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Eosinophils play an important role in the pathogenesis of asthma. Several pro-inflammatory responses of eosinophils are primed in vivo in this disease. The aim of the present study was to investigate whether regular antiasthma treatment could modulate priming-sensitive cytotoxic mechanisms of human eosinophils. In a randomized, two-centre, double-blind parallel group study, the effect of 8 weeks of treatment with salmeterol xinafoate 50 microg b.i.d., beclomethasone dipropionate 400 microg b.i.d. or both on pulmonary function and the activation of priming-sensitive cytotoxic mechanisms of eosinophils, i.e. degranulation of eosinophil cationic protein (ECP) in serum, and activation of isolated eosinophils in the context of induction of the respiratory burst and release of platelet-activating factor (PAF) were tested. These effects were evaluated in 40 allergic asthmatics before and 24 h after allergen inhalation challenge. Whereas baseline forced expiratory volume in one second (FEV1) improved in all treatment groups, only treatment with a combination of salmeterol and beclomethasone significantly inhibited the allergen-induced increase in serum ECP, and (primed/unprimed) PAF-release, suggesting inhibition of eosinophil priming after allergen challenge. In contrast to the combination therapy, monotherapy with beclomethasone had no influence on allergen-induced PAF-release, suggesting an additional anti-inflammatory effect of salmeterol during combination therapy. Monotherapy with beclomethasone inhibited the prechallenge serum-treated zymosan (STZ) (0.1 mg mL(-1))-induced respiratory burst and the allergen-induced increase in serum ECP levels, reflecting pre- and postchallenge anti-inflammatory effects. During monotherapy with salmeterol, an allergen-induced increase in serum ECP concentration and STZ (0.1 mg x mL(-1))-induced respiratory burst was observed, suggesting that treatment with salmeterol alone had no effect on priming-sensitive eosinophil cytotoxic mechanisms. In conclusion, this study shows that standard asthma therapy leads to inhibition of eosinophil priming of cytotoxic mechanisms in vivo.

Human eosinophils are capable of synthesizing almost exclusively the strongly spasmogenic compound LTC4 when stimulated with either the calcium ionophore A 23187 or opsonized zymosan (OZ). Although PAF-acether in concentrations ranging from 10 nM to 1 microM is hardly capable of inducing significant LTC4 synthesis itself, it significantly enhances the OZ-induced LTC4 formation at a concentration of 1 microM. However, at a concentration of 10 microM, PAF-acether itself is capable of inducing LTC4 formation comparable with that induced by OZ. PAF-acether, at a concentration of 10 microM (and not at a concentration of 1 microM) is also capable of inducing a luminol dependent chemiluminescent response by eosinophils. The PAF-acether antagonist BN 52021 at a concentration of 0.1 mM not only partially inhibited the PAF-acether induced LTC4 formation but also the OZ induced LTC4 formation. Since an equal inhibition is found the inhibitory mode of action of BN 52021 is most likely directed towards a common pathway. Taken together, these results suggest that eosinophils may be triggered by high locally reached concentrations of PAF-acether to release inflammatory and bronchoconstrictive mediators. This may be of importance for the pathogenesis of the allergen induced late phase asthmatic reaction.

Measurements of the level of 1,2-diacylglycerol (1,2-DG during activation of the respiratory burst of human neutrophils by formyl-methionyl-leucyl-phenylalanine (fMLP in the presence of platelet-activating factor (PAF or by opsonized particles show that a correlation between accumulation of 1,2-DG and O 2 consumption does not exist. Inhibition of protein kinase C activity with staurosporine before addition of opsonized particles demonstrates that the first phase of the respiratory burst is not inhibited, whereas the second phase, which is accompanied by a rise in the content of 1,2-DG, is strongly inhibited. This study indicates that accumulation of 1,2-DG cannot be the sole signal for the initiation of the respiratory burst in human neutrophils.

