Publications by authors named "Eva M Sturm"

29 Publications

  • Page 1 of 1

The anti-parasitic drug miltefosine suppresses human eosinophil activation and ameliorates murine allergic inflammation in vivo.

Br J Pharmacol 2021 Mar 2;178(5):1234-1248. Epub 2021 Feb 2.

Division of Pharmacology, Otto Loewi Research Center, Medical University of Graz, Graz, Austria.

Background And Purpose: Miltefosine is an alkylphosphocholine drug with proven effectiveness against various types of parasites and cancer cells. Miltefosine is not only able to induce direct parasite killing but also modulates host immunity, for example by reducing the severity of allergies in patients. To date, there are no reports on the effect of miltefosine on eosinophils, central effector cells involved in allergic inflammation.

Experimental Approach: We tested the effect of miltefosine on the activation of human eosinophils and their effector responses in vitro and in mouse models of eosinophilic migration and ovalbumin-induced allergic lung inflammation.

Key Results: The addition of miltefosine suppressed several eosinophilic effector reactions such as CD11b up-regulation, degranulation, chemotaxis and downstream signalling. Miltefosine significantly reduced the infiltration of immune cells into the respiratory tract of mice in an allergic cell recruitment model. Finally, in a model of allergic inflammation, treatment with miltefosine resulted in an improvement of lung function parameters.

Conclusion And Implications: Our observations suggest a strong modulatory activity of miltefosine in the regulation of eosinophilic inflammation in vitro and in vivo. Our data underline the potential efficacy of miltefosine in the treatment of allergic diseases and other eosinophil-associated disorders and may raise important questions regarding the immunomodulatory effect of miltefosine in patients treated for leishmania infections.
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http://dx.doi.org/10.1111/bph.15368DOI Listing
March 2021

Lysophosphatidylcholines inhibit human eosinophil activation and suppress eosinophil migration in vivo.

Biochim Biophys Acta Mol Cell Biol Lipids 2020 07 11;1865(7):158686. Epub 2020 Mar 11.

Division of Pharmacology, Otto Loewi Research Center, Medical University of Graz, Universitätsplatz 4, 8010 Graz, Austria; BioTechMed-Graz, Graz, Austria. Electronic address:

Eosinophils are important multifaceted effector cells involved in allergic inflammation. Following allergen challenge, eosinophils and other immune cells release secreted phospholipases, generating lysophosphatidylcholines (LPCs). LPCs are potent lipid mediators, and serum levels of LPCs associate with asthma severity, suggesting a regulatory activity of LPCs in asthma development. As of yet, the direct effects of LPCs on eosinophils remain unclear. In the present study, we tested the effects of the major LPC species (16:0, 18:0 and 18:1) on eosinophils isolated from healthy human donors. Addition of saturated LPCs in the presence of albumin rapidly disrupted cholesterol-rich nanodomains on eosinophil cell membranes and suppressed multiple eosinophil effector responses, such as CD11b upregulation, degranulation, chemotaxis, and downstream signaling. Furthermore, we demonstrate in a mouse model of allergic cell recruitment, that LPC treatment markedly reduces immune cell infiltration into the lungs. Our observations suggest a strong modulatory activity of LPCs in the regulation of eosinophilic inflammation in vitro and in vivo.
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http://dx.doi.org/10.1016/j.bbalip.2020.158686DOI Listing
July 2020

Stearoylethanolamide interferes with retrograde endocannabinoid signalling and supports the blood-brain barrier integrity under acute systemic inflammation.

Biochem Pharmacol 2020 04 25;174:113783. Epub 2019 Dec 25.

Otto-Loewi Research Center for Vascular Biology, Immunology and Inflammation, Division of Pharmacology, Medical University Graz, Universitätsplatz 4, 8010 Graz, Austria. Electronic address:

Neuroinflammation plays a prominent role in the onset of demyelinating diseases, major depressive disorder and delayed neurodegeneration. An open question remains whether pharmacological suppression of inflammation can effectively reduce the progression of these states. Bioactive lipid mediators such as N-acylethanolamines (NAEs) have an anti-inflammatory activity and are of pharmacological interest due to their endogenous on-demand production and the existence of distinct biological targets in humans and animals. Here we demonstrate for the first time, that treatment with stearoylethanolamide (SEA), a prevailing endogenously formed NAE, is neuroprotective against LPS-induced neuroinflammation in C57BL/6 male mice. SEA restricted the spreading of peripheral inflammation to the brain, and averted the activation of resident microglia and leukocyte trafficking to the brain parenchyma. Treatment with SEA per se increased the neuronal expression of cannabinoid receptors CB and brain levels of the most potent endogenous CB agonist 2-arachidonoylglycerol in vivo. SEA enhanced the amplitude of synaptic vesicle release, supported the balanced signal-to-noise ratio in glutamate- and GABAergic neurotransmission and decreased the excitotoxic risk associated with higher extracellular glutamate levels under neuroinflammation. The interference of SEA with the endocannabinoid system and presynaptic neurotransmitter release may represent an intrinsic neuroprotective mechanism that is triggered by inflammation and glutamate excitotoxicity. Thus, our data allows to consider SEA for the preventive therapy of acute and late-onset neuroinflammation-associated synaptic dysfunction and neurodegeneration.
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http://dx.doi.org/10.1016/j.bcp.2019.113783DOI Listing
April 2020

Apolipoprotein A-IV acts as an endogenous anti-inflammatory protein and is reduced in treatment-naïve allergic patients and allergen-challenged mice.

Allergy 2020 02 10;75(2):392-402. Epub 2019 Sep 10.

Division of Pharmacology, Otto-Loewi Research Center for Vascular Biology, Immunology and Inflammation, Medical University of Graz, Graz, Austria.

Background: Recent studies pointed to a crucial role for apolipoproteins in the pathogenesis of inflammatory diseases. However, the role of apolipoprotein-IV (ApoA-IV) in allergic inflammation has not been addressed thoroughly thus far.

Objective: Here, we explored the anti-inflammatory effects and underlying signaling pathways of ApoA-IV on eosinophil effector function in vitro and in vivo.

Methods: Migratory responsiveness, Ca -flux and apoptosis of human peripheral blood eosinophils were assessed in vitro. Allergen-driven airway inflammation was assessed in a mouse model of acute house dust mite-induced asthma. ApoA-IV serum levels were determined by ELISA.

Results: Recombinant ApoA-IV potently inhibited eosinophil responsiveness in vitro as measured by Ca -flux, shape change, integrin (CD11b) expression, and chemotaxis. The underlying molecular mechanism involved the activation of Rev-ErbA-α and induced a PI3K/PDK1/PKA-dependent signaling cascade. Systemic application of ApoA-IV prevented airway hyperresponsiveness (AHR) and airway eosinophilia in mice following allergen challenge. ApoA-IV levels were decreased in serum from allergic patients compared to healthy controls.

