Eosinophil Cationic Protein Release Research Articles

Involvement of PAR2 in inflammatory mediator release from human blood eosinophils

Proteinase Activated Receptors (PARs) are the members of G-protein-coupled receptor family and can be cleaved by certain serine proteases to expose a tethered ligand domain, which binds and activates the receptors to initiate multiple signaling cascades. There is some evidence that certain proteases may regulate target cells by activating PARs. There are many studies, in which PARs play important roles in inflammation. One study indicated that PAR2 inhibition and deletion significantly suppressed the degree of inflammation due to decreased IL-6 and IL-1 levels. Another study also showed that PARs activation could mediate reactive oxygen species production and MAPK signaling leading to alveolar inflammation. In addition, platelet-derived CAPN1 can trigger the vascular inflammation associated with diabetes via cleavage of PAR1 and the release of TNF from the endothelial cell surface, and sarsasapogenin may alleviate diabetic nephropathy by the downregulation of PAR1. Another Phellodendron amurense bark extract can suppress the particulate matter-induced Ca2+ influx caused by direct action upon PAR2, alleviating inflammation and maintaining homeostatic levels of cell adhesion components. There are also other two antagonists of I-287 and GB88, which can reduce the PAR2-mediated inflammatory reaction. In this study, we tested expression of PARs and IL-5, IL-6, RANTES and ECP release from human blood eosinophils using different enzymes and PAR agonists. The expression of PARs was assessed in human blood eosinophils by flow cytometry and RT-PCR, and the levels of cytokine and eosinophil cationic protein (ECP) in the cultured supernatants were determined with ELISA kits. Flow cytometry shows that human eosinophils express PAR2 protein and do not express PAR1, PAR3 and PAR4 proteins. RT-PCR analysis revealed expression of PAR2 and PAR3 genes in human eosinophils. Tryptase, trypsin and elastase can induce significant IL-5, IL-6 and ECP release. Trypsin and elastase may also stimulate RANTES secretion, but tryptase cannot induce the RANTES secretion. Tryptase, trypsin and elastase-induced cytokine and ECP release from human blood eosinophils most likely occurs via activation of PAR2.

Regulation and directed inhibition of ECP production by human neutrophils.

Neutrophils are involved in the pathophysiology of allergic asthma, where the Eosinophil Cationic Protein (ECP) is a critical inflammatory mediator. Although ECP production is attributed to eosinophils, we reported that ECP is also present in neutrophils from allergic patients where, in contrast to eosinophils, it is produced in an IgE-dependent manner. Given the key role of ECP in asthma, we investigated the molecular mechanisms involved in ECP production as well as the effects induced by agonists and widely used clinical approaches. We also analyzed the correlation between ECP production and lung function. Neutrophils from allergic asthmatic patients were challenged with allergens, alone or in combination with cytokines, in the presence of cell-signaling inhibitors and clinical drugs. We analyzed ECP levels by ELISA and confocal microscopy. Lung function was assessed by spirometry. IgE-mediated ECP release is dependent on phosphoinositide 3-kinase, the extracellular signal-regulated kinase (ERK1/2) and the production of reactive oxygen species by NADPH-oxidase. Calcineurin phosphatase and the transcription factor NFAT are also involved. ECP release is enhanced by the cytokines interleukin (IL)-5 and granulocyte macrophage-colony stimulating factor, and inhibited by interferon-γ, IL-10, clinical drugs (formoterol, tiotropium and budesonide) and allergen-specific IT. We also found an inverse correlation between asthma severity and ECP levels. Our results suggest the molecular pathways involved in ECP production and potential therapeutic targets. We also provide a new method to evaluate disease severity in asthmatic patients based on the quantification of in vitro ECP production by peripheral neutrophils.

Different levels of mucus inflammatory mediators in nasal polyposis with and without aeroallergen sensitivity.

