Eosinophil Adhesion Research Articles

αMβ2Integrin–Mediated Adhesion and Motility of IL-5–Stimulated Eosinophils on Periostin

Periostin is an extracellular matrix protein that is up-regulated by T helper cell type 2 cytokines in the asthmatic airway and implicated in mouse studies as promoting eosinophil recruitment. We asked whether periostin modulates eosinophil adhesion and motility in vitro. Periostin adsorbed to polystyrene supported adhesion of purified human blood eosinophils stimulated by IL-5, IL-3, or granulocyte/macrophage colony-stimulating factor, but did not support adhesion of eosinophils treated with IL-4 or IL-13. The degree of adhesion depended on the concentrations of periostin during coating and activating cytokine during the adhesion assay. Both full-length periostin and alternatively spliced periostin, lacking C-terminal exons 17, 18, 19, and 21, supported adhesion. Adhesion was inhibited by monoclonal antibody to α(M) or β(2) integrin subunits, but not by antibodies to other eosinophil integrin subunits. Adsorbed periostin also supported α(M)β(2)-dependent random motility of IL-5-stimulated eosinophils with optimal movement at an intermediate coating concentration. In the presence of IL-5, eosinophils adherent on periostin formed punctate structures positive for filamentous actin, gelsolin, and phosphotyrosine. These structures fit the criteria for podosomes, highly dynamic adhesive contacts that are distinct from classical focal adhesions. The results establish α(M)β(2) (CD11b/CD18, Mac-1) as an adhesive and promigratory periostin receptor on cytokine-stimulated eosinophils, and suggest that periostin may function as a haptotactic stimulus able to guide eosinophils to areas of high periostin density in the asthmatic airway.

Airway exposure to staphylococcal enterotoxin A potentiates allergen-induced bone marrow eosinophilia and trafficking to peripheral blood and airways

Bone marrow (BM) eosinopoiesis is a common feature during allergen exposure in atopic individuals. Airway exposure to staphylococcal superantigens aggravates allergic airway disease and increases the output of BM eosinophils. However, the exact mechanisms regulating eosinophil mobilization and trafficking to the peripheral circulation and airways remain to be elucidated. Therefore, this study aimed to investigate the mechanisms determining the BM eosinopoiesis in allergic mice under exposure to staphylococcal enterotoxin A (SEA). Ovalbumin (OVA)-sensitized male BALB/C mice were intranasally exposed to SEA (1 μg), and at 4, 12, 24, and 48 h later animals were challenged with OVA (10 μg, twice a day). Measurement of IL-5, eotaxin, and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels, flow cytometry for CCR3(+), VLA4(+), and CCR3(+)VLA4(+), as well as adhesion assays to VCAM-1 were performed in BM. Prior airway exposure to SEA time dependently increased the BM eosinophil number in OVA-challenged mice. Eosinophils gradually disappear from peripheral blood, being recruited over time to the airways, where they achieve a maximal infiltration at 24 h. SEA exposure increased the levels of IL-5 and eotaxin (but not GM-CSF) in BM of OVA-challenged mice. Marked increases in CCR3(+) and CCR3(+)VLA4(+) expressions in BM eosinophils of OVA-challenged mice were observed, an effect largely reduced by prior exposure to SEA. Adhesion of BM eosinophils to VCAM-1 was increased in OVA-challenged mice, but prior SEA exposure abrogated this enhanced cell adhesion. Accumulation of BM eosinophils by airway SEA exposure takes place through IL-5- and CCR3-dependent mechanisms, along with downregulation of CCR3/VL4 and impaired cell adhesion to VCAM-1.

Molecular mechanisms regulating the synergism between IL-32? and NOD ligands for the induction of adhesion molecules, cytokines and chemokines of eosinophils in allergic inflammation