Conclusion: Our data suggest that ApoA-IV is an endogenous anti-inflammatory protein that potently suppresses effector cell functions in eosinophils. Thus, exogenously applied ApoA-IV may represent a novel pharmacological approach for the treatment of allergic inflammation and other eosinophil-driven disorders.
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http://dx.doi.org/10.1111/all.14022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065107PMC
February 2020

Allergic rhinitis is associated with complex alterations in high-density lipoprotein composition and function.

Biochim Biophys Acta Mol Cell Biol Lipids 2019 10 8;1864(10):1280-1292. Epub 2019 Jun 8.

Division of Pharmacology, Otto Loewi Research Center for Vascular Biology, Immunology and Inflammation, Medical University of Graz, Universitätsplatz 4, 8010 Graz, Austria.; BioTechMed Graz, Mozartgasse 12/II, 8010 Graz, Austria. Electronic address:

Despite strong evidence that high-density lipoproteins (HDLs) modulate the immune response, the role of HDL in allergies is still poorly understood. Many patients with allergic rhinitis (AR) develop a late-phase response, characterized by infiltration of monocytes and eosinophils into the nasal submucosa. Functional impairment of HDL in AR-patients may insufficiently suppress inflammation and cell infiltration, but the effect of AR on the composition and function of HDL is not understood. We used apolipoprotein (apo) B-depleted serum as well as isolated HDL from AR-patients (n = 43) and non-allergic healthy controls (n = 20) for detailed compositional and functional characterization of HDL. Both AR-HDL and apoB-depleted serum of AR-patients showed decreased anti-oxidative capacity and impaired ability to suppress monocyte nuclear factor-κB expression and pro-inflammatory cytokine secretion, such as interleukin (IL)-4, IL-6, IL-8, tumor necrosis factor alpha and IL-1 beta. Sera of AR-patients showed decreased paraoxonase and cholesteryl-ester transfer protein activities, increased lipoprotein-associated phospholipase A2 activity, while lecithin-cholesterol acyltransferase activity and cholesterol efflux capacity were not altered. Surprisingly, apoB-depleted serum and HDL from AR-patients showed an increased ability to suppress eosinophil effector responses upon eotaxin-2/CCL24 stimulation. Mass spectrometry and biochemical analyses showed reduced levels of apoA-I and phosphatidylcholine, but increased levels of apoA-II, triglycerides and lyso-phosphatidylcholine in AR-HDL. The changes in AR-HDL composition were associated with altered functional properties. In conclusion, AR alters HDL composition linked to decreased anti-oxidative and anti-inflammatory properties but improves the ability of HDL to suppress eosinophil effector responses.
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http://dx.doi.org/10.1016/j.bbalip.2019.06.007DOI Listing
October 2019

Butyrate ameliorates allergic airway inflammation by limiting eosinophil trafficking and survival.

J Allergy Clin Immunol 2019 09 11;144(3):764-776. Epub 2019 May 11.

Otto Loewi Research Center for Vascular Biology, Immunology and Inflammation, Division of Pharmacology, Medical University of Graz, Graz, Austria; BioTechMed-Graz, Graz, Austria. Electronic address:

Background: Lung eosinophilia is a hallmark of asthma, and eosinophils are believed to play a crucial role in the pathogenesis of allergic inflammatory diseases. Short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, are produced in high amounts in the gastrointestinal tract by commensal bacteria and can be absorbed into the bloodstream. Although there is recent evidence that SCFAs are beneficial in allergic asthma models, the effect on eosinophils has remained elusive.

Objective: The role of SCFAs was investigated in human eosinophil function and a mouse model of allergic asthma.

Methods: Eosinophils were purified from self-reported allergic or healthy donors. Migration, adhesion to the endothelium, and eosinophil survival were studied in vitro. Ca flux, apoptosis, mitochondrial membrane potential, and expression of surface markers were determined by using flow cytometry and in part by using real-time PCR. Allergic airway inflammation was assessed in vivo in an ovalbumin-induced asthma model by using invasive spirometry.

Results: For the first time, we observed that SCFAs were able to attenuate human eosinophils at several functional levels, including (1) adhesion to the endothelium, (2) migration, and (3) survival. These effects were independent from GPR41 and GPR43 but were accompanied by histone acetylation and mimicked by trichostatin A, a pan-histone deacetylase inhibitor. In vivo butyrate ameliorated allergen-induced airway and lung eosinophilia, reduced type 2 cytokine levels in bronchial fluid, and improved airway hyperresponsiveness in mice.

Conclusion: These in vitro and in vivo findings highlight the importance of SCFAs, especially butyrate as a promising therapeutic agent in allergic inflammatory diseases.
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http://dx.doi.org/10.1016/j.jaci.2019.05.002DOI Listing
September 2019

Involvement of EP2 and EP4 Receptors in Eosinophilic Esophagitis: A Pilot Study.

Dig Dis Sci 2019 10 15;64(10):2806-2814. Epub 2019 Apr 15.

Otto Loewi Research Center, Divison of Pharmacology, Medical University of Graz, Universitätsplatz 4, 8010, Graz, Austria.

Background: The prostaglandin D receptor DP2 has been implicated in eosinophil infiltration and the development of eosinophilic esophagitis (EoE).

Aims And Methods: In this study, we investigated an involvement of PGE (EP1-EP4) and PGD (DP1) receptors in EoE by measuring their expression in peripheral blood eosinophils and esophageal mucosal biopsies of EoE patients and by performing migration and adhesion assays with eosinophils from healthy donors.

Results: Expression of EP2 and EP4, but not EP1 and EP3, was decreased in blood eosinophils of patients with EoE vs. control subjects. Adhesion of eosinophils to esophageal epithelial cells was decreased by EP2 receptor agonist butaprost and EP4 agonist ONO-AE1-329, whereas DP1 agonist BW245C increased adhesion. In chemotaxis assays with supernatant from human esophageal epithelial cells, only ONO-AE1-329 but not butaprost or BW245C inhibited the migration of eosinophils. Expression of EP and DP receptors in epithelial cells and eosinophils was detected in sections of esophageal biopsies from EoE patients by immunohistochemistry. qPCR of biopsies from EoE patients revealed that gene expression of EP4 and DP1 was the highest among PGE and PGD receptors. Esophageal epithelial cells in culture showed high gene expression for EP2 and EP4. Activation of EP2 and EP4 receptors decreased barrier integrity of esophageal epithelial cells in impedance assays.