ObjectivesBiomarker levels in nasal secretions can reflect the inflammatory status of nasal mucosa and evolution of sinus disease. The aim of this study was to evaluate the relationship between local inflammatory mediator production and clinical characteristics of patients with nasal polyposis (NP).MethodsThirty‐one nonaeroallergen sensitized patients with NP (NANP), 29 aeroallergen sensitized patients with NP (ANP), and 30 subjects without inflammation of nasal mucosa as controls (C) entered this prospective, cross‐sectional study. Clinical parameters (symptoms, endoscopic, and radiological findings) were assessed. The concentrations of heat shock protein 70 (HSP70), eosinophil cationic protein (ECP), tryptase, substance P and Clara cell protein 16 (CC16) were measured in the nasal secretion samples of all participants by ELISA method.ResultsOur results showed higher concentrations of HSP70, ECP, and tryptase in ANP than in NANP and C (p < .001 for all markers). On the other hand, levels of CC16 were significantly higher in C than in NANP and ANP groups (p < .001; p < .001, respectively). We found positive correlations between HSP70, ECP, tryptase, and substance P levels and nasal symptom score in patients with NP. Also, HSP70, ECP, tryptase, and substance P showed different levels of positive correlation among themselves, with HSP70 showing highest positive correlation with ECP. Finally, relatively strong negative correlations were found between the levels of CC16 and nasal symptoms, as well as between the CC16 levels and levels of other four mediators in nasal fluid.ConclusionHSP70, ECP, tryptase, and substance P might play a role in the pathogenesis of NP. The results suggest that chronic inflammation in NP involves a self‐sustaining local release of HSP70, ECP, and tryptase, independent of aeroallergen stimulation of the mucosal layer, although the production of these mediators is higher in aeroallergen sensitized NP patients.

Simvastatin inhibits the proliferation of HL-60 clone 15- derived eosinophils by inducing the arrest of the cell cycle in the G1/S phase

Eosinophils and their granular proteins are crucial for combating allergic airway diseases. Eosinophils derived from HL-60 clone 15 (HC15) cells have been established as a feasible alternative cell model for human primary eosinophils. Simvastatin, a cholesterol-lowering agent, has been shown to exhibit anti-inflammatory and anti-allergic effects. Among the granular eosinophil proteins, eosinophil cationic protein (ECP) is the one best recognised in allergic airway diseases. The aim of our study is to investigate the effect and regulatory mechanisms of simvastatin on ECP levels derived from eosinophils. Both HC15 cell counts and ECP levels decreased after simvastatin treatment in the animal and cell models; however, after a cell count adjustment, simvastatin was not observed to exert a significantly inhibitory effect on ECP expression. Real-time polymerase chain reaction and Western blotting analyses demonstrated that simvastatin did not inhibit the intracellular formation or release of ECP. Cell cycle analysis showed that the percentage of HC15 cells in the G1 and S phases significantly increased and decreased, respectively, after simvastatin treatment. Simvastatin inhibited the proliferation of HC15-derived eosinophils by inducing G1/S cell cycle arrest in a dose-dependent manner. Its effect on the cell cycle involved the downregulation of cyclin A but without the presence of mevalonate; therefore, total ECP expression from eosinophils decreased, not by suppressing the actual formation or release of ECP but by arresting the G1/S cell cycle phase and inhibiting subsequent cell proliferation through the mevalonate pathway.

Pathological manifestations in lymphatic filariasis correlate with lack of inhibitory properties of IgG4 antibodies on IgE-activated granulocytes.

Helminth parasites are known to be efficient modulators of their host’s immune system. To guarantee their own survival, they induce alongside the classical Th2 a strong regulatory response with high levels of anti-inflammatory cytokines and elevated plasma levels of IgG4. This particular antibody was shown in different models to exhibit immunosuppressive properties. How IgG4 affects the etiopathology of lymphatic filariasis (LF) is however not well characterized. Here we investigate the impact of plasma and affinity-purified IgG/IgG4 fractions from endemic normals (EN) and LF infected pathology patients (CP), asymptomatic microfilaraemic (Mf+) and amicrofilaraemic (Mf-) individuals on IgE/IL3 activated granulocytes. The activation and degranulation states were investigated by monitoring the expression of CD63/HLADR and the release of granule contents (neutrophil elastase (NE), eosinophil cationic protein (ECP) and histamine) respectively by flow cytometry and ELISA. We could show that the activation of granulocytes was inhibited in the presence of plasma from EN and Mf+ individuals whereas those of Mf- and CP presented no effect. This inhibitory capacity was impaired upon depletion of IgG in Mf+ individuals but persisted in IgG-depleted plasma from EN, where it strongly correlated with the expression of IgA. In addition, IgA-depleted fractions failed to suppress granulocyte activation. Strikingly, affinity-purified IgG4 antibodies from EN, Mf+ and Mf- individuals bound granulocytes and inhibited activation and the release of ECP, NE and histamine. In contrast, IgG4 from CP could not bind granulocytes and presented no suppressive capacity. Reduction of both the affinity to, and the suppressive properties of anti-inflammatory IgG4 on granulocytes was reached only when FcγRI and II were blocked simultaneously. These data indicate that IgG4 antibodies from Mf+, Mf- and EN, in contrast to those of CP, natively exhibit FcγRI/II-dependent suppressive properties on granulocytes. Our findings suggest that quantitative and qualitative alterations in IgG4 molecules are associated with the different clinical phenotypes in LF endemic regions.

