Diagnosing eosinophilic asthma using a multivariate prediction model based on blood granulocyte responsiveness.

BACKGROUND: The identification of inflammatory asthma phenotypes, using sputum analysis, has proven its value in diagnosis and disease monitoring. However due to technical limitations of sputum analysis, there is a strong need for fast and noninvasive diagnostics. This study included the activation state of eosinophils and neutrophils in peripheral blood to phenotype and monitor asthma. OBJECTIVES: To (i) construct a multivariable model using the activation state of blood granulocytes, (ii) compare its diagnostic value with sputum eosinophilia as gold standard and (iii) validate the model in an independent patient cohort. METHODS: Clinical parameters, activation of blood granulocytes and sputum characteristics were assessed in 115 adult patients with asthma (training cohort/Utrecht) and 34 patients (validation cohort/Oxford). RESULTS: The combination of blood eosinophil count, fractional exhaled nitric oxide, Asthma Control Questionnaire, medication use, nasal polyposis, aspirin sensitivity and neutrophil/eosinophil responsiveness upon stimulation with formyl-methionyl-leucyl phenylalanine was found to identify sputum eosinophilia with 90.5% sensitivity and 91.5% specificity in the training cohort and with 77% sensitivity and 71% specificity in the validation cohort (relatively high percentage on oral corticosteroids [OCS]). CONCLUSIONS: The proposed prediction model identifies eosinophilic asthma without the need for sputum induction. The model forms a noninvasive and externally validated test to assess eosinophilic asthma in patients not on OCS.

Steroids induce a disequilibrium of secreted interleukin-1 receptor antagonist and interleukin-1ß synthesis by human neutrophils.

Chronic obstructive pulmonary disease (COPD) is characterised by neutrophilic inflammation in the airways and these neutrophils contribute to the production of inflammatory mediators. Dampening the production of proinflammatory mediators might be an important strategy to treat COPD and glucocorticosteroids are known to do so via inhibition of nuclear factor-κB. However, this pathway is important for the control of pro- and anti-inflammatory genes. We studied the effects of dexamethasone on production and secretion of pro-inflammatory interleukin (IL)-1ß and anti-inflammatory secreted IL-1 receptor antagonist (sIL-1Ra) by human neutrophils activated with tumor necrosis factor (TNF)-α. In vitro, TNF-α-stimulated neutrophils produced significant amounts of IL-1ß and sIL-1Ra; this production was inhibited by dexamethasone. However, synthesis and secretion of sIL-1Ra was inhibited at lower concentrations dexamethasone compared to IL-1ß, which changed the IL-1ß:sIL-1Ra ratio significantly. This altered ratio resulted in a more pro-inflammatory condition, as visualised by increased intercellular adhesion molecule-1 expression on human endothelial cells. In vivo, moderate-to-severe COPD patients using inhaled glucocorticosteroids have decreased plasma sIL-Ra levels compared with mild-to-moderate patients not on glucocorticosteroid treatment. In conclusion, dexamethasone induces a pro-inflammatory shift in the IL-1ß:sIL-1Ra cytokine balance in neutrophils in vitro, which might contribute to a lack of endogenous anti-inflammatory signals to dampen inflammation in vivo.

Mechanisms involved in eosinophil migration. Platelet-activating factor-induced chemotaxis and interleukin-5-induced chemokinesis are mediated by different signals.

Eosinophils play an important role in the pathogenesis of allergic diseases such as allergic asthma. Eosinophil migration in vitro can be divided into directed migration, or chemotaxis, and random migration, or chemokinesis. Here, we studied intracellular signals involved in eosinophil migration in vitro induced by platelet-activating factor (PAF) and interleukin-5 (IL-5), applying a Boyden chamber assay. Migration induced by PAF (10(-11)-10(-6) M) largely consisted of chemotaxis with some chemokinesis, whereas IL-5 (10(-12)-10(-8) M) induced chemokinesis only. Eosinophils were depleted from intracellular and extracellular Ca2+ to study the role of Ca2+ as a second messenger. Ca2+ depletion did not change PAF-induced chemotaxis, however, IL-5-induced chemokinesis was inhibited. Interestingly, PAF, but not IL-5, induced changes in [Ca2+]i. This rise originated mainly from internal stores. Inhibition of protein kinase A by H-89 and protein kinase C by GF 109203X had no effect on both forms of eosinophil migration. Addition of the protein kinase inhibitor staurosporine significantly inhibited IL-5-induced chemokinesis. Inhibition of tyrosine kinases by herbimycin A completely blocked IL-5-induced chemokinesis. PAF and IL-5-induced actin polymerization was studied to compare migratory responses with a migration-associated intracellular response. Ca2+ depletion significantly enhanced PAF-induced (10(-8) M) actin polymerization, whereas IL-5-induced actin polymerization was not influenced. Addition of staurosporine led to an increase in F-actin. Subsequent addition of PAF or IL-5 resulted in an additive increase in F-actin content. In summary, both forms of eosinophil migration are protein kinase A and protein kinase C independent. In contrast to PAF-induced chemotaxis, Il-5-induced chemokinesis was found to be completely Ca2+ and tyrosine kinase dependent.