Conclusions: Activation of EP2 and EP4 receptors may inhibit eosinophil recruitment to the esophageal mucosa. However, their activation could negatively affect esophageal barrier integrity suggesting that eosinophilic rather than epithelial EP2 and EP4 have a protective role in EoE.
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http://dx.doi.org/10.1007/s10620-019-05623-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6744386PMC
October 2019

Modulation of neurosecretion and approaches for its multistep analysis.

Biochim Biophys Acta Gen Subj 2018 12 6;1862(12):2701-2713. Epub 2018 Aug 6.

The Department of Neurochemistry, Palladin Institute of Biochemistry, NAS of Ukraine, 9 Leontovycha Street, 01030 Kyiv, Ukraine. Electronic address:

Background: Neurosecretion is the multistep process occurring in separate spatial and temporal cellular boundaries which complicates its comprehensive analysis. Most of the research are focused on one distinct stage of synaptic vesicle recycling. Here, we describe approaches for complex analysis of synaptic vesicle (SV) endocytosis and separate steps of exocytosis at the level of presynaptic bouton and highly purified SVs.

Methods: Proposed fluorescence-based strategies and analysis of neurotransmitter transport provided the advantages in studies of exocytosis steps. We evaluated SV docking/tethering, their Ca-dependent fusion and release of neurotransmitters gamma-aminobutyric acid (GABA) and glutamate in two animal models.

Results: Approaches enabled us to study: 1) endocytosis/Ca-dependent release of fluorescent carbon nanodots (CNDs) during stimulation of nerve terminals; 2) the action of levetiracetam, modulator of SV glycoprotein SV2, on fusion competence of SVs and stimulated release of GABA and glutamate; 3) impairments of several steps of neurosecretion under vitamin D deficiency.

Conclusions: Our algorithm enabled us to verify the method validity for multidimensional analysis of SV turnover. By increasing SV docking and the size of readily releasable pool (RRP), levetiracetam is able to selectively enhance the stimulated GABA secretion in hippocampal neurons. Findings suggest that SV2 regulates RRP through impact on the number of docked/primed SVs.

General Significance: Methodology can be widely applied to study the stimulated neurosecretion in presynapse, regulation of SV docking, their Ca-dependent fusion with target membranes, quantitative analysis of expression of neuron-specific proteins, as well as for testing the efficiency of pre-selected designed neuroactive substances.
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http://dx.doi.org/10.1016/j.bbagen.2018.08.004DOI Listing
December 2018

DP1 receptor signaling prevents the onset of intrinsic apoptosis in eosinophils and functions as a transcriptional modulator.

J Leukoc Biol 2018 07 1;104(1):159-171. Epub 2018 Apr 1.

Otto Loewi Research Center for Vascular Biology, Immunology and Inflammation, Division of Pharmacology, Medical University of Graz, Graz, Austria.

Prostaglandin (PG) D is the ligand for the G-protein coupled receptors DP1 (D-type prostanoid receptor 1) and DP2 (also known as chemoattractant receptor homologous molecule, expressed on Th2 cells; CRTH2). Both, DP1 and DP2 are expressed on the cellular surface of eosinophils; although it has become quite clear that PGD induces eosinophil migration mainly via DP2 receptors, the role of DP1 in eosinophil responses has remained elusive. In this study, we addressed how DP1 receptor signaling complements the pro-inflammatory effects of DP2. We found that PGD prolongs the survival of eosinophils via a DP1 receptor-mediated mechanism that inhibits the onset of the intrinsic apoptotic cascade. The DP1 agonist BW245c prevented the activation of effector caspases in eosinophils and protected mitochondrial membranes from depolarization which-as a consequence-sustained viability of eosinophils. DP1 activation in eosinophils enhanced the expression of the anti-apoptotic gene BCL-X , but also induced pro-inflammatory genes, such as VLA-4 and CCR3. In HEK293 cells that overexpress recombinant DP1 and/or DP2 receptors, activation of DP1, but not DP2, delayed cell death and stimulated proliferation, along with induction of serum response element (SRE), a regulator of anti-apoptotic, early-response genes. We conclude that DP1 receptors promote the survival via SRE induction and induction of pro-inflammatory genes. Therefore, targeting DP1 receptors, along with DP2, may contribute to anti-inflammatory therapy in eosinophilic diseases.
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http://dx.doi.org/10.1002/JLB.3MA1017-404RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6032830PMC
July 2018

G protein-coupled receptor GPR55 promotes colorectal cancer and has opposing effects to cannabinoid receptor 1.

Int J Cancer 2018 01 21;142(1):121-132. Epub 2017 Sep 21.

Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Graz, Austria.

The putative cannabinoid receptor GPR55 has been shown to play a tumor-promoting role in various cancers, and is involved in many physiological and pathological processes of the gastrointestinal (GI) tract. While the cannabinoid receptor 1 (CB ) has been reported to suppress intestinal tumor growth, the role of GPR55 in the development of GI cancers is unclear. We, therefore, aimed at elucidating the role of GPR55 in colorectal cancer (CRC), the third most common cancer worldwide. Using azoxymethane (AOM)- and dextran sulfate sodium (DSS)-driven CRC mouse models, we found that GPR55 plays a tumor-promoting role that involves alterations of leukocyte populations, i.e. myeloid-derived suppressor cells and T lymphocytes, within the tumor tissues. Concomitantly, expression levels of COX-2 and STAT3 were reduced in tumor tissue of GPR55 knockout mice, indicating reduced presence of tumor-promoting factors. By employing the experimental CRC models to CB knockout and CB /GPR55 double knockout mice, we can further show that GPR55 plays an opposing role to CB . We report that GPR55 and CB mRNA expression are differentially regulated in the experimental models and in a cohort of 86 CRC patients. Epigenetic methylation of CNR1 and GPR55 was also differentially regulated in human CRC tissue compared to control samples. Collectively, our data suggest that GPR55 and CB play differential roles in colon carcinogenesis where the former seems to act as oncogene and the latter as tumor suppressor.
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http://dx.doi.org/10.1002/ijc.31030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5679368PMC
January 2018

Prostaglandins and Their Receptors in Eosinophil Function and As Therapeutic Targets.

Front Med (Lausanne) 2017 19;4:104. Epub 2017 Jul 19.

Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Graz, Austria.