Eosinophil granular protein deposits have been demonstrated in lesional atopic dermatitis skin. This suggests active tissue infiltration of eosinophils. To find an explanation for the tissue influx of eosinophils, eosinophil migration was studied in vitro by means of a microchemotaxis assay. Eosinophils from the circulation of patients with atopic dermatitis showed an altered capacity to respond to chemotactic stimuli in vitro compared with eosinophils from healthy donors. Eosinophils from patients with atopic dermatitis had significantly increased migratory responses toward dose ranges of N -formyl-methionyl-leucyl-phenylalanine, neutrophil-activating factor, platelet-activating factor, and platelet factor 4. Eosinophils from normal individuals did not respond to N -formyl-methionyl-leucyl-phenylalanine and neutrophil activating factor and responded only slightly to platelet factor 4. The migratory responses toward tumor necrosis factor-4 and complement factor C5a were identical in both groups. Interleukin-5, an eosinophil-selective cytokine, is a strong modulator of the migratory responses to these chemotaxins in eosinophils from normal donors. A migratory response toward N -formyl-methionyl-leucyl-phenylalanine and neutrophil-activating factor was induced by interleukin-5, whereas the migratory response toward platelet-activating factor and platelet factor 4 was markedly potentiated. In contrast, the response to complement fragment C5a was only slightly influenced. Our findings indicate that the increased migratory responsiveness of eosinophils from patients with atopic dermatitis to various chemotaxins reflects in vivo "priming" of eosinophils, presumably by circulating cytokines such as interleukin-5. This in vivo "priming" is not optimal because it can be further potentiated by renewed contact with interleukin-5.

The NADPH oxidase of human eosinophils, measured in the cell-free system, shows the same characteristics as the enzyme from human neutrophils. All proteins required for activity of the enzyme are expressed in eosinophils at a higher level than in neutrophils. Eosinophils isolated from patients with chronic granulomatous disease show the same molecular defects as the neutrophils fom these patients.

Phosphatidylinositol 3-kinase (PI3K) and its effector protein kinase B (PKB/c-akt) have been implicated as critical mediators of cytokine-induced survival signals. In this study, we have utilized an IL-5 dependent hematopoietic cell line (TF-1) to investigate the signaling events involved in cytokine-dependent erythroblast survival. We demonstrate that IL-5 rescues TF-1 cells from apoptosis through a PI3K/PKB-dependent signaling pathway. Cytokine-withdrawal leads to activation of the Forkhead transcription factor FOXO3a and subsequent expression of the pro-apoptotic Bcl-2 family member Bim. Bim is itself sufficient to induce apoptosis in these cells. Importantly, activation of an inducible active FOXO3a mutant is alone sufficient for upregulation of Bim expression and induction of apoptosis. These data define a mechanism by which survival factors inhibit the default apoptotic pathway and can regulate TF-1 erythroblast survival.

Addition of arachidonic acid (50 microM) to purified human eosinophils leads to the formation of considerable amounts of LTC4 [11.3 +/- 1.3) x 10(6) molecules/cell, mean +/- SEM, n = 10), 15-HETE [412 +/- 142) x 10(6) molecules/cell, mean +/- SEM, n = 3) and 15-series leukotrienes [35 +/- 15) x 10(6) molecules/cell, mean +/- SEM, n = 3). The ratio of the amounts of LTC4 and 15-lipoxygenase products was found to be strongly dependent on the arachidonic acid concentration, being relatively large at low arachidonic acid concentrations and very small at high arachidonic acid concentrations. Platelet activating factor (1 microM) was able to enhance significantly the production of LTC4 but not that of 15-lipoxygenase products. As arachidonic acid was found to be capable of inducing a fast, transient rise in the cytosolic free Ca2+ concentration, this explains, at least partly, its ability to induce the Ca2+-dependent formation of LTC4.

Erythrocytes from adults and newborn infants (at term and premature) were separated by Percoll density gradient centrifugation into four fractions of increasing density. Glycolytic enzymes, especially the age-dependent ones, hexokinase (EC 2.7.1.1, HK), pyruvate kinase (EC 2.7.1.40, PK), and glucose-6-phosphate dehydrogenase (EC 1.1.1.49, G6PD) were studied during reticulocyte maturation and further red cell senescence. Analysis of the fraction with lowest density showed an almost linear and steep decline of HK, PK, and G6PD activity with a decreasing number of reticulocytes. In the next three fractions of increasing density, the activity decline was far less. These data are therefore illustrative for a biphasic activity decay pattern of HK, PK, and G6PD during both adult and neonatal red cell aging. The strong decline in HK activity could not be ascribed to the disappearance of a particulate (mitochondrial) bound fraction of the enzyme during reticulocyte maturation. All hexokinase activity in human reticulocytes was found to be cytosolic in contrast with rabbit reticulocytes in which 70% of HK activity was particulate.