Human platelets secrete chemotactic activity for eosinophils.

Thrombin-stimulated platelets liberate factors that induce chemotaxis of eosinophils and raise their cytosolic Ca2+ content ([Ca2+]i). The sources of this activity are the dense- and alpha-granules because inhibition of prostaglandin endoperoxide/thromboxane A2 formation and the platelet-activating factor receptor-antagonist WEB 2086 have no effect. Platelets from patients with Storage-Pool Deficiency show about 60% of the normal chemotactic activity with little effect on [Ca2+]i, whereas completely degranulated platelets fail to affect eosinophils. In concentrations secreted by the platelets, adenosine diphosphate (ADP), and platelet factor 4 have no effect, whereas adenosine triphosphate (ATP) induces a strong chemotactic response and increases [Ca2+]i. However, apart from ATP other modulating factors must be involved as platelet releasates induce more chemotaxis than ATP alone. Thus, platelets secrete factors that activate eosinophils and may contribute to inflammatory and allergic processes.

RANTES- and interleukin-8-induced responses in normal human eosinophils: effects of priming with interleukin-5.

We report that responses of normal human eosinophils toward the chemokines RANTES and interleukin-8 (IL-8) are modulated and upregulated by priming with IL-5. In a modified Boyden chamber assay, we studied migratory responses toward the members of the chemokine family RANTES, monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1 alpha (MIP-1 alpha) (C-C subfamily), and IL-8, platelet factor-4 (PF-4), and neutrophil-activating peptide-2 (NAP-2) (C-x-C subfamily). These chemokines were also studied in terms of actin polymerization and ([Ca2+]i)-mobilizing properties, intracellular signals that are thought to play a role during migratory responses. We found that eosinophils showed significant migratory responses toward RANTES and IL-8 at concentrations of 10(-9) to 10(-7) mol/L only after priming with IL-5 (10 pmol/L). At these concentrations, PF-4, NAP-2, MCP-1, and MIP-1 alpha induced no significant migratory responses after priming. Unprimed eosinophils only showed a significant migratory response toward RANTES (10(-6) mol/L). Changes in [Ca2+]i were found after addition of RANTES, MIP-1 alpha, and NAP-2 (10 nmol/L) to unprimed eosinophils. RANTES (10(-9) to 10(-7) mol/L) significantly induced actin polymerization both in primed and unprimed eosinophils, whereas IL-8 (10(-9) to 10(-8) mol/L) and MIP-1 alpha (10(-8) mol/L) only induced actin polymerization after priming with IL-5. NAP-2, PF-4, and MCP-1 did not affect actin polymerization. These findings are further evidence for the hypothesis that cytokines like IL-5 and locally secreted chemokines like RANTES and IL-8 are both at the basis of specific eosinophil influx into the allergic inflammatory locus.

Modulation of eosinophil chemotaxis by interleukin-5.

Eosinophilia and eosinophil function are regulated by cytokines such as granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and interleukin-5 (IL-5). We have investigated the modulatory role of IL-5 on N-formyl-methionyl-leucyl-phenylalanine (FMLP), neutrophil-activating factor (NAF/IL-8), platelet factor 4 (PF4), and cytokine-induced chemotaxis of eosinophils from normal individuals. These eosinophils show a small chemotactic response toward PF4 but not to NAF/IL-8 and FMLP. Preincubation of eosinophils with low concentrations of IL-5 caused significantly increased responses toward PF4 and induced a significant chemotactic response toward FMLP and NAF/IL-8. In marked contrast, IL-5 (or IL-3) priming of eosinophils from normal donors resulted in a strong inhibition of GM-CSF-induced chemotaxis. A similar decrease in the chemotactic response toward GM-CSF was observed in eosinophils derived from allergic asthmatic individuals. This finding suggests that the latter eosinophils may have had a prior exposure to IL-5 (or IL-3). Washing of the cells after priming did not abrogate the inhibition of the GM-CSF response. Our data indicate that at low concentrations IL-5 is an important modulator of eosinophil chemotaxis, causing selective upregulation or downregulation of chemotactic responses toward different agents.