Of the known prostanoid receptors, human eosinophils express the prostaglandin D (PGD) receptors DP1 [also D-type prostanoid (DP)] and DP2 (also chemoattractant receptor homologous molecule, expressed on Th2 cells), the prostaglandin E receptors EP2 and EP4, and the prostacyclin (PGI) receptor IP. Prostanoids can bind to either one or multiple receptors, characteristically have a short half-life , and are quickly degraded into metabolites with altered affinity and specificity for a given receptor subtype. Prostanoid receptors signal mainly through G proteins and naturally activate signal transduction pathways according to the G protein subtype that they preferentially interact with. This can lead to the activation of sometimes opposing signaling pathways. In addition, prostanoid signaling is often cell-type specific and also the combination of expressed receptors can influence the outcome of the prostanoid impulse. Accordingly, it is assumed that eosinophils and their (patho-)physiological functions are governed by a sensitive prostanoid signaling network. In this review, we specifically focus on the functions of PGD, PGE, and PGI and their receptors on eosinophils. We discuss their significance in allergic and non-allergic diseases and summarize potential targets for drug intervention.
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http://dx.doi.org/10.3389/fmed.2017.00104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5515835PMC
July 2017

Eosinophils Contribute to Intestinal Inflammation via Chemoattractant Receptor-homologous Molecule Expressed on Th2 Cells, CRTH2, in Experimental Crohn's Disease.

J Crohns Colitis 2016 Sep 29;10(9):1087-95. Epub 2016 Feb 29.

Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Graz, Austria

Background And Aims: Prostaglandin [PG] D2 activates two receptors, DP and CRTH2. Antagonism of CRTH2 has been shown to promote anti-allergic and anti-inflammatory effects. We investigated whether CRTH2 may play a role in Crohn's disease [CD], focusing on eosinophils which are widely present in the inflamed mucosa of CD patients and express both receptors.

Methods: Using the 2,4,6-trinitrobenzenesulfonic acid [TNBS]-induced colitis model, involvement of CRTH2 in colitis was investigated by pharmacological antagonism, immunohistochemistry, Western blotting, immunoassay, and leukocyte recruitment. Chemotactic assays were performed with isolated human eosinophils. Biopsies and serum samples of CD patients were examined for presence of CRTH2 and ligands, respectively.

Results: High amounts of CRTH2-positive cells, including eosinophils, are present in the colonic mucosa of mice with TNBS colitis and in human CD. The CRTH2 antagonist OC-459, but not the DP antagonist MK0524, reduced inflammation scores and decreased TNF-α, IL-1β, and IL-6 as compared with control mice. OC-459 inhibited recruitment of eosinophils into the colon and also inhibited CRTH2-induced chemotaxis of human eosinophils in vitro. Eosinophil-depleted ΔdblGATA knockout mice were less sensitive to TNBS-induced colitis, whereas IL-5 transgenic mice with lifelong eosinophilia were more severely affected than wild types. In addition, we show that serum levels of PGD2 and Δ(12)-PGJ2 were increased in CD patients as compared with control individuals.

Conclusions: CRTH2 plays a pro-inflammatory role in TNBS-induced colitis. Eosinophils contribute to the severity of the inflammation, which is improved by a selective CRTH2 antagonist. CRTH2 may, therefore, represent an important target in the pharmacotherapy of CD.
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http://dx.doi.org/10.1093/ecco-jcc/jjw061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4892354PMC
September 2016

Phosphoinositide-dependent protein kinase 1 (PDK1) mediates potent inhibitory effects on eosinophils.

Eur J Immunol 2015 May 27;45(5):1548-59. Epub 2015 Feb 27.

Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Graz, Austria.

Prostaglandin E2 (PGE2 ) protects against allergic responses via binding to prostanoid receptor EP4, which inhibits eosinophil migration in a PI3K/PKC-dependent fashion. The phosphoinositide-dependent protein kinase 1 (PDK1) is known to act as a downstream effector in PI3K signaling and has been implicated in the regulation of neutrophil migration. Thus, here we elucidate whether PDK1 mediates inhibitory effects of E-type prostanoid receptor 4 (EP4) receptors on eosinophil function. Therefore, eosinophils were isolated from human peripheral blood or differentiated from mouse BM. PDK1 signaling was investigated in shape change, chemotaxis, CD11b, respiratory burst, and Ca(2+) mobilization assays. The specific PDK1 inhibitors BX-912 and GSK2334470 prevented the inhibition by prostaglandin E2 and the EP4 agonist ONO-AE1-329. Depending on the cellular function, PDK1 seemed to act through PI3K-dependent or PI3K-independent mechanisms. Stimulation of EP4 receptors caused PDK1 phosphorylation at Ser396 and induced PI3K-dependent nuclear translocation of PDK1. EP4-induced inhibition of shape change and chemotaxis was effectively reversed by the Akt inhibitor triciribine. In support of this finding, ONO-AE1-329 induced a PI3K/PDK1-dependent increase in Akt phosphorylation. In conclusion, our data illustrate a critical role for PDK1 in transducing inhibitory signals on eosinophil effector function. Thus, our results suggest that PDK1 might serve as a novel therapeutic target in diseases involving eosinophilic inflammation.
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http://dx.doi.org/10.1002/eji.201445196DOI Listing
May 2015

Opposing roles of prostaglandin D2 receptors in ulcerative colitis.

J Immunol 2014 Jul 13;193(2):827-39. Epub 2014 Jun 13.

Institute of Experimental and Clinical Pharmacology, Medical University of Graz, 8010 Graz, Austria;

Proresolution functions were reported for PGD2 in colitis, but the role of its two receptors, D-type prostanoid (DP) and, in particular, chemoattractant receptor homologous molecule expressed on Th2 cells (CRTH2), is less well defined. We investigated DP and CRTH2 expression and function during human and murine ulcerative colitis (UC). Expression of receptors was measured by flow cytometry on peripheral blood leukocytes and by immunohistochemistry and immunoblotting in colon biopsies of patients with active UC and healthy individuals. Receptor involvement in UC was evaluated in a mouse model of dextran sulfate sodium colitis. DP and CRTH2 expression changed in leukocytes of patients with active UC in a differential manner. In UC patients, DP showed higher expression in neutrophils but lower in monocytes as compared with control subjects. In contrast, CRTH2 was decreased in eosinophils, NK, and CD3(+) T cells but not in monocytes and CD3(+)/CD4(+) T cells. The decrease of CRTH2 on blood eosinophils clearly correlated with disease activity. DP correlated positively with disease activity in eosinophils but inversely in neutrophils. CRTH2 internalized upon treatment with PGD2 and 11-dehydro TXB2 in eosinophils of controls. Biopsies of UC patients revealed an increase of CRTH2-positive cells in the colonic mucosa and high CRTH2 protein content. The CRTH2 antagonist CAY10595 improved, whereas the DP antagonist MK0524 worsened inflammation in murine colitis. DP and CRTH2 play differential roles in UC. Although expression of CRTH2 on blood leukocytes is downregulated in UC, CRTH2 is present in colon tissue, where it may contribute to inflammation, whereas DP most likely promotes anti-inflammatory actions.
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http://dx.doi.org/10.4049/jimmunol.1303484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4121674PMC
July 2014

Sensitization to Hymenoptera venoms is common, but systemic sting reactions are rare.