A simple and improved procedure is described for the isolation of human eosinophils from normal individuals with about 2% eosinophils in their peripheral blood. This method comprises a preincubation of a mixed granulocyte preparation with 10 nM fMLP for 10 min at 37° followed by a one-step density centrifugation on isotonic Percoll. The recovery of eosinophils is 49 ± 4% at 89 ± 4% purity. Because of the relatively high rate of recovery, it is now possible to isolate eosinophils from blood samples as small as 20 ml. Because treatment with fMLP may alter the functional activity of the eosinophils, the following metabolic functions were tested: changes in cytosolic free Ca2+, oxygen consumption, chemiluminescence, chemotaxis, and leukotriene C4 formation. We found that 10 nM fMLP does not activate eosinophils in these assays, whereas 1 μM fMLP does (with the exception of chemotaxis). Furthermore, pretreatment of eosinophils with 10 nM fMLP did not influence the response to other stimuli in these assays. The usefulness of this method was evaluated by comparing it with three other previously described procedures. In our hands, only the method presented here enabled us to isolate eosinophils from normal individuals with about 2% eosinophils in their peripheral blood.

Stimulation of purified human eosinophils with 50 microM arachidonic acid leads to the production of leukotriene C4, 15-hydroxy-eicosatetraenoic acid and 15-series leukotrienes. The ratio of the amounts of leukotriene C4 and 15-lipoxygenase products was found to be strongly dependent on the arachidonic acid concentration, being relatively large at low arachidonic acid concentrations and very small at high arachidonic acid concentrations. In the presence of 1 microM platelet-activating factor a significant elevation of leukotriene C4 formation is observed, whereas the formation of 15-lipoxygenase products remains unaltered. As arachidonic acid was found to be capable of inducing a fast, transient rise in the cytosolic free Ca2+ concentration, this explains at least partly its ability to induce the Ca2+-dependent formation of leukotriene C4.

Addition of opsonized particles to human neutrophils in suspension leads to a biphasic elevation in the cytosolic free Ca2+ concentration ([Ca2+]i). The rise in [Ca2+]i during the second phase (greater than 3 min) is pronounced (about 400 nM), in contrast to the rise during the first phase, which is relatively small (less than 100 nM). The second and large rise in [Ca2+]i is brought about by messenger(s) released from the cell after addition of opsonized particles. This second rise in [Ca2+]i is not observed in the presence of the platelet-activating factor (PAF) antagonist WEB 2086, indicating that PAF can act as an intercellular messenger affecting Ca2+ homeostasis in human neutrophils.

Accumulation of monocyte-derived foam cells in focal areas of the atherosclerotic (A.S.-) lesion is one of the key events in early atherogenesis. Using a flow model for the damaged vessel wall, we examined the ability of ECM-bound platelets to induce monocyte tethering and adhesion. Whereas ECM-proteins alone induced monocyte adhesion only at low shear stresses (