Neutrophil autophagy and extracellular DNA traps contribute to airway inflammation in severe asthma.

Autophagy and neutrophil extracellular DNA traps (NETs) are implicated in asthma; however, their roles in asthma pathogenesis have not been elucidated. We compared autophagy and NET production levels from peripheral blood neutrophils (PBNs) of patients with severe asthma (SA) and non-severe asthma (NSA). Additionally, we investigated the inflammatory effects of NETs on human airway epithelial cells (AECs) and peripheral blood eosinophils (PBEs). Peripheral blood neutrophils from patients with SA (n=30) and NSA (n=38) were treated with interleukin (IL)-8 (100ng/mL). Autophagy (light chain 3-II expression) and NET production levels were evaluated by Western blot, immunofluorescence microscopy, and PicoGreen assay. The effects of NETs on AECs were assessed by investigating cell death, cell detachment, expression of occludin and claudin-1, and IL-8 production; the effects of NETs on PBEs were examined by investigating the activation and release of eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). Untreated and IL-8-treated PBNs from the SA group produced higher autophagy and NET levels compared with those from the NSA group (P<0.01). IL-8 increased autophagy and NET levels in PBNs from the SA group, but not from the NSA group. NET levels were correlated with autophagy levels in PBNs (P<0.001). IL-8-induced NET production levels negatively were correlated with FEV1/FVC (r=-0.700, P=0.016). NETs induced cell death, detachment, degradation of occludin and claudin-1, and IL-8 production from AECs. Higher levels of NET-induced ECP and EDN were released from PBEs in SA compared with NSA groups. Neutrophil autophagy and NETs could enhance asthma severity by damaging airway epithelium and triggering inflammatory responses of AECs and PBEs. Modulating neutrophil autophagy and NET production may be a new target therapy for SA.

Helicobacter pylori outer membrane vesicle proteins induce human eosinophil degranulation via a β2 Integrin CD11/CD18- and ICAM-1-dependent mechanism.

Eosinophil cationic protein (ECP), a cytotoxic protein contained in eosinophils granules, can contribute to various inflammatory responses. Although Helicobacter pylori infection increases infiltration of eosinophils, the mechanisms of eosinophil degranulation by H. pylori infection are largely unknown. The goal of this study was to investigate the role of H. pylori outer membrane vesicles (OMVs) in modulating eosinophil degranulation. We found that eosinophils treated with H. pylori OMVs released significantly more ECP compared with untreated controls. In addition, eosinophils cocultured with OMV-preexposed primary gastric epithelial cells exhibited significantly increased ECP release. Similarly, eosinophils cocultured with culture supernatant (CM) from primary gastric epithelial cells exposed to OMVs (OMV-CM) released significantly higher amounts of ECP compared with eosinophils cocultured with CM from unexposed control cells. Furthermore, OMVs and OMV-CM both induced the upregulation of ICAM-1 on gastric epithelial cells and β2 integrin CD11b on eosinophils. In addition, both transduction of ICAM-1 shRNA into gastric epithelial cells and treatment with neutralizing mAbs to CD18 significantly decreased OMV-mediated or OMV-CM-mediated release of ECP. These results suggest that the eosinophil degranulation response to H. pylori OMVs occurs via a mechanism that is dependent on both β2 integrin CD11/CD18 and ICAM-1.