J Allergy Clin Immunol 2014 Jun 22;133(6):1635-43.e1. Epub 2013 Dec 22.

Department of Dermatology, Division of Environmental Dermatology and Venerology, Medical University of Graz, Graz, Austria.

Background: Sensitization to Hymenoptera venom without systemic sting reactions (SSRs) is commonly observed in the general population. Clinical relevance for a future sting has not yet been investigated.

Objective: We aimed to evaluate the effect of these debatable sensitizations with deliberate sting challenges and to monitor serologic changes for up to 2 years.

Methods: One hundred thirty-one challenges with bees and wasps were performed in 94 subjects with a hitherto irrelevant sensitization. The clinical outcome was recorded, and results of specific IgE (sIgE) determinations, skin tests, and basophil activation tests were correlated to the sting reaction. sIgE levels were monitored in reactors and nonreactors after 3 hours, 1 week, 4 weeks, and 1 year.

Results: Only 5 (5.3%) patients had SSRs, but 41 (43.6%) had large local reactions (LLRs) after the sting. Compared with the general population, there was a 9.5-fold higher risk for LLRs but not for SSRs. Three hours after the sting, sIgE levels slightly decreased, but none of the 94 subjects' results turned negative. After 1 week, sIgE levels already increased, increasing up to 3.5-fold (range, 0.2- to 34.0-fold) baseline levels after 4 weeks. To assess the clinical relevance of this increase, we randomly selected 18 patients for a re-sting. Again, 50% had an LLR, but none had an SSR.

Conclusion: Although sensitization to Hymenoptera venoms was common, the risk of SSRs in sensitized subjects was low in our study. The sIgE level increase after the sting was not an indicator for conversion into symptomatic sensitization. Currently available tests were not able to distinguish between asymptomatic sensitization, LLRs, and SSRs.
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http://dx.doi.org/10.1016/j.jaci.2013.10.046DOI Listing
June 2014

Protocols for identifying, enumerating, and assessing mouse eosinophils.

Methods Mol Biol 2013 ;1032:59-77

Inflammation Immunobiology Section, Laboratory of Allergic Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.

Eosinophils are prominent in allergic diseases, and their effector functions are studied in numerous gene-deleted and transgenic mouse models. However, mouse eosinophils and human eosinophils are not structurally or functionally equivalent, and assays designed to evaluate the properties of human eosinophils may or may not be reliable or effective in experiments targeting their murine counterparts. In this chapter, we emphasize methods focused on detection, isolation, and functional assessment of eosinophils from mouse tissue and present a protocol that promotes the growth and differentiation of eosinophils from unselected mouse bone marrow progenitors. Overall, these protocols provide a scaffold on which the relative contributions of mouse eosinophils can be evaluated.
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http://dx.doi.org/10.1007/978-1-62703-496-8_5DOI Listing
March 2014

Chemotaxis of bone marrow derived eosinophils in vivo: a novel method to explore receptor-dependent trafficking in the mouse.

Eur J Immunol 2013 Aug 14;43(8):2217-28. Epub 2013 Jun 14.

Inflammation Immunobiology Section, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.

Here, we describe a novel method via which ex vivo cultured mouse bone marrow derived eosinophils (bmEos) can be adoptively transferred into recipient mice in order to study receptor-dependent recruitment to lung tissue in vivo. Intratracheal instillation of recombinant human eotaxin-2 (hCCL24) prior to introduction of bmEos via tail vein injection resulted in an approximately fourfold increase in Siglec F-positive/CD11c-negative eosinophils in the lungs of eosinophil-deficient ΔdblGATA recipient mice compared with controls. As anticipated, bmEos generated from CCR3-gene-deleted mice did not migrate to the lung in response to hCCL24 in this model, indicating specific receptor dependence. BmEos generated from GFP-positive BALB/c mice responded similarly to hCCL24 in vitro and were detected in lung tissue of BALB/c WT as well as BALB/c ΔdblGATA eosinophil-deficient recipient mice, at approximately fourfold (at 5 h post-injection) and approximately threefold (at 24 h postinjection) over baseline, respectively. Comparable results were obtained with GFP-positive C57BL/6 bmEos responding to intratracheal hCCL24 in C57BL/6 ΔdblGATA recipient mice. The use of ex vivo cultured bmEos via one or more of these methods offers the possibility of manipulating bmEos prior to transfer into a WT or gene-deleted recipient host. Thus, this chemotaxis model represents a novel and robust tool for pharmacological studies in vivo.
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http://dx.doi.org/10.1002/eji.201343371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3786166PMC
August 2013

Inconsistent results of diagnostic tools hamper the differentiation between bee and vespid venom allergy.

PLoS One 2011 15;6(6):e20842. Epub 2011 Jun 15.

Division of Environmental Dermatology and Venerology, Department of Dermatology, Medical University of Graz, Graz, Austria.

Background: Double sensitization (DS) to bee and vespid venom is frequently observed in the diagnosis of hymenoptera venom allergy, but clinically relevant DS is rare. Therefore it is sophisticated to choose the relevant venom for specific immunotherapy and overtreatment with both venoms may occur. We aimed to compare currently available routine diagnostic tests as well as experimental tests to identify the most accurate diagnostic tool.

Methods: 117 patients with a history of a bee or vespid allergy were included in the study. Initially, IgE determination by the ImmunoCAP, by the Immulite, and by the ADVIA Centaur, as well as the intradermal test (IDT) and the basophil activation test (BAT) were performed. In 72 CAP double positive patients, individual IgE patterns were determined by western blot inhibition and component resolved diagnosis (CRD) with rApi m 1, nVes v 1, and nVes v 5.

Results: Among 117 patients, DS was observed in 63.7% by the Immulite, in 61.5% by the CAP, in 47.9% by the IDT, in 20.5% by the ADVIA, and in 17.1% by the BAT. In CAP double positive patients, western blot inhibition revealed CCD-based DS in 50.8%, and the CRD showed 41.7% of patients with true DS. Generally, agreement between the tests was only fair and inconsistent results were common.

Conclusion: BAT, CRD, and ADVIA showed a low rate of DS. However, the rate of DS is higher than expected by personal history, indicating that the matter of clinical relevance is still not solved even by novel tests. Furthermore, the lack of agreement between these tests makes it difficult to distinguish between bee and vespid venom allergy. At present, no routinely employed test can be regarded as gold standard to find the clinically relevant sensitization.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0020842PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3115969PMC
November 2011

Interaction of eosinophils with endothelial cells is modulated by prostaglandin EP4 receptors.