Event Abstract Back to Event Molecular mechanisms regulating the synergism between IL-32γ and NOD ligands for the induction of adhesion molecules, cytokines and chemokines of eosinophils in allergic inflammation Chun K. Wong1*, Jie Dong1 and Christopher W. Lam1, 2 1 The Chinese University of Hong Kong, Department of Chemical Pathology, Hong Kong, SAR China 2 Macau University of Science and Technology, Macau Institute for Applied Research in Medicine and Health, Macao, SAR China Background: Key intracytosolic pattern recognition receptors in innate immune responses for bacterial infections are nucleotide binding oligomerization domain (NOD) like receptors (NLR). IL-32 is novel pro-inflammatory cytokine that are associated with viral and bacterial infections, and allergic asthma. We elucidated the synergistic effect of IL-32gamma and NOD-mediated the activation of human eosinophils, the principal effector cells for allergic inflammation and the underlying mechanisms. Methodology: Expression of adhesion molecules and chemokines was quantitated by flow cytometry and multiplex assay, respectively. Phosphorylation of signaling molecules and caspase-1 activity was analysed by Western blot. Results: IL-32γ was more capable in activating eosinophils than its isotype variant IL-32beta, and the internalization of IL-32γ is dispensable for this effect. IL-32γ and NOD1 ligand iE-DAP or NOD2 ligand MDP exhibit significant up-regulation of cell surface expression of CD18 and intercellular adhesion molecule-1 on eosinophils, and the adherence of eosinophils on dermal fibroblasts/bronchial epithelial cells. In addition, IL-32γ exhibit synergistic effect with iE-DAP and MDP on the induction of allergic inflammation-related IL-1beta, chemokines CXCL8, CCL3 and CCL4, and IL-1beta, IL-6, CXCL8, TNF-alpha, CCL3 and CCL4 (all p<0.05). Synergism between IL-32γ and NOD depended on the activation of intracellular caspase 1, ERK, p38 MAPK and NF-kappaB in eosinophils. Conclusion: The above study provides immunological mechanisms by which bacterial infection-mediated activation of NOD1,2 together with novel pro-inflammatory IL-32 in synergizing allergic inflammation via the activation of eosinophils. Acknowledgements This work was supported by the Research Grant Committee General Research Fund, Hong Kong (Project ref. no. CUHK 476411) Keywords: allergic inflammation, Eosinophils, IL-32, NLR, Signal Transduction Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Wong CK, Dong J and Lam CW (2013). Molecular mechanisms regulating the synergism between IL-32γ and NOD ligands for the induction of adhesion molecules, cytokines and chemokines of eosinophils in allergic inflammation. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00161 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 11 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Prof. Chun K Wong, The Chinese University of Hong Kong, Department of Chemical Pathology, Hong Kong, Hong Kong, SAR China, ck-wong@cuhk.edu.hk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Chun K Wong Jie Dong Christopher W Lam Google Chun K Wong Jie Dong Christopher W Lam Google Scholar Chun K Wong Jie Dong Christopher W Lam PubMed Chun K Wong Jie Dong Christopher W Lam Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Dynamics and function of eosinophils’ CD48 molecules in allergic rhinitis and in response to eotaxin stimulation

Background : The functional expression of eosinophil's CD48 molecules that act in the form of ligand–receptor pairs with 2B4 (CD244), in allergic rhinitis (AR) and in response to eotaxin stimulation, is studied. Methodology : Nasal allergen provocation, flow cytometry, adhesion assay, confocal microscopy and westren blot were used in the current study. Results : Herein, it is shown that following a single nasal allergen challenge with the house dust mite (HDM), the percentage of peripheral blood eosinophils expressing total CD48 molecules increased in 100% of AR patients ( n =10) sensitized to HDM and lasted up to 24 h, while the surface expression increased in 20% of these patients and lasted for 6 h. After challenge, a significant increase of eosinophil adhesion and intercellular adhesion to collagen type IV coated slides was observed. Eotaxin induced the surface expression of CD48 in eosinophils from normal subjects and AR patients. This was sensitive to genistein, a tyrosine kinase inhibitor. Eotaxin-induced eosinophil adhesion was blocked in a dose-dependent fashion by anti-CD48 Ab. Eosinophil stimulation with functional grade purified C1.7 Ab induced F-actin increase and eosinophil shape changes that were sensitive to genistein. Western blot analysis identified low-molecular-weight tyrosine kinase protein residues phosphorylation by eotaxin and C1.7 Ab. Conclusion: These results identify the CD48–2B4 complex to be involved in eosinophil functional responses, making them novel targets for therapeutic approaches to eosinophilic inflammation of the airway. Keywords: eosinophils; intercellular adhesion; adhesion; allergic rhinitis; CD48; 2B4 Citation: Advances in Cellular and Molecular Otolaryngology 2013, 1 : 22389 - http://dx.doi.org/10.3402/acmo.v1i0.22389

Integrin Activation States and Eosinophil Recruitment in Asthma

Eosinophil arrest and recruitment to the airway in asthma are mediated, at least in part, by integrins. Eosinophils express α4β1, α6β1, αLβ2, αMβ2, αXβ2, αDβ2, and α4β7 integrins, which interact with counter-receptors on other cells or ligands in the extracellular matrix. Whether a given integrin-ligand pair mediates cell adhesion and migration depends on the activation state of the integrin. Integrins exist in an inactive bent, an intermediate-activity extended closed, and a high-activity extended open conformation. Integrin activation states can be monitored by conformation-specific monoclonal antibodies (mAbs). Studies in mice indicate that both β1 and β2 integrins mediate eosinophil recruitment to the lung. In vitro studies indicate that α4β1 and αMβ2 are the principal integrins mediating eosinophil adhesion, including to vascular cell adhesion molecule-1 and the novel αMβ2 ligand periostin. In vivo, blood eosinophils have intermediate-activity β1 integrins, as judged by mAb N29, apparently resulting from eosinophil binding of P-selectin on the surface of activated platelets, and have a proportion of their β2 integrins in the intermediate conformation, as judged by mAb KIM-127, apparently due to exposure to low concentrations of interleukin-5 (IL-5). Airway eosinophils recovered by bronchoalveolar lavage (BAL) after segmental antigen challenge have high-activity β1 integrins and high-activity αMβ2 that does not require IL-5. Here we review information on how the activation states of eosinophil β1 and β2 integrins correlate with measurements of eosinophil recruitment and pulmonary function in asthma. Blood eosinophil N29 reactivity is associated with decreased lung function under various circumstances in non-severe asthma and KIM-127 with BAL eosinophil numbers, indicating that intermediate-activity α4β1 and αMβ2 of blood eosinophils are important for eosinophil arrest and consequently for recruitment and aspects of asthma.

