Eosinophil Adhesion Research Articles

The role of IL-36γ and its regulation in eosinophilic inflammation in allergic rhinitis

BackgroundAllergic rhinitis (AR) is characterized by eosinophilic inflammation. However, the function and regulation of eosinophils in AR are largely unknown. This study aimed to explore the expression and role of interleukin-36 (IL-36) cytokines in AR. MethodsSixty AR patients and 20 control subjects were recruited in this study. The mRNA and protein expression of serum IL-36 family cytokines and IL-36R in AR were detected by quantitative RT-PCR and enzyme-linked immunosorbent assay ELISA, respectively. IL-36R expression and regulation by eosinophils and the role of IL-36γ in the survival, adhesion, migration and activation of eosinophils were performed in purified eosinophils. Human nasal epithelial cell line was cultured and treated with different stimulators and IL-36γ was measured. ResultsThe mRNA and protein expression of serum IL-36 cytokines and IL-36R were significantly higher in AR compared with control, especially in asthmatic patients. Among the IL-36 cytokines, the expression of IL-36γ was the highest. The expression of IL-36R by eosinophils were significantly increased compared with normal controls and was up-regulated by recombinant IL-17, IL-25, IL-33 and Dermatophagoides pteronyssinus group 1. The IL-36γ promote the survival, adhesion, migration and activation of eosinophils. Human nasal epithelial cells can secrete IL-36γ after treated with recombinant IL-17, IL-25, IL-33. ConclusionsHigh expression of IL-36γ exaggerates eosinophilic inflammation in AR by promoting the survival, adhesion, and activation of eosinophils.

Dermatophagoides farinae Upregulates the Effector Functions of Eosinophils through αMβ2-Integrin and Protease-Activated Receptor-2

Background: Even in subjects who are not sensitized to house dust mite (HDM), allergic symptoms can be aggravated by exposure to dust, suggesting that innate immune responses may be involved in these processes. Since eosinophils express pattern recognition receptors, HDM may directly upregulate eosinophil functions through these re ceptors. The objective of this study was to examine whether Dermatophagoides farinae (Df), a representative HDM, or Der f 1, a major allergen of Df, modifies the effector functions of eosinophils. Methods: Eosinophils isolated from the blood of healthy donors or allergic patients were stimulated with Df extract or Der f 1, and their adhesion to recombinant human intercellular adhesion molecule (ICAM)-1 was measured using eosinophil peroxidase assays. Generation of the eosinophil superoxide anion (O₂^–) was examined based on the superoxide dismutase-inhibitable reduction of cytochrome C. Eosinophil-derived neurotoxin (EDN) concentrations in cell media were measured by ELISA as a marker of degranulation. Results: Df extract or Der f 1 directly induced eosinophil adhesion to ICAM-1, O₂^– generation, and EDN release. Anti-αM- or anti-β₂-integrin antibodies or protease-activated receptor (PAR)-2 antagonists suppressed the eosinophil adhesion, O₂^– generation, and EDN release induced by Df extract or Der f 1. Eosinophils from allergic patients showed higher adhesion to ICAM-1 than those from healthy donors. Conclusions: These findings suggested that Df extract and Der f 1 directly activate eosinophil functions through αMβ₂-integrin and PAR-2. Eosinophil activation by HDM may play roles in the aggravation of allergic symptoms, not only in HDM-sensitized patients, but also in nonsensitized patients.

Role of Leptin/Osteopontin Axis in the Function of Eosinophils in Allergic Rhinitis with Obesity.

Background Allergic rhinitis (AR) is characterized by tissue and blood eosinophilia. Previous studies showed enhanced eosinophilia in allergic rhinitis patients with obesity, suggesting an association between obesity and eosinophilia. However, the interaction and mechanism between obesity and eosinophilia is still unclear. Methods We recruited thirty AR children and 30 controls in this study. Expression of leptin and osteopontin (OPN) proteins in serum was detected, and correlation analysis with eosinophilia was performed. The effect of leptin or OPN on eosinophil apoptosis, adhesion, migration, and activation of eosinophil was examined. Ovalbumin-sensitized mice were established to prove the role of obesity on eosinophil regulation by leptin and OPN. Results We found that upregulated serum and nasal leptin and OPN expression in AR were positively correlated with eosinophilia and eosinophil cationic protein levels. Leptin or OPN inhibited eosinophil apoptosis, demonstrated as inhibited DNA fragmentation and phosphatidylserine (PS) redistribution (P < 0.05). Leptin and OPN promote expression of cluster of differentiation 18 (CD-18) and intercellular adhesion molecule 1 (ICAM-1) and inhibit expression of ICAM-1 and L-selectin by eosinophils, which contribute to the adhesion of eosinophils. Leptin and OPN mediated migration and activation of eosinophil through phosphatidylinositol-3-OH kinase (PI3K) pathway. Obese AR mice presented with more severe eosinophilia and symptoms compared with nonobese AR mice or control mice. Immunochemistry staining of leptin and OPN of nasal turbinate in obese AR mice was also stronger than those in nonobese AR mice or control mice. Anti-OPN, anti-leptin, and anti-α4 treatments reduce nasal eosinophilia inflammation and clinical symptoms in model mice. Conclusion Our results suggested that in an obese state, upregulation of leptin and OPN regulates apoptosis, adhesion, migration, and activation of eosinophils, and this process may be mediated by the PI3K and anti-α4 pathways.