The antimicrobial peptide LL‐37 induces synthesis and release of cysteinyl leukotrienes from human eosinophils – implications for asthma

Eosinophils and their products, including leukotrienes and eosinophil cationic protein (ECP), are well-known mediators of inflammation and tissue damage in asthma. The antimicrobial peptide LL-37 exhibits a variety of immunomodulatory activities. However, the role of LL-37 in asthma has not been fully addressed. Here, we aim to investigate the effect of LL-37 on inducing inflammatory mediators in human eosinophils, probe the underlying mechanisms, and search for a clinical correlate. Primary eosinophils were isolated from peripheral blood. Leukotriene and ECP levels were measured using EIAs or ELISAs. Activation of leukotriene-synthesizing enzymes and signaling kinases was analyzed by Western blot or immunofluorescent imaging. LL-37/its proform hCAP18 expression was analyzed by Western blot. LL-37, via formyl peptide receptor-2 (FPR-2), triggered the release of cysteinyl leukotrienes (cys-LTs) from eosinophils. The release was more prominent in cells primed with the eosinophilopoietic cytokine GM-CSF or IL-5 or cells from asthmatic patients. LL-37 stimulates lipid body formation and activates cys-LT-synthesizing enzymes by multiple mechanisms: enhancing cPLA(2) activity by pERK1/2-mediated phosphorylation and inducing intracellular translocation and assembly of 5-LO and LTC(4) S at perinuclear locations and lipid bodies. In addition to cys-LTs, LL-37 enhances ECP release from eosinophils via pERK1/2. The expression of hCAP18 and its release following leukotriene stimulation are significantly higher in eosinophils from asthmatics. This study identifies LL-37 as an eosinophil-activating peptide that triggers release of inflammatory mediators. The clinical correlation suggests that LL-37/hCAP18 and its signaling pathway represent potential therapeutic targets for this disease.

Priming of eosinophils by GM-CSF is mediated by protein kinase CbetaII-phosphorylated L-plastin.

The priming of eosinophils by cytokines leading to augmented response to chemoattractants and degranulating stimuli is a characteristic feature of eosinophils in the course of allergic inflammation and asthma. Actin reorganization and integrin activation are implicated in eosinophil priming by GM-CSF, but their molecular mechanism of action is unknown. In this regard, we investigated the role of L-plastin, an eosinophil phosphoprotein that we identified from eosinophil proteome analysis. Phosphoproteomic analysis demonstrated the upregulation of phosphorylated L-plastin after eosinophil stimulation with GM-CSF. Additionally, coimmunoprecipitation studies demonstrated a complex formation of phosphorylated L-plastin with protein kinase CβII (PKCβII), GM-CSF receptor α-chain, and two actin-associated proteins, paxilin and cofilin. Inhibition of PKCβII with 4,5-bis(4-fluoroanilino)phtalimide or PKCβII-specific small interfering RNA blocked GM-CSF-induced phosphorylation of L-plastin. Furthermore, flow cytometric analysis also showed an upregulation of α(M)β(2) integrin, which was sensitive to PKCβII inhibition. In chemotaxis assay, GM-CSF treatment allowed eosinophils to respond to lower concentrations of eotaxin, which was abrogated by the above-mentioned PKCβII inhibitors. Similarly, inhibition of PKCβII blocked GM-CSF induced priming for degranulation as assessed by release of eosinophil cationic protein and eosinophil peroxidase in response to eotaxin. Importantly, eosinophil stimulation with a synthetic L-plastin peptide (residues 2-19) phosphorylated on Ser(5) upregulated α(M)β(2) integrin expression and increased eosinophil migration in response to eotaxin independent of GM-CSF stimulation. Our results establish a causative role for PKCβII and L-plastin in linking GM-CSF-induced eosinophil priming for chemotaxis and degranulation to signaling events associated with integrin activation via induction of PKCβII-mediated L-plastin phosphorylation.