Glucocorticoids are efficient antiinflammatory agents, and their effects include transcriptional repression of several cytokines and adhesion molecules. Whereas glucocorticoids down-regulate the expression of genes relevant during inflammation, nuclear factor (NF)-kappa B/Rel proteins function as important positive regulators of these genes. The expression of intercellular adhesion molecule-1 (ICAM-1), which plays an essential role in recruitment and migration of leukocytes to sites of inflammation, is also down-regulated by glucocorticoids. We found that a functional NF-kappa B site in the ICAM-1 promoter, which can be activated by either 12-O-tetradecanoylphorbol-13-acetate or tumor necrosis factor-alpha (TNF alpha), is also the target for glucocorticoids. In this report we present evidence that the ligand-activated glucocorticoid receptor (GR) is able to repress RelA-mediated activation of the ICAM-1 NF-kappa B site. Conversely, transcriptional activation by GR via a glucocorticoid response element is specifically repressed by RelA, but not by other NF-kappa B/Rel family members. Mutational analysis of GR demonstrates that the DNA binding domain and the ligand binding domain are required for the functional repression of NF-kappa B activation. Despite the importance of the DNA binding domain, we found that the transcriptional repression of NF-kappa B, mediated by GR, is not caused by binding of GR to the ICAM-1 NF-kappa B element, but by a physical interaction between the GR and RelA protein. The repressive effect of GR on NF-kappa B-mediated activation was not shared by other steroid/thyroid receptors. Only the progesterone receptor, which belongs to the same subfamily as GR and which possesses high homology with GR, was able to repress NF-kappa B-mediated transcription. These studies highlight a possible molecular mechanism that can explain the antiinflammatory effects of glucocorticoid treatment during inflammation.

The cytokines granulocyte-macrophage colony-stimulating factor (GM- CSF), interleukin (IL)-3, and IL-5 are important modulators of eosinophilia and eosinophil function. Eosinophil chemotaxis is known to be particularly sensitive for cytokine priming. In the present study, we compared chemotactic responses of eosinophils derived from peripheral blood of allergic asthmatics to responses of eosinophils from peripheral blood of healthy individuals. Eosinophils from allergic asthmatics exhibited a markedly increased sensitivity in their chemotactic response toward platelet-activating factor (PAF) compared with eosinophils from normal donors. In contrast, C5a-induced eosinophil chemotaxis between both groups was similar. This in vivo- primed phenotype could be mimicked in vitro, by preincubating eosinophils from peripheral blood of healthy individuals with picomolar concentrations of either GM-CSF, IL-3, or IL-5. The chemotactic response of eosinophils derived from the circulation of allergic asthmatic patients toward GM-CSF was significantly lower compared with the response of eosinophils of healthy individuals. Our data strongly suggest that release of cytokines may be an important in vivo priming mechanism for eosinophils in the circulation of allergic asthmatic patients. Such an in vivo priming can subsequently result in selective upregulation and downregulation of chemotactic responses toward various chemoattractants release in the lung tissue.

The age dependent behaviour of 11, mainly glycolytic, red blood cell enzymes in 26 patients with various haematological disorders has been investigated after separation of red blood cells by discontinuous density gradient centrifugation. The frequency of enzyme deficiencies in the old cells of these patients was significantly increased in comparison with the unseparated cells, 29 and 13 deficiencies, respectively. Particularly hexokinase activity, although normal or even increased in unseparated cells, was found deficient in old cells in seven cases. In addition, an increased number of phosphofructokinase deficiencies was observed in the patients' old cells (eight cases) as compared to the unseparated cells (three cases). However, the red blood cells of the majority of these patients were found to contain increased enzyme activities, irrespective of cell age. Enzyme activities in the youngest cell population did not correlate with the reticulocyte count. Cases of high pyruvate kinase and hexokinase activities were studied for kinetical, electrophoretical and immunological properties of the respective enzymes, but no abnormalities could be demonstrated, indicating an increased synthesis of these enzymes.

Critically ill post-surgical, post-trauma and/or septic patients are characterised by severe inflammation. This immune response consists of both a pro- and an anti-inflammatory component. The pro-inflammatory component contributes to (multiple) organ failure whereas occurrence of immune paralysis predisposes to infections. Strikingly, infectious complications arise in these patients despite the presence of a clear neutrophilia. We propose that dysfunction of neutrophils potentially increases the susceptibility to infections or can result in the inability to clear existing infections. Under homeostatic conditions these effector cells of the innate immune system circulate in a quiescent state and serve as the first line of defence against invading pathogens. In severe inflammation, however, neutrophils are rapidly activated, which affects their functional capacities, such as chemotaxis, phagocytosis, intra-cellular killing, NETosis, and their capacity to modulate adaptive immunity. This review provides an overview of the current understanding of neutrophil dysfunction in severe inflammation. We will discuss the possible mechanisms of downregulation of anti-microbial function, suppression of adaptive immunity by neutrophils and the contribution of neutrophil subsets to immune paralysis.