Epigenetic Regulation of Galectin-3 Expression by β1 Integrins Promotes Cell Adhesion and Migration

Introduction of the integrin β1- but not the β3-subunit in GE11 cells induces an epithelial-to-mesenchymal-transition (EMT)-like phenomenon that is characterized by the loss of cell-cell contacts, cell scattering, increased cell migration and RhoA activity, and fibronectin fibrillogenesis. Because galactose-binding lectins (galectins) have been implicated in these phenomena, we investigated whether galectins are involved in the β1-induced phenotype. We examined 9 galectins and, intriguingly, found that the expression of galectin-3 (Gal-3) is specifically induced by β1 but not by β3. Using β1-β3 chimeric integrins, we show that the induction of Gal-3 expression requires the hypervariable region in the extracellular domain of β1, but not its cytoplasmic tail. Furthermore, Gal-3 expression does not depend on RhoA signaling, serum factors, or any of the major signal transduction pathways involving protein kinase C (PKC), p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase-1/-2 (ERK-1/2), phosphatidylinositol-3-OH kinase (PI3-K), or Src kinases. Instead, Gal-3 expression is controlled in an epigenetic manner. Whereas DNA methylation of the Lgals3 promoter maintains Gal-3 silencing in GE11 cells, expression of β1 causes its demethylation, leading to transcriptional activation of the Lgals3 gene. In turn, Gal-3 expression enhances β1 integrin-mediated cell adhesion to fibronectin (FN) and laminin (LN), as well as cell migration. Gal-3 also promotes β1-mediated cell adhesion to LN and Collagen-1 (Col)-1 in cells that endogenously express Gal-3 and β1 integrins. In conclusion, we identify a functional feedback-loop between β1 integrins and Gal-3 that involves the epigenetic induction of Gal-3 expression during integrin-induced EMT and cell scattering.

Interferon-alpha inhibits airway eosinophila and hyperresponsiveness in an animal asthma model

BackgroundAsthma is characterized by a chronic inflammatory process involving high numbers of inflammatory cells and mediators which have multiple inflammatory effects on the airway. Interferon (IFN)-alpha, which is used widely for treating chronic hepatitis C, is reported to have an effect on patients with Churg-Strauss syndrome. Therefore, it may also be suitable for patients with severe asthma.ObjectiveWe studied the effect of IFN-alpha on airway eosinophilia in a guinea pig model of asthma and the expression of adhesion molecules on human eosinophils and vascular endothelial cells.MethodsAfter antigen challenge, airway hyperresponsiveness and airway eosinophilia were measured in a guinea pig asthma model with or without airway IFN-alpha administration. Expression of adhesion molecules on eosinophils and cultured human umbilical vein endothelial cells (HUVECs) was also evaluated with or without IFN-alpha.ResultsIFN-alpha inhibited eosinophil recruitment into the tracheal wall and improved airway hyperresponsiveness in sensitized guinea pigs. IFN-alpha also significantly suppressed IL-1 beta-induced intercellular adhesion molecule-1 (ICAM-1) expression on HUVECs. However, IFN-alpha did not suppress platelet-activating factor-induced macrophage antigen-1 expression on human eosinophils. IFN-alpha significantly inhibited eosinophil adhesion to IL-1 beta-induced HUVECs and migration through IL-1 beta induced HUVECs.ConclusionThe findings suggest that the modulation of ICAM-1 in lung with pre-existing inflammation following treatment with IFN-alpha may be a novel and selective treatment for control of chronic airway inflammation and hyperresponsiveness associated with asthma.

5,7‐Dihydroxy‐3,4,6‐trimethoxyflavone inhibits intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 via the Akt and nuclear factor‐κB‐dependent pathway, leading to suppression of adhesion of monocytes and eosinophils to bronchial epithelial cells