PI3-Kinase Regulates Eosinophil and Neutrophil Degranulation in Patients with Allergic Rhinitis and Allergic Asthma Irrespective of Allergen Challenge Model

The PI3K pathway plays a major role in many vital cell processes. Our primary aim was to investigate signalling through PI3K for in vitro degranulation from allergen-primed eosinophils and neutrophils in allergic rhinitis and allergic asthma after seasonal and experimental allergen challenge. Nine patients with allergic rhinitis, eight with allergic asthma and four controls were studied during birch pollen season and after nasal and bronchial allergen challenge. Primed blood eosinophils and neutrophils were stimulated for in vitro degranulation with C3b-coated Sephadex particles, after prior incubation with Wortmannin, a PI3K inhibitor. The released amounts of eosinophil cationic protein (ECP), eosinophil peroxidase (EPO) and myeloperoxidase (MPO) were measured by radioimmunoassay. Wortmannin (10(-6) to 10(-9) M) inhibited ECP, EPO and MPO release in a dose-dependent manner in allergic rhinitis and allergic asthma in all three allergen challenge models. Inhibition of ECP release tended to be lower in the asthmatics in all allergen challenge models, statistically significant compared to the controls during season for 10(-8) M Wortmannin (p=0.01). A clear propensity towards less inhibition in the rhinitic patients was seen after nasal and bronchial challenge compared to seasonal exposure, significant for ECP (10(-8) M Wortmannin; p=0.034 and 0.002, respectively). Signalling through PI3K is clearly involved in ECP, EPO and MPO release in allergic rhinitis and allergic asthma irrespective of allergen challenge model. Allergic asthma demonstrated less inhibition of ECP release via PI3K during pollen season, indicating that other pathways play a greater role in eosinophil degranulation in allergic asthma than allergic rhinitis.

Patients with allergic rhinitis and allergic asthma share the same pattern of eosinophil and neutrophil degranulation after allergen challenge

BackgroundPatients with allergic rhinitis and allergic asthma demonstrate comparable local and systemic eosinophil inflammation, and yet they present with different clinical pictures. Less is even known about the contribution of neutrophil inflammation in allergic diseases. The aim of the study was to examine the propensity and selectivity of granule release from primed systemic eosinophils and neutrophils in allergic rhinitis and allergic asthma after seasonal and experimental allergen exposure. We hypothesize that the dissimilar clinical manifestations are due to diverse eosinophil and neutrophil degranulation.MethodsNine birch pollen allergic patients with rhinitis, eight with asthma and four controls were studied during pollen season and after nasal and bronchial allergen challenge. Eosinophils and neutrophils were incubated in vitro with assay buffer and opsonized Sephadex particles for spontaneous and C3b-induced granule protein release. The released amount of eosinophil cationic protein (ECP), eosinophil peroxidase (EPO) and myeloperoxidase (MPO) was measured by specific radioimmunoassay.ResultsC3b-induced degranulation resulted in increased release of ECP and MPO from primed blood eosinophils and neutrophils in both allergic rhinitis and allergic asthma during pollen season and after both nasal and bronchial challenge (p-values 0.008 to 0.043). After bronchial challenge, the ECP release was significantly higher in the rhinitic group compared to the asthmatic group [19.8 vs. 13.2%, (p = 0.010)]. The propensity for EPO release was weak in all challenge models but followed the same pattern in both allergic groups.ConclusionsSystemically activated eosinophils and neutrophils have similar patterns of degranulation after allergen exposure in allergic rhinitis and allergic asthma. The released amount of ECP, EPO and MPO was similar in all allergen challenge models in both allergic groups. Our results indicate that other mechanisms than the magnitude of eosinophil and neutrophil inflammation or the degranulation pattern of the inflammatory cells determines whether or not an allergic patient develops asthma.

Human Eosinophils Exert TNF-α and Granzyme A-Mediated Tumoricidal Activity toward Colon Carcinoma Cells

Peripheral blood and tissue eosinophilia is a prominent feature in allergic diseases and helminth infections. In cancer patients, tumor-associated tissue eosinophilia is frequently observed. Tumor-associated tissue eosinophilia can be associated with a favorable prognosis, notably in colorectal carcinoma. However, underlying mechanisms of eosinophil contribution to antitumor responses are poorly understood. We have in this study investigated the direct interactions of human eosinophils with Colo-205, a colorectal carcinoma cell line, and show that eosinophils induce apoptosis and directly kill tumor cells. Using blocking Abs, we found that CD11a/CD18 complex is involved in the tumoricidal activity. Coculture of eosinophils with Colo-205 led to the release of eosinophil cationic protein and eosinophil-derived neurotoxin as well as TNF-α secretion. Moreover, eosinophils expressed granzyme A, which was released upon interaction with Colo-205, whereas cytotoxicity was partially inhibited by FUT-175, an inhibitor of trypsin-like enzymatic activity. Our data present the first demonstration, to our knowledge, that granzyme A is a cytotoxic mediator of the eosinophil protein arsenal, exerting eosinophil tumoricidal activity toward Colo-205, and provide mechanistic evidence for innate responses of eosinophil against tumor cells.