High-affinity receptors for interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) are composed of two distinct subunits, a ligand-specific  chain and a common β chain (βc). Whereas the mouse has two homologous β subunits (βc and βIL-3), in humans, only a single β chain is identified. We describe here the isolation and characterization of the gene encoding the human IL-3/IL-5/GM-CSF receptor β subunit. The gene spans about 25 kb and is divided into 14 exons, a structure very similar to that of the murine βc/βIL-3 genes. Surprisingly, we also found the remnants of a second βc chain gene directly downstream of βc. We identified a functional promoter that is active in the myeloid cell lines U937 and HL-60, but not in HeLa cells. The proximal promoter region, located from −103 to +33 bp, contains two GGAA consensus binding sites for members of the Ets family. Single mutation of those sites reduces promoter activity by 70% to 90%. The 5′ element specifically binds PU.1, whereas the 3′ element binds a yet-unidentified protein. These findings, together with the observation that cotransfection of PU.1 and other Ets family members enhances βc promoter activity in fibroblasts, reinforce the notion that GGAA elements play an important role in myeloid-specific gene regulation.

Neutrophil activation is a multistep process. In vitro activation of neutrophils with semiphysiological activators is optimal only after preactivation or priming with cytokines, chemotaxins, and/or bacterial products. Until now, no antibodies have been developed that can distinguish between resting and (cytokine) primed neutrophils with a sufficient dynamic range necessary for screening clinical samples. We have isolated two human phage antibodies, designated MoPhab A17 and A27, from a synthetic bacteriophage antibody library. These phage antibodies recognize epitopes that are upregulated on neutrophils present in whole blood treated with low priming concentrations of cytokines, such as GM-CSF and TNF-α. This induction was time- and concentration-dependent and optimal at concentrations that are sufficient for priming functional responses in neutrophils: GM-CSF (10 pM) and TNF-α (100 IU/ml). PMNs, isolated from the peripheral blood of chronic obstructive pulmonary disease (COPD) patients with a clinical exacerbation, exhibited a partial in vivo primed phenotype. These antibodies promise to be an ideal tool to monitor disease activity in whole blood of patients with inflammatory diseases.

SummarySurface-bound platelets support selectin-mediated rolling and β2-integrin-mediated firm adhesion of neutrophils (PMN) under flow conditions. We examined which ligands on platelets mediate this firm adhesion. Surface-bound platelets express ICAM-2 and GPIIbIIIa-bound fibrinogen, which are ligands for LFA-1 and MAC-1. In a well defined model for vessel wall injury, blood from an afibrinogenemic patient was perfused over ECM-coated coverslips to obtain fibrinogen-free platelet surfaces. At high shear rates, PMN-adhesion to fibrinogen-free platelet surfaces decreased compared to fibrinogen-containing controls. Under these conditions, firm adhesion and not rolling was blocked demonstrating the importance of fibrinogen in this process. In addition, MAC-1 and LFA-1 on PMN and ICAM-2 on platelets played a role in firm adhesion; the effect of blocking antibodies was most evident at high shear. The effects of fibrinogen depletion and ICAM-2 blocking were additive. In conclusion, multiple redundant ligands, like ICAM-2 and fibrinogen, induce firm and shear resistant PMN adhesion to platelets under flow conditions. Individually these ligands become critical at higher shear. Blocking of two or more interactions also interferes with low shear adhesion.