Eur J Immunol 2011 Aug 4;41(8):2379-89. Epub 2011 Jul 4.

Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Graz, Austria.

Eosinophil extravasation across the endothelium is a key feature of allergic inflammation. Here, we investigated the role of PGE(2) and its receptor, E-type prostanoid receptor (EP)-4, in the regulation of eosinophil interaction with human pulmonary microvascular endothelial cells. PGE(2) and the EP4 receptor agonist ONO AE1-329 significantly reduced eotaxin-induced eosinophil adhesion to fibronectin, and formation of filamentous actin and gelsolin-rich adhesive structures. These inhibitory effects were reversed by a selective EP4 receptor antagonist, ONO AE3-208. PGE(2) and the EP4 agonist prevented the activation and cell-surface clustering of β2 integrins, and L-selectin shedding of eosinophils. Under physiological flow conditions, eosinophils that were treated with the EP4 agonist showed reduced adhesion to endothelial monolayers upon stimulation with eotaxin, as well as after TNF-α-induced activation of the endothelial cells. Selective activation of EP1, EP2, and EP3 receptors did not alter eosinophil adhesion to endothelial cells, whereas the EP4 antagonist prevented PGE(2) from decreasing eosinophil adhesion. Finally, eosinophil transmigration across thrombin- and TNF-α-activated endothelial cells was effectively reduced by the EP4 agonist. These data suggest that PGE(2) -EP4 signaling might be protective against allergic responses by inhibiting the interaction of eosinophils with the endothelium and might hence be a useful therapeutic option for controlling inappropriate eosinophil infiltration.
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http://dx.doi.org/10.1002/eji.201141460DOI Listing
August 2011

Inhibitory effect of prostaglandin I2 on bone marrow kinetics of eosinophils in the guinea pig.

J Leukoc Biol 2011 Aug 17;90(2):285-91. Epub 2011 May 17.

Institute for Experimental and Clinical Pharmacology, Medical University of Graz, Graz, Austria.

Enhanced eosinophil trafficking from bone marrow to the tissue is a hallmark of allergic diseases. We have shown previously that PGI(2) markedly attenuates the locomotion of human eosinophils in vitro. Here, we set out to determine the effect of PGI(2) on the trafficking of bone marrow eosinophils in the guinea pig. Shape change of bone marrow eosinophils was determined by flow cytometry, and chemotaxis assays were performed using a transwell migration system. Eosinophil release from bone marrow of guinea pigs was investigated in the isolated, perfused hind-limb preparation. We found that PGI(2) prevented the mobilization of eosinophils from bone marrow and attenuated the shape change and chemotactic responses of bone marrow eosinophils. These effects were mimicked by iloprost and were prevented by the IP antagonist CAY10441 and the adenylyl cyclase inhibitor SQ22536. Immunohistochemical staining of bone marrow confirmed the expression of IPs by bone marrow eosinophils. The rate-limiting enzyme of PGI(2) formation, PGIS, was found in large mononuclear cells. These data show that IP activation negatively modulates the mobilization and locomotion of bone marrow eosinophils and might therefore also protect against exaggerated recruitment of eosinophils to inflammatory sites.
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http://dx.doi.org/10.1189/jlb.0211087DOI Listing
August 2011

EP4 receptor stimulation down-regulates human eosinophil function.

Cell Mol Life Sci 2011 Nov 2;68(21):3573-87. Epub 2011 Mar 2.

Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Universitaetsplatz 4, 8010 Graz, Austria.

Accumulation of eosinophils in tissue is a hallmark of allergic inflammation. Here we observed that a selective agonist of the PGE(2) receptor EP4, ONO AE1-329, potently attenuated the chemotaxis of human peripheral blood eosinophils, upregulation of the adhesion molecule CD11b and the production of reactive oxygen species. These effects were accompanied by the inhibition of cytoskeletal rearrangement and Ca(2+) mobilization. The involvement of the EP4 receptor was substantiated by a selective EP4 antagonist, which reversed the inhibitory effects of PGE(2) and the EP4 agonist. Selective kinase inhibitors revealed that the inhibitory effect of EP4 stimulation on eosinophil migration depended upon activation of PI 3-kinase and PKC, but not cAMP. Finally, we found that EP4 receptors are expressed by human eosinophils, and are also present on infiltrating leukocytes in inflamed human nasal mucosa. These data indicate that EP4 agonists might be a novel therapeutic option in eosinophilic diseases.
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http://dx.doi.org/10.1007/s00018-011-0642-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3192285PMC
November 2011

CD203c-based basophil activation test in allergy diagnosis: characteristics and differences to CD63 upregulation.

Cytometry B Clin Cytom 2010 Sep 14;78(5):308-18. Epub 2010 Apr 14.

Institute of Experimental and Clinical Pharmacology, Center of Molecular Medicine, Medical University of Graz, Austria.

Background: The basophil activation test (BAT) based on CD203c upregulation has been validated as a reliable tool for the diagnosis of IgE-mediated allergies. Nevertheless, CD203c-based BAT is hardly comparable with that of CD63-based tests, as the mechanisms of CD203c versus CD63 induction differ considerably. The aim of the present study was to identify potent influencing factors of the CD203c-based BAT and to emphasize differences between CD63 and CD203c detection.

Methods: CD203c-based BAT was determined in 82 healthy controls and in 79 allergic patients. The effects of interleukin (IL)-3 and degranulation enhancing substances were investigated and compared with CD63 upregulation. Furthermore, the influence of different storage conditions and incubation times was evaluated and the impact of antiallergic drugs on the test results was assessed.

Results: CD203c and CD63 expression was rapidly upregulated reaching a maximum after 20-30 min. Basophil CD203c upregulation assayed after storage times up to 48 h declined already after 4 h. IL-3 treatment increased CD203c and CD63 baseline levels and decreased basophil CD203c responses in a dose-dependent manner. In contrast, cytochalasin B and latrunculin B did not affect CD203c responses but decreased CD63-based BAT. Finally, therapeutic concentrations of dimetindene and desloratadine did not affect CD203c upregulation.

Conclusion: CD203c-based basophil activation test should be performed preferentially within 4 h after taking the blood samples. Priming and degranulation-enhancing factors are not required for CD203c-based BAT. In contrast to skin testing, CD203c-based BAT can be performed in patients undergoing antiallergic treatment. © 2010 International Clinical Cytometry Society.
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http://dx.doi.org/10.1002/cyto.b.20526DOI Listing
September 2010

Endothelium-derived prostaglandin I(2) controls the migration of eosinophils.

J Allergy Clin Immunol 2010 May 11;125(5):1105-13. Epub 2010 Feb 11.

Institute of Experimental and Clinical Pharmacology, Medical University Graz, A-8010 Graz, Austria.