C3b‐induced eosinophil degranulation involves PI3‐kinases and is inhibited by protein kinase C activity

Selective release of individual eosinophil granule proteins has been demonstrated in eosinophilic conditions and in vitro using different stimuli. The aim of this study was to investigate if selective release of eosinophil cationic protein (ECP), eosinophil protein X/eosinophil derived-neurotoxin (EPX/EDN) and eosinophil peroxidase (EPO) could be due to the involvement of different signal transduction pathways. Peripheral blood granulocytes from healthy donors were incubated with Wortmannin, LY294002, Genistein, Staurosporine, GÖ6976 or PD98059 prior to the induction of degranulation by C3b. The released amounts of ECP, EPO and EPX/EDN were determined by immunoassays, and related to the total cell content of respective protein. Wortmannin caused a significant, dose-dependent inhibition of all three granule proteins. LY294002 (10⁻⁶ M) also inhibited the release of all proteins. Genistein (10⁻⁶ M) inhibited the release of ECP, whereas the release of EPO was increased. However, there was a tendency towards similar concentration-dependent patterns of release of all three proteins. Staurosporine (10⁻⁷ M), GÖ6976 (10⁻⁶ M) and PD98059 (10⁻⁵ M) caused an increased release of the three proteins. PI3-kinases play an important role in the C3b-induced release of ECP, EPO and EPX/EDN, whereas protein kinase C seems to have inhibitory effects on C3b-induced degranulation.

Cysteinyl leukotrienes acting via granule membrane-expressed receptors elicit secretion from within cell-free human eosinophil granules

Cysteinyl leukotrienes (cysLTs) are recognized to act via receptors (cysLTRs) expressed on cell surface plasma membranes. Agents that block cysLT(1) receptor (cysLT(1)R) are therapeutics for allergic disorders. Eosinophils contain multiple preformed proteins stored within their intracellular granules. Cell-free eosinophil granules are present extracellularly as intact membrane-bound organelles in sites associated with eosinophil infiltration, including asthma, rhinitis, and urticaria, but have unknown functional capabilities. We evaluated the expression of cysLTRs on eosinophil granule membranes and their functional roles in eliciting protein secretion from within eosinophil granules. We studied secretory responses of human eosinophil granules isolated by subcellular fractionation. Granules were stimulated with cysLTs, and eosinophil cationic protein and cytokines were measured in the supernatants. Receptor expression on granule membranes and eosinophils was evaluated by flow cytometry and Western blot. We report that receptors for cysLTs, cysLT(1)R, cysLT(2) receptor, and the purinergic P2Y12 receptor, are expressed on eosinophil granule membranes. Leukotriene (LT) C(4) and extracellularly generated LTD(4) and LTE(4) stimulated isolated eosinophil granules to secrete eosinophil cationic protein. MRS 2395, a P2Y12 receptor antagonist, inhibited cysLT-induced eosinophil cationic protein release. Montelukast, likely not solely as an inhibitor of cysLT(1)R, inhibited eosinophil cationic protein release elicited by LTC(4) and LTD(4) as well as by LTE(4). These studies identify previously unrecognized sites of localization, the membranes of intracellular eosinophil granule organelles, and function for cysLT-responsive receptors that mediate cysteinyl leukotriene-stimulated secretion from within eosinophil granules, including those present extracellularly.