Increased expression of the intercellular adhesion molecule-1 (ICAM-1) on cells present in the airways has been suggested to play a role in the pathogenesis of asthma by enhancing airway inflammation. We used the monocytic U937 cell line, both undifferentiated and differentiated to a macrophage-like phenotype, and the bronchial epithelial cell line NCI-H292 as cellular model systems for human monocytes/macrophages and bronchial epithelial cells, respectively, and studied the effects of 12-O-tetradecanoyl phorbol-13-acetate (TPA) and dexamethasone on ICAM-1 expression. Both cell lines expressed the ICAM-1 protein constitutively. In addition, TPA- or 1,25-dihydroxyvitamin D3-mediated differentiation of the U937 cell line into a macrophage-like phenotype was associated with increased expression of ICAM-1. In both cell lines, two ICAM-1 mRNA transcripts were found, and expression was stimulated to a similar degree within 1 to 2 h after addition of TPA. In both cell lines, the anti-inflammatory corticosteroid dexamethasone repressed both constitutive and TPA-stimulated ICAM-1 expression, within 3 h of its addition. In the presence of cycloheximide, a marked superinduction of ICAM-1 was observed, while the repressive effect of dexamethasone remained, supporting the hypothesis that dexamethasone acts directly at the transcriptional level.

We have identified a patient with a number of neutrophil dysfunctions. The patient was a female baby who lived for 8 months. During her life, she developed severe bacterial infections and showed omphalitis, impaired wound healing, and a pronounced leukocytosis. She was not a patient with leukocyte adhesion deficiency, because all leukocyte CD18 complex proteins were expressed at normal levels. Yet, neutrophil polarization and chemotaxis to platelet-activating factor, leukotriene B4, or formyl-methionyl-leucyl-phenylalanine (FMLP) were completely absent. We found a strong defect in actin polymerization in response to chemotactic stimuli, but only a retarded or even normal reaction with other stimuli. This indicates that the cellular dysfunctions were not due to an intrinsic defect in actin metabolism. Instead, the regulation of actin polymerization with chemotactic stimuli seemed to be defective. We concentrated on FMLP-induced responses in the patient

Eosinophil-derived neurotoxin (EDN) found in the granules of human eosinophils is a cationic ribonuclease toxin. Expression of the EDN gene (RNS2) in eosinophils is dependent on proximal promoter sequences in combination with an enhancer located in the first intron. We further define here the active region of the intron using transfections in differentiated eosinophilic HL60 cells. We show that a region containing a tandem PU.I binding site is important for intronic enhancer activity. This region binds multiple forms of transcription factor PU.I as judged by gel-shift analysis and DNA affinity precipitation. Importantly, introducing point mutations in the PU.I site drastically reduces the intronic enhancer activity, showing the importance of PU.I for expression of EDN in cells of the eosinophilic lineage.

It is unclear whether the presence and severity of atopic dermatitis (AD) is predictive for the occurrence and severity of early and late asthmatic responses to inhaled allergens. The aim of this study was to compare the bronchial effects of allergen inhalation challenge in allergic asthma (AA) and atopic dermatitis. We therefore studied these responses in: nine patients with mild-to-moderate AA without AD; eight patients with mild-to-moderate AA and mild AD; eight patients with severe AD and mild AA; and eight patients with severe AD without AA. Allergen challenge was performed by inhaling doubling doses until the dose provoking a 20% fall in forced expiratory volume in one second (PC20) was reached. The late asthmatic reaction (LAR) was defined as a fall of >20% in peak expiratory flow (PEF) between 3 and 8 h after the challenge. All but four of the patients with severe AD without AA developed an early asthmatic response (EAR). A LAR was seen in all patients with severe AD and mild AA, in four patients with mild AD and mild-to-moderate AA, and in three patients with mild-to-moderate AA without AD. The LAR was most pronounced in patients with a combination of mild AA and severe AD. This could be explained, in part, by a decreased skin sensitivity in these patients, which made the Cockcroft formula for prediction of PC20 allergen less accurate in such patients. We conclude that patients with severe atopic dermatitis and mild asthma are at risk for developing pronounced late asthmatic responses after allergen exposure. This suggests that eosinophils activated in atopic dermatitis also predispose to airway inflammation.

Signal transducers and activators of transcription (STATs) belong to a family of transcription factors that were originally identified as mediators of cytokine-induced gene expression. Recent evidence, however, has shown that certain members of the STAT family, including STAT3, are also involved in cellular transformation. Here we show that STAT5 also plays a role in cellular transformation by the BCR-Abl oncogene. In BCR-Abl transformed K562 cells, STAT5A and 5B are constitutively phosphorylated on tyrosine and are transcriptionally active. Moreover, expression of a dominant negative form of STAT5 shows that active STAT5 is necessary for the growth in soft agar of these cells. These results show that besides STAT3, STAT5 can also be involved in cellular transformation.