5,7-Dihydroxy-3',4',6'-trimethoxyflavone (eupatilin), the active pharmacological ingredient from Artemisia asiatica Nakai (Asteraceae), is reported to have a variety of anti-inflammatory properties in intestinal epithelial cells. However, little information is known about the molecular mechanism of eupatilin-induced attenuation of bronchial epithelial inflammation. This study investigates the role of eupatilin in the adhesion of inflammatory cells such as monocytes and eosinophils to bronchial epithelial cells. Stimulation of a human bronchial epithelial cell line (BEAS-2B) with tumour necrosis factor-α (TNF-α) increased the expression of surface adhesion molecules, including intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), in which eupatilin significantly inhibited the expression of those adhesion molecules in a dose-dependent manner. Eupatilin suppressed the TNF-α-induced activation of IκBα and nuclear factor-κB (NF-κB) signals in BEAS-2B cells. The IκB kinase (IKK) activation was also significantly reduced in eupatilin-pre-treated BEAS-2B and primary normal human bronchial epithelial (NHBE) cells. However, eupatilin did not influence AP-1 activity in TNF-α-stimulated cells. Suppression of NF-κB signalling induced by eupatilin resulted in the inhibition of the expression of adhesion molecules and the adhesion of monocytes and eosinophils to BEAS-2B cells. Furthermore, eupatilin suppressed the phosphorylation of Akt in TNF-α-stimulated BEAS-2B and NHBE cells, leading to down-regulation of NF-κB activation and adhesion molecule expression and finally to suppression of the inflammatory cell adhesion to epithelial cells. These results suggest that eupatilin can inhibit the adhesion of inflammatory cells to bronchial epithelial cells via a signalling pathway, including activation of Akt and NF-κB, as well as expression of adhesion molecules.

Response to Treatment in Individuals with Late‐Onset Asthma

Despite the fact that the onset of asthma can occur at any age, there have been few studies specifically assessing whether the therapeutic effects of asthma medications vary according to age of onset.1, 2 The present study was constructed to compare the responses to a combination of fluticasone (FP) and salmeterol (SAL) in terms of symptoms, airflow obstruction, and airway inflammation between individuals with asthma with early (<40) and late (≥65) onset. Various factors, including current medications, smoking history, asthma duration, comorbid conditions, adherence, and inhalation technique, may modify response to asthma treatment.1, 2 This prospective parallel-group study was designed to control for these modifiers. Never-smoking untreated individuals from both age groups whose asthma duration was <3 years were included. Individuals were excluded if they had other pulmonary or cardiac diseases or had been treated with any form of corticosteroid in the 8 weeks before the study. Hydrofluoroalkane (HFA)-FP/SAL (250/50 μg) was administered twice daily, and instructions were repeated monthly to all participants. Asthma control questionnaire (ACQ), spirometry, and fraction of exhaled nitric oxide (FENO) were assessed before and 3 months after HFA-FP/SAL therapy. FENO was measured using an online electrochemical nitric oxide analyzer (NIOX MINO; Aerocrine AB, Solna, Sweden) according to standard procedures.3, 4 Statistical analysis was performed using Mann–Whitney U-tests. The local ethics committee approved this trial, which was registered with the university hospital Medical Information Network (UMIN000006291). Informed written consent was obtained from each participant. Participants consisted of 16 younger individuals (seven male, nine female, 14 with atopy) with an average onset age of 27.4, and 16 elderly individuals (eight male, eight female, eight with atopy) with an average onset age of 72.3. At baseline, individuals with late onset had lower forced expiratory volume in 1-s percentage of predicted (%FEV1; 89.3 ± 7.3%) than those with early onset (80.9 ± 12.8%, P = .03), but no differences in ACQ score (1.3 ± 0.5 vs 1.4 ± 0.6, P = .7) of FENO (50 ± 18 vs 57 ± 38 ppb, P = .48) were seen between the two age groups. As shown in Figure 1, HFA-FP/SAL therapy significantly improved ACQ,%FEV1, and FENO equally in participants with early and late onset, and there was no significant difference in these outcomes between the groups 3 months after treatment (0.2 ± 0.3 vs 0.3 ± 0.3, P = .45; 94.7 ± 8.4% vs 90.1 ± 12.5%, P = .14; and 26 ± 11 vs. 24 ± 18 ppb, P = .99, respectively). Two points from the preliminary data should be emphasized. First, untreated individuals with late-onset asthma have the same degree of airway inflammation detected using FENO as those with early onset. Second, individuals with early- and late-onset asthma respond equally well to HFA-FP/SAL therapy in terms of asthma symptoms, airflow obstruction, and airway inflammation. Because inhaled corticosteroids are capable of reducing airway inflammation, thereby reducing symptoms and improving lung function, the present study indicates that airway inflammation is an important therapeutic target in persistent asthma regardless of the age of onset. It has been shown that peripheral blood eosinophil adhesion and chemotactic activities were comparable in individuals with early and late-onset asthma, and the percentages of sputum eosinophils were similar in the two age groups.5 There is also evidence that regular use of inhaled corticosteroids improves asthma-related outcomes in elderly adults.6-9 The findings of the current study suggest that regular antiinflammatory treatment and instructions are an important therapeutic strategy in asthma for all ages, and the implementation of this strategy can be expected to improve health outcomes in elderly adults. The authors thank Mr. Brent Bell for reading the manuscript. This study was supported by Grant 22591097 from the Japan Society for the Promotion of Science. No potential conflicts of interest exist with any companies whose products or services may be discussed in this article. Conflict of Interest: The editor in chief has reviewed the conflict of interest checklist provided by the authors and has determined that the authors have no financial or any other kind of personal conflicts with this paper. Author Contributions: Kazuto Matsunaga: study design and preparation of manuscript. Masakazu Ichinose: interpretation of data. Keiichiro Akamatsu and Tsunahiko Hirano: acquisition of subjects and data. Sponsor's Role: None.