Therapeutic Surfactants Modulate the Viability of Eosinophils and Induce Inflammatory Mediator Release

Background: There is increasing interest in testing surfactant preparations for asthma therapy. Previously, Curosurf was demonstrated to increase inflammation in allergic asthmatics. So far, little is known about the immunomodulatory effects of therapeutic surfactants, in particular concerning the interaction of surfactant components with eosinophils as key effector cells of the allergic airway inflammation. The aim of the present study was to determine the effect of different therapeutic surfactants on cellular functions of eosinophils. Methods: Eosinophils were isolated from peripheral blood of atopic volunteers and incubated with the natural animal-derived surfactants Curosurf or Alveofact or the synthetic recombinant human surfactant Venticute at different concentrations for up to 42 h. Results: Curosurf and Venticute modulated the viability of eosinophils. While incubation with Curosurf increased the number of necrotic eosinophils after 1, 20 and 42 h, Venticute increased the number of apoptotic and necrotic cells after 1 h, but there were no differences compared with control cells at later time points. All surfactant preparations increased the levels of eosinophil cationic protein after 20 h and, in addition, Curosurf enhanced eosinophil cationic protein release after 42 h. The supernatant of eosinophils induced chemotaxis against autologous eosinophils, and the presence of Curosurf, but not Alveofact or Venticute, augmented the chemotactic effect. Chemotaxis was partly blocked by inhibition of eotaxin but not by inhibition of leukotrienes or platelet-activating factor. Conclusions: Therapeutic surfactants differ in their effects on eosinophil viability and the accompanying release of inflammatory mediators and chemotactic signals. Proinflammatory effects were most pronounced for the natural surfactant Curosurf.

Parent psychological states predict changes in inflammatory markers in children with asthma and healthy children

Previous research has shown that parent mental health is associated with asthma morbidity in children. However, the biological pathways explaining these relationships are not known. The present study tested whether parent psychological characteristics could predict longitudinal changes in inflammatory markers in children with asthma and a comparison group of healthy children.For this, 33 healthy children (17m/16f; 13.5years) and 50 children with asthma (37m/13f; 13.3years) were assessed at two time points on average 208days apart. Parent depression (CES-D) and perceived stress (PSS) were assessed at baseline, child depression (CDI) and anxiety (RCMAS) at follow-up. Asthma-relevant inflammatory markers eosinophil cationic protein (ECP) and stimulated interleukin-4 (IL-4) production were measured at baseline and at follow-up.Hierarchical regression analyses controlling for asthma severity and medication use revealed that higher levels of parental perceived stress at baseline were associated with greater increases over time in children’s IL-4 production (β=.29, p=.019) as well as ECP release (β=.27, p=.004). Additionally, higher levels of parental depression at baseline were associated with increases in ECP over time (β=.19, p=.046). There was no evidence that these associations were mediated by child depression or anxiety.These results demonstrate that parental stress and depression at baseline predict increases in children’s inflammatory profiles over a six month period. This pattern appeared in both children with asthma and healthy children, and was not due to effects on child psychological states. These changes in inflammatory makers may represent one biological mechanism underlying the association between parental distress and child asthma morbidity.

Neutrophils as a Novel Source of Eosinophil Cationic Protein in IgE-Mediated Processes

The production of eosinophil cationic protein (ECP) in IgE-mediated diseases has been associated mainly with eosinophils, although no IgE-dependent ECP release has been observed in these cells. Because there is increasing evidence of neutrophil participation in allergic processes, we have examined whether human neutrophils from allergic patients were able to produce ECP by an IgE-dependent mechanism. After challenge with specific Ags to which the patients were sensitized, ECP release was detected in the culture medium. Furthermore, intracellular protein was detected by flow cytometry, immunofluorescence staining, and Western blotting. Expression at both mRNA and de novo protein synthesis were detected, respectively, by RT-PCR and radiolabeling with (35)S. Ag effect was mimicked by cell treatment with anti-IgE Abs or Abs against FcepsilonRI and galectin-3 (FcepsilonRI>galectin-3), but not against FcepsilonRII. These observations represent a novel view of neutrophils as possible source of ECP in IgE-dependent diseases.