Background: Enhanced eosinophil migration from the blood into the tissue is a hallmark of allergic diseases. Prostaglandin (PG) I(2) is the major prostanoid released by endothelial cells. Mice deficient in PGI(2) receptors (IPs) show exaggerated eosinophilic inflammation in response to allergen.

Objective: We set out to determine the role of PGI(2) in eosinophil trafficking.

Methods: Human lung microvascular endothelial cells and purified human eosinophils were used to study adhesion and transendothelial migration. Morphologic studies were performed with fluorescence microscopy.

Results: PGI(2) markedly attenuated the migration of eosinophils through cell-free filters but had no effect on neutrophil migration. The inhibitory effect of PGI(2) on eosinophils was prevented by the IP antagonist Cay10441 and the adenylyl cyclase inhibitor SQ22536. Similarly, PGI(2) prevented the adhesion of eosinophils to fibronectin and the rapid upregulation and activation of the adhesion molecule CD11b. IP expression on eosinophils was confirmed by means of flow cytometry and Western blotting. Furthermore, when endothelial cells were treated with the COX inhibitor diclofenac to abolish PGI(2) production, adhesion of eosinophils to endothelial monolayers and subsequent transendothelial migration were markedly enhanced. Similarly, the IP antagonist enhanced eosinophil adhesion to endothelial cells. Inhibition of PGI(2) biosynthesis decreased the electrical resistance of endothelial monolayers and compromised the texture of adherent junctions, as visualized by means of VE-cadherin and F-actin staining.

Conclusion: We propose that endothelium-derived PGI(2) might be fundamental for the maintenance of the endothelial barrier function against infiltrating cells. These results suggest that selective IP agonists might have beneficial effects in allergic inflammation.
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http://dx.doi.org/10.1016/j.jaci.2009.12.002DOI Listing
May 2010

Prostaglandin E2 inhibits eosinophil trafficking through E-prostanoid 2 receptors.

J Immunol 2008 Nov;181(10):7273-83

Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Graz, Austria.

The accumulation of eosinophils in lung tissue is a hallmark of asthma, and it is believed that eosinophils play a crucial pathogenic role in allergic inflammation. Prostaglandin (PG) E(2) exerts anti-inflammatory and bronchoprotective mechanisms in asthma, but the underlying mechanisms have remained unclear. In this study we show that PGE(2) potently inhibits the chemotaxis of purified human eosinophils toward eotaxin, PGD(2), and C5a. Activated monocytes similarly attenuated eosinophil migration, and this was reversed after pretreatment of the monocytes with a cyclooxygenase inhibitor. The selective E-prostanoid (EP) 2 receptor agonist butaprost mimicked the inhibitory effect of PGE(2) on eosinophil migration, whereas an EP2 antagonist completely prevented this effect. Butaprost, and also PGE(2), inhibited the C5a-induced degranulation of eosinophils. Moreover, selective kinase inhibitors revealed that the inhibitory effect of PGE(2) on eosinophil migration depended upon activation of PI3K and protein kinase C, but not cAMP. In animal models, the EP2 agonist butaprost inhibited the rapid mobilization of eosinophils from bone marrow of the in situ perfused guinea pig hind limb and prevented the allergen-induced bronchial accumulation of eosinophils in OVA-sensitized mice. Immunostaining showed that human eosinophils express EP2 receptors and that EP2 receptor expression in the murine lungs is prominent in airway epithelium and, after allergen challenge, in peribronchial infiltrating leukocytes. In summary, these data show that EP2 receptor agonists potently inhibit eosinophil trafficking and activation and might hence be a useful therapeutic option in eosinophilic diseases.
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http://dx.doi.org/10.4049/jimmunol.181.10.7273DOI Listing
November 2008

Prostaglandin H2 induces the migration of human eosinophils through the chemoattractant receptor homologous molecule of Th2 cells, CRTH2.

J Leukoc Biol 2009 Jan 3;85(1):136-45. Epub 2008 Oct 3.

Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Universitaetsplatz 4. A-8010 Graz, Austria.

The major mast cell product PGD2 is released during the allergic response and stimulates the chemotaxis of eosinophils, basophils, and Th2-type T lymphocytes. The chemoattractant receptor homologous molecule of Th2 cells (CRTH2) has been shown to mediate the chemotactic effect of PGD2. PGH2 is the common precursor of all PGs and is produced by several cells that express cyclooxygenases. In this study, we show that PGH2 selectively stimulates human peripheral blood eosinophils and basophils but not neutrophils, and this effect is prevented by the CRTH2 receptor antagonist (+)-3-[[(4-fluorophenyl)sulfonyl] methyl amino]-1,2,3,4-tetrahydro-9H-carbazole-9-acetic acid (Cay10471) but not by the hematopoietic PGD synthase inhibitor 4-benzhydryloxy-1-[3-(1H-tetrazol-5-yl)-propyl]piperidine (HQL79). In chemotaxis assays, eosinophils showed a pronounced migratory response toward PGH2, but eosinophil degranulation was inhibited by PGH2. Moreover, collagen-induced platelet aggregation was inhibited by PGH2 in platelet-rich plasma, which was abrogated in the presence of the D-type prostanoid (DP) receptor antagonist 3-[(2-cyclohexyl-2-hydroxyethyl)amino]-2,5-dioxo-1-(phenylmethyl)-4-imidazolidine-heptanoic acid (BWA868c). Each of these effects of PGH2 was enhanced in the presence of plasma and/or albumin. In eosinophils, PGH2-induced calcium ion (Ca2+) flux was subject to homologous desensitization with PGD2. Human embryo kidney (HEK)293 cells transfected with human CRTH2 or DP likewise responded with Ca2+ flux, and untransfected HEK293 cells showed no response. These data indicate that PGH2 causes activation of the PGD2 receptors CRTH2 and DP via a dual mechanism: by interacting directly with the receptors and/or by giving rise to PGD2 after catalytic conversion by plasma proteins.
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http://dx.doi.org/10.1189/jlb.0608387DOI Listing
January 2009

The role of the prostaglandin D2 receptor, DP, in eosinophil trafficking.

J Immunol 2007 Oct;179(7):4792-9

Institute of Experimental and Clinical Pharmacology, Medical University Graz, Graz, Austria.