Staphylococcus aureus directly activates eosinophils via platelet-activating factor receptor

Colonization by SA is associated with exacerbation of AD. Eosinophilic inflammation is a cardinal pathological feature of AD, but little is known about possible direct interaction between SA and eosinophils. PAFR appears to be involved in phagocytosis of Gram-positive bacteria by leukocytes. The objective of this study was to investigate whether SA directly induces eosinophil effector functions via PAFR in the context of AD pathogenesis. Peripheral blood eosinophils were cultured with heat-killed SA, and EDN release, superoxide generation, and adhesion to fibronectin-coated plates were measured. Cytokines, released in the supernatants, were quantified by multiplex bead immunoassays. FISH-labeled SA was incubated with eosinophils and visualized by confocal laser-scanning microscopy. PAFR-blocking peptide and PAFR antagonists were tested for inhibitory effects on SA-induced reactions. SA induced EDN release and superoxide generation by eosinophils in a dose-dependent manner. IL-5 significantly enhanced SA-induced EDN release. IL-5 and IL-17A significantly enhanced SA-induced superoxide generation. SA enhanced eosinophil adhesion to fibronectin, which was blocked by anti-CD49d, and induced eosinophil secretion of various cytokines/chemokines (IL-2R, IL-9, TNFR, IL-1 β, IL-17A, IP-10, TNF-α, PDGF-bb, VEGF, and FGF-basic). After incubation of eosinophils with SA, FISH-labeled SA was visualized in the eosinophils' cytoplasm, indicating phagocytosis. A PAFR-blocking peptide and two PAFR antagonists completely inhibited those reactions. In conclusion, SA directly induced eosinophil activation via PAFR. Blockade of PAFR may be a novel, therapeutic approach for AD colonized by SA.

Lactoferrin Regulates an Axis Involving CD11b and CD49d Integrins and the Chemokines MIP-1α and MCP-1 in GM-CSF-Treated Human Primary Eosinophils

Eosinophils are multifunctional immune cells that contribute to innate and adaptive immune/repair responses. Lactoferrin (LF) is an iron-binding protein indicated to alter cell adhesion and immune function by receptor-mediated interactions or by participating in redox mechanisms. The eosinophil adhesion molecules, αMβ2 and α4β1, are differentially expressed following exposure to the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) and various redox agents. We hypothesized that LF can alter the function and production of proteins involved in adhesion/migration. Utilizing eosinophil peroxidase activity or fluorescent labeling adhesion assays, LF reduced GM-CSF-induced eosinophil adhesion in the presence of fibronectin or vascular adhesion molecule-1 compared with GM-CSF treatment alone. Flow cytometric analysis of eosinophil αM (CD11b) and α4 (CD49d) integrins revealed that cotreatments (24 h) with LF plus GM-CSF induced a significant increase in CD11b compared with control and GM-CSF treatments but a significant decrease in CD49d compared with control and GM-CSF treatments. These changes in CD11b and CD49d levels were significantly correlated with the increased production of chemokines (macrophage inflammatory Protein-1α, monocyte chemotactic protein-1) and an identified increase in S100A9 production. Thus, LF release at sites of inflammation may alter eosinophil recruitment/activation and possibly the progression of diseases such as cancer and asthma where significant eosinophil influx has been described.

The p110δ subunit of PI3K regulates bone marrow-derived eosinophil trafficking and airway eosinophilia in allergen-challenged mice

Trafficking and recruitment of eosinophils during allergic airway inflammation is mediated by the phosphatidylinositol 3-kinase (PI3K) family of signaling molecules. The role played by the p110δ subunit of PI3K (PI3K p110δ) in regulating eosinophil trafficking and recruitment was investigated using a selective pharmacological inhibitor (IC87114). Treatment with the PI3K p110δ inhibitor significantly reduced murine bone marrow-derived eosinophil (BM-Eos) adhesion to VCAM-1 as well as ICAM-1 and inhibited activation-induced changes in cell morphology associated with reduced Mac-1 expression and aberrant cell surface localization/distribution of Mac-1 and α4. Infused BM-Eos demonstrated significantly decreased rolling and adhesion in inflamed cremaster muscle microvessels of mice treated with IC87114 compared with vehicle-treated mice. Furthermore, inhibition of PI3K p110δ significantly attenuated eotaxin-1-induced BM-Eos migration and prevented eotaxin-1-induced changes in the cytoskeleton and cell morphology. Knockdown of PI3K p110δ with siRNA in BM-Eos resulted in reduced rolling, adhesion, and migration, as well as inhibition of activation-induced changes in cell morphology, validating its role in regulating trafficking and migration. Finally, in a mouse model of cockroach antigen-induced allergic airway inflammation, oral administration of the PI3K p110δ inhibitor significantly inhibited airway eosinophil recruitment, resulting in attenuation of airway hyperresponsiveness in response to methacholine, reduced mucus secretion, and expression of proinflammatory molecules (found in inflammatory zone-1 and intelectin-1). Overall, these findings indicate the important role played by PI3K p110δ in mediating BM-Eos trafficking and migration by regulating adhesion molecule expression and localization/distribution as well as promoting changes in cell morphology that favor recruitment during inflammation.