Analysis of signal transduction pathways in human eosinophils activated by chemoattractants and the T-helper 2-derived cytokines interleukin-4 and interleukin-5.

Activation and recruitment of eosinophils in allergic inflammation is in part mediated by chemoattractants and T-helper 2 (Th2)-derived cytokines. However, little is known concerning the signal transduction mechanisms by which this activation occurs. We have investigated tyrosine kinase-mediated activation of phosphatidylinositol 3-kinase (PI3K) and compared this with the activation of the p21ras-ERK signaling pathway in human eosinophils. The related cytokines interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF), all induced PI3K activity detected in antiphosphotyrosine immunoprecipitates. Furthermore, the chemoattractants platelet-activating factor (PAF), RANTES, and C5a were also able to induce phosphotyrosine-associated PI3K activity. Protein kinase B (PKB) is a downstream target of PI3K activation by growth factors. Induction of PKB phosphorylation in human eosinophils was transiently induced on activation with the cytokines IL-4 and IL-5, as well as the chemoattractants PAF, C5a, and RANTES showing a broad activation profile. Surprisingly, analysis of the activation of the mitogen-activated protein (MAP) kinases p44(ERK1) and p42(ERK2), showed that ERK2, but not ERK1, was transiently activated in human eosinophils after stimulation with IL-5 or PAF. Activation kinetics correlated with activation of p21ras by both cytokines and chemoattractants as measured by a novel assay for guanosine triphosphate (GTP)-loading. Finally, using specific inhibitors of both the p21ras-ERK and PI3K signaling pathways, a role was demonstrated for PI3K, but not p21ras-ERK, in activation of the serum-treated zymosan (STZ)-mediated respiratory burst in IL-5 and PAF-primed eosinophils. In summary, these data show that in human eosinophils, Th2-derived cytokines differentially activate both PI3K and MAP kinase signal transduction pathways with distinct functional consequences showing complex regulation of eosinophil effector functions.

Human eosinophils constitutively express a functional interleukin-4 receptor: interleukin-4 -induced priming of chemotactic responses and induction of PI-3 kinase activity.

Similar to interleukin-3 (IL-3), IL-5, and granulocyte macrophage colony-stimulating factor (GM-CSF), IL-4 can be secreted by several cell types involved in allergic inflammatory reactions, and therefore can affect eosinophil function similarly. In this study, we investigated the presence of an IL-4 receptor (IL-4R) on human eosinophils. When two different monoclonal antibodies (mAbs) against the IL-4R alpha-chain (IL-4Ralpha) were used, fluorescent-activated cell sorter analysis revealed the presence of an IL-4Ralpha on both eosinophils of normal donors and atopic dermatitis patients. In addition, the expression of the IL-2R gamma-chain, a functional component of the IL-4R in some cell types, was demonstrated. The IL-4Ralpha appeared to be expressed constitutively, and stimulation with cytokines IL-2, IL-3, IL-5, GM-CSF, and interferon-gamma did not further increase IL-4Ralpha expression. Evidence for an IL-4Ralpha was further substantiated by mRNA analysis. Both Northern blot analysis and reverse transcriptase/polymerase chain reaction revealed the presence of mRNA for the IL-4Ralpha in eosinophils from normal individuals and AD patients. Furthermore, we demonstrated that both IL-4 and IL-13 were capable of inducing PI-3 kinase activity in human eosinophils. Because this activation could be inhibited by an IL-4Ralpha mAb, we conclude that both cytokines can activate human eosinophils through binding to a receptor complex comprising the IL-4Ralpha and-yet to be identified-associated proteins. In addition, the involvement of IL-4 in functional responses was studied. IL-4 appeared to "prime" eosinophils to respond chemotactically toward regulated on activation, normal T cells expressed and secreted, but did not affect platelet-activating factor-induced chemotaxis. Taken together, these data show the presence of a functional IL-4R on human eosinophils.

Eosinophil chemotactic activity in bronchoalveolar lavage from idiopathic pulmonary fibrosis is dependent on cytokine priming of eosinophils.