Prostaglandin (PG) D2 is a major mast cell product that acts via two receptors, the D-type prostanoid (DP) and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) receptors. Whereas CRTH2 mediates the chemotaxis of eosinophils, basophils, and Th2 lymphocytes, the role of DP has remained unclear. We report in this study that, in addition to CRTH2, the DP receptor plays an important role in eosinophil trafficking. First, we investigated the release of eosinophils from bone marrow using the in situ perfused guinea pig hind limb preparation. PGD2 induced the rapid release of eosinophils from bone marrow and this effect was inhibited by either the DP receptor antagonist BWA868c or the CRTH2 receptor antagonist ramatroban. In contrast, BWA868c did not inhibit the release of bone marrow eosinophils when this was induced by the CRTH2-selective agonist 13,14-dihydro-15-keto-PGD2. In additional experiments, we isolated bone marrow eosinophils from the femoral cavity and found that these cells migrated toward PGD2. We also observed that BWA868c inhibited this response to a similar extent as ramatroban. Finally, using immunohistochemistry we could demonstrate that eosinophils in human bone marrow specimens expressed DP and CRTH2 receptors at similar levels. Eosinophils isolated from human peripheral blood likewise expressed DP receptor protein but at lower levels than CRTH2. In agreement with this, the chemotaxis of human peripheral blood eosinophils was inhibited both by BWA868c and ramatroban. These findings suggest that DP receptors comediate with CRTH2 the mobilization of eosinophils from bone marrow and their chemotaxis, which might provide the rationale for DP antagonists in the treatment of allergic disease.
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http://dx.doi.org/10.4049/jimmunol.179.7.4792DOI Listing
October 2007

Hierarchy of eosinophil chemoattractants: role of p38 mitogen-activated protein kinase.

Eur J Immunol 2006 Sep;36(9):2401-9

Institute of Experimental and Clinical Pharmacology, Medical University Graz, Graz, Austria.

Several chemoattractants can regulate the recruitment of eosinophils to sites of inflammation, but the hierarchy among them is unknown. We observed here that eosinophil chemotaxis towards eotaxin or 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) was amplified up to sixfold in the presence of prostaglandin (PG) D2. This effect was only seen in eosinophils, and not in neutrophils or basophils. Pretreatment with the chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH2) antagonist ramatroban prevented the PGD2 enhancement of eosinophil migrations. In contrast, eotaxin or 5-oxo-ETE inhibited the migration of eosinophils towards PGD2. 5-oxo-ETE enhanced the chemotaxis to eotaxin, while eotaxin had no effect on 5-oxo-ETE-induced migration. 5-oxo-ETE induced the phosphorylation of p38 mitogen-activated protein kinase, and inhibition of p38 mitogen-activated protein kinase by SB-202190 converted the effect of 5-oxo-ETE on the chemotaxis to PGD2 from inhibition to enhancement. The presence of blood or plasma markedly decreased the sensitivity of eosinophils to eotaxin or 5-oxo-ETE, while responses to PGD2 were unaltered. In conclusion, PGD2 might be an initial chemoattractant, since it maintains its potency in the circulation and augments the responsiveness of eosinophils to other chemoattractants. In contrast, eotaxin seems to be an end-point chemoattractant, since it has reduced efficacy in blood and is capable of down-modulating eosinophil responsiveness to other chemoattractants.
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http://dx.doi.org/10.1002/eji.200535672DOI Listing
September 2006

5-Oxo-6,8,11,14-eicosatetraenoic acid is a potent chemoattractant for human basophils.

J Allergy Clin Immunol 2005 Nov 3;116(5):1014-9. Epub 2005 Oct 3.

Department of Experimental and Clinical Pharmacology, Medical University of Graz, Austria.

Background: 5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a chemoattractant for eosinophils and neutrophils, and the messenger RNA for its receptor, the oxo-eicosatetraenoic acid receptor (OXE), has been detected in several tissues.

Objectives: This study aimed at clarifying the role of 5-oxo-ETE in the regulation of basophil function.

Methods: Basophil responses were determined in assays of flow-cytometric shape change, Ca(2+) flux, chemotaxis, and histamine release. Messenger RNA for OXE was detected by real-time PCR.

Results: We observed that human eosinophils were 3 to 10 times more sensitive to 5-oxo-ETE than neutrophils in flow-cytometric shape change and Ca(2+) flux assays, as estimated from the half-maximal responses of the cells. Basophils responded to 5-oxo-ETE in the shape change assay with a sensitivity similar to that of eosinophils. 5-Oxo-ETE was a weak inducer of Ca(2+) flux in basophils and did not cause histamine release but was a highly effective chemoattractant for basophils in the low nanomolar concentration range in a pertussis toxin-sensitive manner. In agreement with these functional studies, the messenger RNA for the 5-oxo-ETE receptor, OXE, was detectable in basophils as in monocytes, eosinophils, and neutrophils, but not in fibroblasts. Specimens from sinus mucosa, tonsils, and adenoids also contained detectable levels of messenger RNA for OXE.

Conclusion: Our data suggest that 5-oxo-ETE is potentially involved in the regulation of basophil recruitment and might hence be a useful therapeutic target in atopic disease.
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http://dx.doi.org/10.1016/j.jaci.2005.08.001DOI Listing
November 2005

Stem cell factor stimulates the chemotaxis, integrin upregulation, and survival of human basophils.

J Allergy Clin Immunol 2005 Oct 2;116(4):820-6. Epub 2005 Aug 2.

Department of Experimental and Clinical Pharmacology, Medical University of Graz, Austria.

Background: Little is known about the mechanisms that regulate the selective recruitment of basophils to sites of allergic inflammation.

Objective: Here we examine the role of stem cell factor (SCF) in the regulation of basophil function.

Methods: Human basophils were isolated from peripheral blood, and their migration was investigated in chemotaxis assays. Apoptosis was detected by means of annexin V and propidium iodide staining. The expression of cell-surface molecules was measured by means of flow cytometry.

Results: SCF amplified the chemotactic responsiveness of human peripheral blood basophils to the chemoattractants eotaxin, monocyte chemotactic protein 2 and macrophage inflammatory protein 1alpha, and C5a, without being chemotactic or chemokinetic by itself. SCF synergized with chemoattractants in causing basophil upregulation of the integrin CD11b, and this effect was inhibited by a c-kit antibody, the tyrosine kinase inhibitor imatinib mesylate (STI-571), and a phosphatidylinositol 3 kinase inhibitor but not by inhibitors of p38 mitogen-activated protein kinase or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase. Basophils bound fluorescence-labeled SCF and expressed its receptor, c-kit, which was markedly upregulated in culture for 24 to 48 hours in the presence of IL-3. Moreover, SCF prolonged basophil survival in concert with IL-3 by delaying apoptosis. These effects of SCF were selective for basophils because chemotaxis and CD11b upregulation of eosinophils or neutrophils were unchanged.

Conclusion: SCF might be an important selective modulator of basophil function through a phosphatidylinositol 3 kinase-dependent pathway.
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http://dx.doi.org/10.1016/j.jaci.2005.06.008DOI Listing
October 2005