Possible Involvement of Sphingosine-1-Phosphate/Gi/RhoA Pathways in Adherence of Eosinophils to Pulmonary Endothelium

Sphingosine-1-phosphate (S1P), a lysophospholipid released from inflammatory cells, causes cell migration by increasing cytokines and chemokines. This study was designed to determine whether S1P causes adherence of eosinophils to pulmonary endothelial cells via enhancement of adhesion molecule expression. Expression of VCAM-1 and ICAM-1 was assessed by RT-PCR and Western blot analysis in human pulmonary microvasucular endothelial cells (HPMVECs). The number of adherent eosinophils to HPMVECs was calculated according to adhesion assay. Pre-treatment of HPMVECs with S1P increased mRNA and protein expression of VCAM-1, in contrast, did not dramatically increase those expression of ICAM-1. The maximal expression of these adhesion molecules in mRNA and protein was observed 4 and 8h after exposure to S1P, respectively. Pre-treatment with S1P also activated RhoA, a monomeric G protein; the ability of S1P to enhance the expression of VCAM-1 was attenuated by RhoA related inhibitors such as Y-27632, C3 exoenzyme, and GGTI-286. The effects of S1P on VCAM-1 were attenuated by pre-incubation with pertussis toxin, which catalyzes the ADP-ribosylation of G(i), a heterotrimeric G protein. After HPMVECs were treated with S1P, adhesion of human eosinophilic leukemic cell line (EoL-1) cells to HPMVECs was enhanced in a concentration-dependent manner. Augmented adherence of EoL-1 cells by S1P was also attenuated by Y-27632 and pertussis toxin. S1P causes adherence of eosinophils to pulmonary endothelium via RhoA activation. S1P may act as a lipid mediator in asthma. The RhoA/Rho-kinase pathway may be a therapeutic target for preventing eosinophil infiltration to the airway.

Therapeutic targeting of eosinophil adhesion and accumulation in allergic conjunctivitis.

Considerable evidence indicates that eosinophils are important effectors of ocular allergy. Increased worldwide prevalence of allergic eye pathologies has stimulated the identification of novel drug targets, including eosinophils and adhesion molecules. Accumulation of eosinophils in the eye is a key event in the onset and maintenance of allergic inflammation and is mediated by different adhesion molecules. Antihistamines with multiple mechanisms of action can be effective during the early and late phases of allergic conjunctivitis by blocking the interaction between β1 integrins and vascular cell adhesion molecule (VCAM)-1. Small molecule antagonists that target key elements in the process of eosinophil recruitment have been identified and reinforce the validity of α4β1 integrin as a therapeutic target. Glucocorticoids are among the most effective drugs for ocular allergy, but their use is limited by adverse effects. Novel dissociated glucocorticoids can prevent eosinophil accumulation and induce apoptosis of eosinophils, making them promising candidates for ophthalmic drugs. This article reviews recent understanding of the role of adhesion molecules in eosinophil recruitment in the inflamed conjunctiva along with effective treatments for allergic conjunctivitis.

IFN-γ-inducible protein of 10 kDa upregulates the effector functions of eosinophils through β2integrin and CXCR3

BackgroundEosinophils play an important role in the pathogenesis of bronchial asthma and its exacerbation. Recent reports suggest the involvement of IFN-γ-inducible protein of 10 kDa (IP-10) in virus-induced asthma exacerbation. The objective of this study was to examine whether CXCR3 ligands including IP-10 modify the effector functions of eosinophils.MethodsEosinophils isolated from the blood of healthy donors were stimulated with CXCR3 ligands and their adhesion to rh-ICAM-1 was then measured using eosinophil peroxidase assays. The generation of eosinophil superoxide anion (O2-) was examined based on the superoxide dismutase-inhibitable reduction of cytochrome C. Eosinophil-derived neurotoxin (EDN) release was evaluated to determine whether CXCR3 ligands induced eosinophil degranulation. Cytokine and chemokine production by eosinophils was examined using a Bio-plex assay.ResultsEosinophil adhesion to ICAM-1 was significantly enhanced by IP-10, which also significantly induced eosinophil O2- generation in the presence of ICAM-1. Both the enhanced adhesion and O2- generation were inhibited by an anti-β2 integrin mAb or an anti-CXCR3 mAb. Other CXCR3 ligands, such as monokine induced by IFN-γ (Mig) and IFN-inducible T cell α chemoattractant (I-TAC), also induced eosinophil adhesion and O2- generation in the presence of ICAM-1. IP-10, but not Mig or I-TAC, increased the release of EDN. IP-10 increased the production of a number of cytokines and chemokines by eosinophils.ConclusionsThese findings suggest that CXCR3 ligands such as IP-10 can directly upregulate the effector functions of eosinophils. These effects might be involved in the activation and infiltration of eosinophils in the airway of asthma, especially in virus-induced asthma exacerbation.