Increased numbers of eosinophils have been found in bronchoalveolar lavage (BAL) fluid obtained from patients with idiopathic pulmonary fibrosis (IPF). This suggests the presence of one or more cytokines in the lung tissue of patients with IPF, which are involved in the induction of migration of eosinophils towards the pulmonary compartment. To evaluate this hypothesis, we studied migratory responses of blood eosinophils towards BAL fluid. Migratory responses were tested by means of a modified Boyden chamber assay in 21 patients with IPF and 14 healthy controls. Experiments were performed with unprimed eosinophils and in vitro primed eosinophils (preincubated with 10(-11) M granulocyte macrophage-colony-stimulating factor). Changes in intracellular free Ca2+ ([Ca2+]i) in eosinophils in response to BAL fluid were also investigated, to characterize putative chemotaxins further. Chemotactic responses of eosinophils were observed towards BAL fluid from both patients with IPF and controls, provided that the eosinophils were primed. No changes in [Ca2+]i in eosinophils were detected in response to BAL fluid. Furthermore, neither a blocking antibody against interleukin-8 nor one against regulated on activation, normal T-cell, expressed and secreted (RANTES) influenced the response. Since a chemotactic response of in vitro primed eosinophils was also observed towards bronchoalveolar lavage fluid from normals, it was concluded, that in idiopathic pulmonary fibrosis, apart from the presence of a chemotactic factor in the lung tissue, other mechanisms such as priming of eosinophils in the peripheral blood are responsible for the extravasation of eosinophils into the pulmonary compartment. As no changes in [Ca2+]i were observed in the eosinophils after incubation with bronchoalveolar lavage fluid, the chemotaxin responsible for the migratory responses is probably not one of the known eosinophil-activating chemokines.

Tyrosine phosphorylation-dependent activation of phosphatidylinositide 3-kinase occurs upstream of Ca2+-signalling induced by Fcgamma receptor cross-linking in human neutrophils.

The effect of wortmannin on IgG-receptor (FcgammaR)-mediated stimulation of human neutrophils was investigated. The Ca2+ influx induced by clustering of both Fcgamma receptors was inhibited by wortmannin, as was the release of Ca2+ from intracellular stores. Wortmannin also inhibited, with the same efficacy, the accumulation of Ins(1,4,5)P3 observed after FcgammaR stimulation, but did not affect the increase in Ins(1,4,5)P3 induced by the chemotactic peptide, formyl-methionine-leucine-phenylalanine. Because wortmannin is, in the concentrations used here, an inhibitor of PtdIns 3-kinase, these results suggested a role for PtdIns 3-kinase upstream of Ca2+ signalling, induced by FcgammaR cross-linking. Support for this notion was obtained by investigating the effect of another inhibitor of PtdIns 3-kinase, LY 294002, and by studying the kinetics of PtdIns 3-kinase activation. We found translocation of PtdIns 3-kinase to the plasma membrane and increased PtdIns 3-kinase activity in the membrane as soon as 5 s after FcgammaR cross-linking, even before the onset of the Ca2+ response. Moreover, the translocation of PtdIns 3-kinase to the plasma membrane was inhibited by co-cross-linking of either FcgammaRIIa and FcgammaRIIIb with the tyrosine phosphatase, CD45, indicating a requirement for protein tyrosine phosphorylation in the recruitment of PtdIns 3-kinase to the plasma membrane. Taken together, our results suggest a role for PtdIns 3-kinase in early signal transduction events after FcgammaR cross-linking in human neutrophils.

Eosinophil priming by cytokines: from cellular signal to in vivo modulation.

Eosinophils play an important role in the effector phase of allergic inflammation. This review will focus on the conversion of the unprimed eosinophil phenotype in the peripheral blood of normal individuals to the primed phenotype found in the peripheral blood and tissues of allergic patients, a phenomenon called priming. Recent data on the signals initiated after cytokine receptor activation on eosinophils will be reviewed.

Comparison of the roles of mitogen-activated protein kinase kinase and phosphatidylinositol 3-kinase signal transduction in neutrophil effector function.

Although it is known that many stimuli can activate mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinases (PI3K) in human neutrophils, little is known concerning either the mechanisms or function of this activation. We have utilized a selective inhibitor of MAPK kinase (MEK), PD098059, and two inhibitors of PI3K, wortmannin and LY294002, to investigate the roles of these kinases in the regulation of neutrophil effector functions. Granulocyte/macrophage colony-stimulating factor, platelet-activating factor (PAF) and N-formylmethionyl-leucyl-phenylalanine are capable of activating both p44ERK1 and p42ERK2 MAPKs and phosphotyrosine-associated PI3K in human neutrophils. The activation of extracellular signal-related protein kinases (ERKs) is correlated with the activation of p21ras by both tyrosine kinase and G-protein-coupled receptors as measured by a novel assay for GTP loading. Wortmannin and LY294002 inhibit, to various degrees, superoxide generation, neutrophil migration and PAF release. Incubation with PD098059, however, inhibits only the PAF release stimulated by serum-treated zymosan. This demonstrates that, while neither MEK nor ERK kinases are involved in the activation of respiratory burst or neutrophil migration, inhibition of PAF release suggests a potential role in the activation of cytosolic phospholipase A2. PI3K isoforms, however, seem to have a much wider role in regulating neutrophil functioning.