Eosinophils were isolated from normal human blood by separation over Percoll gradients, which resulted in eosinophil suspensions of a purity higher than 95% and recoveries of about 65%. Normal human eosinophils were found to respond to formyl-methionyl-leucyl-phenylalanine (fMLP) at concentrations greater than 10(-7) mol/L with an increase in the concentration of intracellular free calcium, oxygen consumption, nitroblue tetrazolium reduction, and chemiluminescence. The maximal response of eosinophils to fMLP was lower than that of neutrophils isolated from the same blood samples and required at least ten times as much fMLP as was needed for neutrophils. Low fMLP concentrations (approximately 10(-8) mol/L), which in themselves did not stimulate O2 consumption by either eosinophils or neutrophils, primed these cells to respond to a suboptimal concentration of another stimulus. Purification of eosinophils after treatment of whole blood with fMLP showed that these eosinophils had lost their ability to respond to fMLP. We conclude that normal eosinophils do respond to fMLP and that therefore fMLP should not be used to isolate eosinophils.

After interaction with so-called priming agents, the respiratory burst in human granulocytes does not become activated, but is enhanced upon subsequent stimulation with the chemoattractant FMLP. Investigating the mechanism of the priming reaction, we found that a transient rise in the cytosolic free calcium concentration [( Ca2+]i) suffices to irreversibly prime human granulocytes. Thus, platelet-activating factor (PAF) induced a transient increase in [Ca2+]i and primed the cells to an enhanced respiratory burst upon subsequent interaction with FMLP. Artificially, the transient rise in [Ca2+]i was mimicked by addition and subsequent removal of the Ca2+ ionophore ionomycin; this treatment too, primed the respiratory burst of the granulocytes. The priming induced by ionomycin was completely abolished when [Ca2+]i changes were buffered during exposure of the cells to the ionophore. The priming induced by PAF was only partially inhibited under [Ca2+]i-buffering conditions during priming, indicating that multiple pathways exist in the priming of granulocytes by PAF.

Background A robust inflammatory response occurs in the hours and days following cerebral ischemia. However, little is known about the immediate innate immune response in the first minutes after an ischemic insult in humans. We utilized the use of circulatory arrest during cardiac surgery to assess this. Methods Twelve neonates diagnosed with an aortic arch obstruction underwent cardiac surgery with cardiopulmonary bypass and approximately 30 minutes of deep hypothermic circulatory arrest (DHCA, representing cerebral ischemia). Blood samples were drawn from the vena cava superior immediately after DHCA and at various other time points from preoperatively to 24 hours after surgery. The innate immune response was assessed by neutrophil and monocyte count and phenotype using FACS, and concentrations of cytokines IL-1β, IL-6, IL-8, IL-10, TNFα, sVCAM-1 and MCP-1 were assessed using multiplex immunoassay. Results were compared to a simultaneously drawn sample from the arterial cannula. Twelve other neonates were randomly allocated to undergo the same procedure but with continuous antegrade cerebral perfusion (ACP). Results Immediately after cerebral ischemia (DHCA), neutrophil and monocyte counts were higher in venous blood than arterial (P = 0.03 and P = 0.02 respectively). The phenotypes of these cells showed an activated state (both P