The Major Phagocyte Peroxidase Product HOSCN Stimulates PMN and Eosinophil Adhesion to Endothelium by p38 MAPK-Dependent Enhancement of CD11b/18-ICAM-1 Binding,

Abstract 3205Hypothiocyanous acid (HOSCN) is the predominant physiologic product of eosinophil peroxidase (EPO) and a major (50%) product of myeloperoxidase (MPO), a product of H2O2 oxidation of the pseudohalide substrate thiocyanate (SCN-). We have previously shown that this sulfhydryl-specific, cell-permeant oxidant, unlike HOCl, can potently induce expression of several NF-kB regulated genes, including the adhesion molecules ICAM-1 and VCAM-1, in human umbilical vein endothelial cells (HUVEC) through a transcriptional mechanism reflecting NF-κB activation. We here tested the hypothesis that HOSCN generated within activated PMN and eosinophils (EO) promotes their adhesion to ICAM-1 and VCAM-1 by enhancing the avidity for their leukocyte integrin counterligands, CD11b/18 (Mac-1) and CD49d/29 (VLA-4), respectively. We isolated human PMN and EO and tested the influence of reagent HOSCN upon their adhesion to purified human ICAM-1 and VCAM-1 immobilized in the bottom of bovine serum albumin (BSA)-blocked of tissue culture plate wells using a method based on crystal violet staining of nuclei and spectrophotometric assay of acetic acid extracts of adherent cells. 3–150 μM HOSCN enhanced adhesion to ICAM-1 of EO 10–18-fold over baseline binding and PMN 2–3-fold. This increase was inhibited >90% by both CBRM1/5, a blocking antibody against CD11b/18 and M1/70, which blocks ICAM-1, showing the specificity of this interaction. Similar concentrations of H2O2 did not significantly influence EO and PMN adhesion. HOSCN did not affect PMN or EO binding to immobilized VCAM-1. Pretreatment of EO with 33–150 μM HOSCN for 15 min prior to washing out HOSCN and layering them over TNF-α-activated HUVEC monolayers for 30 min stimulated a 5–15-fold increase in both EO and PMN adhesion that was > 75% inhibited by blocking antibodies against ICAM-1 and VCAM-1 (EO only) as well as CD11b. Assessing the potential mechanism behind HOSCN enhancing of CD11b/18-dependent EO and PMN adhesion, flow cytometry experiments showed HOSCN did not induce total cell surface expression of EO CD11b/18, but confocal microscopy showed prominent clustering of CD11b 20 min after exposure. Hypothesizing that the “stress” kinase p38 MAPK plays a role in HOSCN promotion of EO adhesion, we probed western blots whole cell lysates of EO exposed 1–10 min to 75 μM HOSCN for activated phospho-p38 MAPK and found a pronounced (10x baseline) but evanescent (1–2 min long) increase starting at 2 min. 1 μM SB203580, a potent and specific p38 MAPK inhibitor, blocked by >80% EO and PMN HOSCN stimulation of adhesion to both immobilized ICAM-1 and activated HUVEC. Collectively, these data show that HOSCN is a uniquely effective phagocyte oxidant inducer of both EO and PMN binding to ICAM-1 by a mechanism depending upon activation of p38 MAPK and enhancement of the CD11b/18-ICAM-1 interaction. This, in turn, suggests that endogenous MPO- and EPO-generated HOSCN may play a hitherto unsuspected physiologic autocrine amplification role in the regulation of adhesion and, hence, inflammation. In view of this, p38 MAPK inhibitors may have promise as therapeutic inhibitors in pathologic human inflammatory states, especially allergic diseases involving EOs. Disclosures:No relevant conflicts of interest to declare.

2B4 (CD244) is Involved in Eosinophil Adhesion and Chemotaxis, and its Surface Expression is Increased in Allergic Rhinitis after Challenge

A role for the subtypes of CD2 Ig superfamily receptors has been recently demonstrated in eosinophilic inflammation in experimental asthma and atopic asthmatics. We investigated the functions of 2B4 (CD244) molecules in eosinophil adhesion and chemotaxis, and correlated the results to the pathophysiology of allergic rhinitis (AR). Herein, we show that agonistic stimulation of 2B4 by C1.7, the anti-human 2B4 functional grade purified antibody, resulted in significant increase of eosinophils and eosinophil cell line (Eol-1 cells) adhesion to collagen type IV, and random migration. These functions were associated with tyrosine kinase phosphorylation of several protein residues of low molecular weight. Flow cytometry (FACS) experiments demonstrated that Eol-1 cells, normal peripheral blood eosinophils and eosinophils from AR patients, express surface 2B4 molecules. In vitro AR model demonstrated that the CC-chemokine receptor CCR3 stimulation by eotaxin induced significant increase in the expression of surface 2B4 in eosinophils and Eol-1 cells. Immunofluorescence confocal microscopy images showed that eotaxin induces also redistribution of 2B4 molecules towards the pseudopods in eosinophils and Eol-1 cells, changing their shape. Blocking of 2B4 molecules by the corresponding neutralizing antibody inhibited eotaxin induced Eol-1-adhesion, chemotaxis and the cytoskeleton changes. Pretreatment of Eol-1 cells with 1 microM genistein blocked eotaxin-induced Eol-1 adhesion, chemotaxis and 2B4 up-regulated expression. In vivo correlation demonstrated the expression of 2B4 molecules in eosinophils from AR patients to be significantly increased, after nasal provocation challenge. These results identify a novel role for 2B4 molecules in eosinophil functional migratory response and may point to a novel tyrosine kinase-mediated ligation between CCR3 receptor and 2B4 co-receptor in eosinophil chemotaxis. If so, then 2B4 molecules would be a novel target for therapeutic modalities in diseases characterized by eosinophilia such as AR.

