinophils with GM-CSF, IL-3, or IL-5 enhanced the respiratory burst induced by opsonized particles. In order to gain more insight into the intracellular mechanism(s) involved in cytokine priming, the role of [Ca2+], and tyrosine kinases was studied. Optimal priming concentrations of GM-CSF, IL-3, and IL-5 did not induce a rise in [Ca2+]i, and Ca2+-depleted eosinophils ([Ca2+]i < 20 nM) were still primed after preincubation with these cytokines. GM-CSF, IL-3, and IL-5 induced phosphorylation of two proteins (102 and 122 kd) on tyrosine residues, as deduced from Western blot analysis with an antiphospho- tyrosine monoclonal antibody (4G10). This cytokine- stimulated tyrosine phosphorylation was not inhibited under Ca2+-depleted conditions. In conclusion, this study demonstrates that GM-CSF, IL-3, and IL-5 priming of the opsonized particle-induced respiratory burst in human eosinophils is completely Ca2+ independent. Moreover the tyrosine phosphorylation of a 102-kd and a 122-kd protein is Ca2+ independent, suggesting that this event might be involved in cytokine priming. J. Leukoc. Biol. 53: 347–353; 1993.

Background: Successful translation of pre-clinical treatment strategies into clinical therapies depends on reproducible, conclusive models. To date, two distinctive procedures for the induction of myocardial ischemia-reperfusion to test novel therapies for myocardial infarction (MI) are commonly used. The first method applies coronary artery ligation via an open chest procedure, requiring invasive surgery. Alternatively, the coronary artery is occluded by balloon inflation via a transluminal, minimally invasive procedure. In the current study we hypothesized that invasive surgery as for open chest procedures reduces myocardial infarct size and preserves cardiac function post-MI. Methods: Pigs (n=14) were subjected to 75-minute balloon occlusion followed by 3 days of reperfusion. In 7 pigs, the balloon occlusion was preceded by medial sternotomy (MS) that is normally performed for open chest coronary artery ligation. In the remaining 7 pigs the same procedure was performed without sternotomy (NS). Cardiac damage was determined by histological infarct size, 3D-echocardiography and troponin I measurements. The area at risk (AAR) was assessed through Evans blue injection. Circulating leukocyte numbers were measured at baseline, ischemia and reperfusion. Results: Heart rate and blood pressure did not differ significantly between the two groups during the procedure. Also the area at risk (AAR) was comparable between groups (19.8±1.6% (NS) vs. 19.7±3.4% (MS), p=ns). We observed a significantly smaller infarct size as a percentage of the AAR in pigs subjected to sternotomy (67.6±10.2% (NS) vs. 49.3±14.8% (MS), p=0.024), culminating in a significantly higher left ventricular ejection fraction (39.5±3.8% (NS) vs. 47.9±7.1% (MS), p=0.022). Furthermore, troponin I levels 1 hour after reperfusion were significantly different between the two groups (127.6±100.4 ng/mL (NS) vs. 24.5±13.1 (MS) ng/mL, p=0.030). Leukocyte count after 30 minutes of reperfusion was higher in the MS group than the NS group (14.5±3.1*106 cell/mL (NS) vs. 19.0±3.0*106 cell/mL (MS); p=0.023). Conclusion: Invasive surgery required for an open chest temporary coronary artery ligation reduces infarct size compared to minimally invasive surgery in a porcine model of myocardial infarction, despite increased leukocyte numbers. This confounding effect should be taken into account when assessing therapeutic efficacy for novel treatment strategies to treat myocardial infarction.

Background: Revascularization after myocardial infarction (MI) induces a strong inflammatory response resulting in an increase of circulating neutrophils. Neutrophil quantities have found to be a good predictor for future adverse events. In this study, we hypothesized that the extent of morphological changes in circulating neutrophils also reflects the myocardial damage after MI and therefore relates to outcome. Methods: One hundred seven STEMI patients treated between 2009 and 2012 with at least one white blood cell count determined by an automated hematology analyzer within 24 hours after PCI were selected. This analyzer differentiates between leukocyte subsets based on morphological characteristics derived from light scatter patterns. Neutrophil morphology was compared with simultaneously measured creatine kinase (CK). Pigs (n=9) were subjected to left anterior descending artery (LAD) occlusion for 75 minutes followed by 3 days of reperfusion. Blood was collected at baseline, during ischemia and at multiple time-points during reperfusion followed by whole-blood analysis with the same hematology analyzer as above. Cardiac damage was determined by histological infarct size, 3D-echocardiography and Troponin measurements. Coronary blood sampling was performed to determine differences in neutrophil morphology between simultaneously sampled arterial and venous coronary blood. Results: In STEMI patients, a significant increase in neutrophil axial light loss (ALL) over time was seen (p