Background Systemic inflammation in response to a femur fracture and the additional fixation is associated with inflammatory complications, such as acute respiratory distress syndrome and multiple organ dysfunction syndrome. The injury itself, but also the additional procedure of femoral fixation induces a release of pro-inflammatory cytokines such as interleukin-6. This results in an aggravation of the initial systemic inflammatory response, and can cause an increased risk for the development of inflammatory complications. Recent studies have shown that administration of the serum protein C1-esterase inhibitor can significantly reduce the release of circulating pro-inflammatory cytokines in response to acute systemic inflammation. Objective Attenuation of the surgery-induced additional systemic inflammatory response by perioperative treatment with C1-esterase inhibitor of trauma patients with a femur fracture. Methods The study is designed as a double-blind randomized placebo-controlled trial. Trauma patients with a femur fracture, Injury Severity Score ≥ 18 and age 18-80 years are included after obtaining informed consent. They are randomized for administration of 200 U/kg C1-esterase inhibitor intravenously or placebo (saline 0.9%) just before the start of the procedure of femoral fixation. The primary endpoint of the study is Δ interleukin-6, measured at t = 0, just before start of the femur fixation surgery and administration of C1-esterase inhibitor, and t = 6, 6 hours after administration of C1-esterase inhibitor and the femur fixation. Conclusion This study intents to identify C1-esterase inhibitor as a safe and potent anti-inflammatory agent, that is capable of suppressing systemic inflammation in trauma patients. This might facilitate early total care procedures by lowering the risk of inflammation in response to the surgical intervention. This could result in increased functional outcomes and reduced health care related costs. Trial registration clinicaltrials.gov NCT01275976 (January 12th 2011)

Binding of interferon-γ (IFN-γ) to its heterodimeric receptor induces activation of the tyrosine kinases JAK1 and JAK2 followed by tyrosine phosphorylation of STAT1α. Selective activation of STAT1α at the IFN-γ receptor is achieved by specific interaction between a cytosolic tyrosine motif including Y440 in the IFN-γ receptor α-chain and the SH2 domain of STAT1α. We demonstrate that, in addition to STAT1α, STAT3 is also activated by IFN-γ in human neutrophils. The activation of STAT3 was not found in human eosinophils, monocytes, and HL-60 cells, although the STAT3 protein was expressed in these cells. The cell type-specific activation of STAT3 by IFN-γ was also observed in neutrophils that are differentiated in vitro from human CD34+ hematopoietic stem cells. These results indicate that a single cytokine receptor can activate different STAT family members in a cell-specific manner, which might result in cell-specific gene transcription. J. Leukoc. Biol. 65: 391–396; 1999.

Background Inflammation, both local and systemic, is a hallmark of chronic obstructive pulmonary disease (COPD). Inflammatory mediators such as TNFα and GM-CSF are secreted by lung epithelium, alveolar macrophages and other inflammatory cells and are thought to be important contributors in the pathogenesis of COPD. Indeed, neutrophils are activated by these cytokines and these cells are one of the major inflammatory cell types recruited to the pulmonary compartment of COPD patients. Furthermore, these inflammatory mediators are found in the peripheral blood of COPD patients and, therefore, we hypothesized that TNFα/GM-CSF-induced protein profiles can be found in peripheral neutrophils of COPD patients. Methods Using fluorescence 2-dimensional difference gel electrophoresis we investigated differentially regulated proteins in peripheral neutrophils from COPD patients and healthy age-matched control subjects. Furthermore, protein profiles from COPD patients were compared with those of neutrophils of healthy age-matched controls that were stimulated with TNFα and/or GM-CSF in vitro. Protein gels were compared using DeCyder 7.0 software. Results We identified 7 significantly regulated protein spots between peripheral neutrophils from COPD patients and age-matched healthy control subjects. Stimulation of peripheral neutrophils with TNFα, GM-CSF or TNFα + GM-CSF in vitro resulted in 13, 20 and 22 regulated protein spots, respectively. However, these cytokine-induced protein differences did not correspond with the protein differences found in neutrophils from COPD patients. Conclusion These results show that neutrophils from COPD patients have a unique protein profile compared to neutrophils from healthy age-matched controls. Furthermore, the neutrophil profiles of COPD patients do not reflect putative dominant signals induced by TNFα, GM-CSF or their combination. Our results indicate that systemic neutrophil responses in COPD patients are caused by a unique but subtle interplay between multiple inflammatory signals.

Background Identification of an inflammatory asthma phenotype currently requires sputum induction. This technique is invasive, has a high variability, is time-consuming and a burden for patients. Therefore, there is a strong need for a routine blood test to establish the inflammatory phenotype of asthma. We designed a clinical cohort study (AIR-study, NCT01611012) in 115 asthma patients visiting the outpatient clinic to compare the results of such a blood test to sputum analysis. This abstracts focuses at the preliminary results in 21 patients.