Collagenous Colitis: Rare Case of Seasonal and Environmental Exacerbation of Collagenous Colitis Responding to Montelukast

Purpose: Background: Collagenous colitis (CC) is a cause of chronic diarrhea characterized by grossly normal mucosa and a sub-epithelial band of collagen. We report a case of CC showing an exacerbation pattern to environmental change and an excellent response to treatment with montelukast in the setting of biopsy proven CC with increased submucosal eosinophils. Case: A 70 year old relatively healthy white male with a baseline history of allergic rhinitis presented with a three month history of profuse watery diarrhea. Colon biopsy revealed CC. He responded to pepto-bismol and two years later to asacol on such an exacerbation. A year later he developed refractory diarrhea to Pepto-Bismol, loperemide, asacol, cholestyramine and he refused steroids. Colon biopsy revealed CC with increased eosinophils. WBC count was normal with a normal differential and normal ESR. Stool microscopy and serology was negative for Giardia, Cryptospora, Salmonella, Shigella, E. coli O157 and Clostridium difficile toxin. Serum IgA, tissue trans-glutaminase, IBD-7panel and liver function panel were unremarkable. Skin testing for an extensive food panel was unrevealing. The diarrhea responded well to a cromolyn taper and continual montelukast therapy. Thereafter, episodes of diarrhea recurred each time montelukast was stopped. One year post montelukast treatment colon biopsy revealed decreased number of eosinophils with a persistent collagen band. Interestingly, this patient would have no recurrences in winter when he vacationed in Florida prior to starting montelukast. Discussion: The seasonal pattern of his symptoms occurring in summer in Rochester, New York and resolution of diarrhea from his Florida vacation in winter may suggest an unknown environmental allergen or an infectious etiology. The pathogenesis may be similar to sub-epithelial fibrosis caused in irritant induced asthma (irla) by eosinophil induced inflammation. Colon perfusion models in CC and bronchial washings in irla show increased eosinophilic cationic protein, fibroblast growth factor. Interestingly, CC reverses completely with diverting ileostomies, pointing to luminal factors causing CC. This patient's sustained response to treatment with montelukast may be explained by the fact that montelukast decreases the levels of inducible nitric oxide synthase (iNOS) and may alter eosinophil adhesion as shown in experimental models. Conclusion: The etiology of CC is speculative. CC may respond to montelukast therapy in steroid resistant/averse patients especially in the setting of increased eosinophils on histology and patients having other systemic allergy symptoms. Well powered studies are necessary to compare it head-on to budesonide.

GM-CSF Production by Glioblastoma Cells Has a Functional Role in Eosinophil Survival, Activation, and Growth Factor Production for Enhanced Tumor Cell Proliferation

Medicinal interventions of limited efficacy are currently available for the treatment of glioblastoma multiforme (GBM), the most common and lethal primary brain tumor in adults. The eosinophil is a pivotal immune cell in the pathobiology of atopic disease that is also found to accumulate in certain tumor tissues. Inverse associations between atopy and GBM risk suggest that the eosinophil may play a functional role in certain tumor immune responses. To assess the potential interactions between eosinophils and GBM, we cultured human primary blood eosinophils with two separate human GBM-derived cell lines (A172, U87-MG) or conditioned media generated in the presence or absence of TNF-α. Results demonstrated differential eosinophil adhesion and increased survival in response to coculture with GBM cell lines. Eosinophil responses to GBM cell line-conditioned media included increased survival, activation, CD11b expression, and S100A9 release. Addition of GM-CSF neutralizing Abs to GBM cell cultures or conditioned media reduced eosinophil adhesion, survival, and activation, linking tumor cell-derived GM-CSF to the functions of eosinophils in the tumor microenvironment. Dexamethasone, which has been reported to inhibit eosinophil recruitment and shrink GBM lesions on contrast-enhanced scans, reduced the production of tumor cell-derived GM-CSF. Furthermore, culture of GBM cells in eosinophil-conditioned media increased tumor cell viability, and generation of eosinophil-conditioned media in the presence of GM-CSF enhanced the effect. These data support the idea of a paracrine loop between GM-CSF-producing tumors and eosinophil-derived growth factors in tumor promotion/progression.

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

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.