Inhibitory effects of N‐acetylcysteine on the functional responses of human eosinophils in vitro

Oxidative stress appears to be relevant in the pathogenesis of inflammation in allergic diseases like bronchial asthma. Eosinophils are oxidant-sensitive cells considered as key effectors in allergic inflammation. The aim of this work was to study the effects of the clinically used antioxidant N-acetyl-L-cysteine (NAC) on the functional responses of human-isolated eosinophils. Human eosinophils were purified from the blood of healthy donors by a magnetic bead separation system. The effects of NAC were investigated on the generation of reactive oxygen species (chemiluminescence and flow cytometry), Ca(2+) signal (fluorimetry), intracellular glutathione (GSH; flow cytometry), p47(phox)-p67(phox) translocation (Western blot) and eosinophil cationic protein (ECP) release (radioimmunoassay). NAC (0.1-1 mm) inhibited the extracellular generation of oxygen species induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and eotaxin (in the presence of IL-5) with -logIC(50) values of 3.61+/-0.03 and 3.36+/-0.09, respectively. Also, the intracellular generation of hydrogen peroxide was virtually abolished by NAC (0.5-1 mm). NAC (1 mm) did not alter the fMLP-induced Ca(2+) signal but augmented the eosinophil content of reduced GSH and inhibited p47(phox)-p67(phox) translocation. NAC inhibited the release of ECP ( approximately 90% inhibition at 1 mm) from fMLP-activated eosinophils. Inhibition by NAC of human eosinophil functions in vitro is potentially useful in the treatment of allergic inflammation.

Eosinophil activity in Schistosoma mansoni infections in vivo and in vitro in relation to plasma cytokine profile pre- and posttreatment with praziquantel.

Eosinophil activity in vivo and in vitro was studied in relation to infection intensities and plasma cytokine profiles of 51 Schistosoma mansoni-infected Ugandan fishermen before treatment and 24 h and 3 weeks posttreatment. Blood eosinophil numbers significantly declined 24 h posttreatment, but significant eosinophilia had developed by 3 weeks posttreatment. Cellular eosinophil cationic protein (ECP) content increased significantly during the transient eosinopenia but was significantly reduced 3 weeks later. No similar reduction in cellular eosinophil protein X (EPX) content was seen. Before treatment, S. mansoni infection intensity was positively correlated with 24-h boosts in plasma interleukin-5 (IL-5) and IL-6 levels, which were in turn negatively correlated with the posttreatment fall in eosinophil numbers. Significant correlations were observed between pretreatment infection intensities and plasma IL-10 and eotaxin levels. Treatment induced significant fluctuations in plasma IL-5, IL-6, IL-10, tumor necrosis factor alpha (TNF-alpha), and eotaxin levels. Optimal relative release of ECP and EPX in vitro was detected in S. mansoni soluble egg antigen-stimulated cultures during transient eosinopenia. Our data suggest that blood eosinophils are activated during S. mansoni infection and that treatment induces a burst in released antigens, causing increased production of IL-5, IL-6, IL-10, and eotaxin; a drop in TNF-alpha levels; and a transient sequestration of eosinophils, which leaves fewer degranulated eosinophils in the circulation 24 h posttreatment, followed by the development of eosinophilia 3 weeks later. During these events, it appears that preferential release of ECP occurs in vivo. Moreover, it is possible that infection intensity-dependent levels of plasma IL-10 may be involved in the prevention of treatment-induced anaphylactic reactions.

Steroids reduce local inflammatory mediator secretion and mucosal permeability in collagenous colitis patients

To study the effect of oral steroids upon clinical response and rectal mucosa secretion of eosinophil cationic protein (ECP), myeloperoxidase (MPO), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and albumin in patients with collagenous colitis (CC). A segmental perfusion technique was used to collect perfusates from rectum of CC patients once before and twice (one and four weeks) after the start of steroid treatment. Clinical data was monitored and ECP, MPO, bFGF, VEGF and albumin concentrations were analyzed by immunochemical methods in perfusates and in serum. Steroids reduced the number of bowel movements by more than five times within one week and all patients reported improved subjective well-being at wk 1 and 4. At the same time, the median concentrations of ECP, bFGF, VEGF and albumin in rectal perfusates decreased significantly. MPO values were above the detection limit in only 3 patients before treatment and in none during treatment. VEGF, bFGF, ECP and albumin concentrations correlated with each other with the exception of ECP and albumin. A decrease of serum ECP and VEGF concentrations was also seen even if the overtime reduction was not significant. Oral steroid treatment in CC patients induced a simultaneous reduction of bowel movements and rectal release of ECP, bFGF, VEGF and albumin, suggesting that these polypeptides and increased mucosal permeability are important components of the pathophysiology in collagenous colitis.