Involvement and Possible Role of Eosinophils in Asthma Exacerbation.

Eosinophils are involved in the development of asthma exacerbation. Recent studies have suggested that sputum and blood eosinophil counts are important factors for predicting asthma exacerbation. In severe eosinophilic asthma, anti-interleukin (IL)-5 monoclonal antibody decreases blood eosinophil count and asthma exacerbation frequency. However, even in the absence of IL-5, eosinophilic airway inflammation can be sufficiently maintained by the T helper (Th) 2 network, which comprises a cascade of vascular cell adhesion molecule-1/CC chemokines/eosinophil growth factors, including granulocyte-macrophage colony-stimulating factor (GM-CSF). Periostin, an extracellular matrix protein and a biomarker of the Th2 immune response in asthma, directly activates eosinophils in vitro. A major cause of asthma exacerbation is viral infection, especially rhinovirus (RV) infection. The expression of intercellular adhesion molecule (ICAM)-1, a cellular receptor for the majority of RVs, on epithelial cells is increased after RV infection, and adhesion of eosinophils to ICAM-1 can upregulate the functions of eosinophils. The expressions of cysteinyl leukotrienes (cysLTs) and CXCL10 are upregulated in virus-induced asthma. CysLTs can directly provoke eosinophilic infiltration in vivo and activate eosinophils in vitro. Furthermore, eosinophils express the CXC chemokine receptor 3, and CXCL10 activates eosinophils in vitro. Both eosinophils and neutrophils contribute to the development of severe asthma or asthma exacerbation. IL-8, which is an important chemoattractant for neutrophils, is upregulated in some cases of severe asthma. Lipopolysaccharide (LPS), which induces IL-8 from epithelial cells, is also increased in the lower airways of corticosteroid-resistant asthma. IL-8 or LPS-stimulated neutrophils increase the transbasement membrane migration of eosinophils, even in the absence of chemoattractants for eosinophils. Therefore, eosinophils are likely to contribute to the development of asthma exacerbation through several mechanisms, including activation by Th2 cytokines, such as IL-5 or GM-CSF or by virus infection-related proteins, such as CXCL10, and interaction with other cells, such as neutrophils.

Control of cytokine-driven eosinophil migratory behavior by TGF-beta-induced protein (TGFBI) and periostin.

Periostin, which is induced by interleukin (IL)-13, is an extracellular matrix (ECM) protein that supports αMβ2 integrin-mediated adhesion and migration of IL-5-stimulated eosinophils. Transforming growth factor (TGF)-β-induced protein (TGFBI) is a widely expressed periostin paralog known to support monocyte adhesion. Our objective was to compare eosinophil adhesion and migration on TGFBI and periostin in the presence of IL-5-family cytokines. Eosinophil adhesion after 1 h and random motility over 20 h in the presence of various concentrations of IL-5, IL-3, or granulocyte macrophage-colony stimulating factor (GM-CSF) were quantified in wells coated with various concentrations of TGFBI or periostin. Results were compared to video microscopy of eosinophils. Cytokine-stimulated eosinophils adhered equivalently well to TGFBI or periostin in a coating concentration-dependent manner. Adhesion was blocked by anti-αMβ2 and stimulated at the lowest concentration by GM-CSF. In the motility assay, periostin was more potent than TGFBI, the coating-concentration effect was bimodal, and IL-3 was the most potent cytokine. Video microscopy revealed that under the optimal coating condition of 5 μg/ml periostin, most eosinophils migrated persistently and were polarized and acorn-shaped with a ruffling forward edge and granules gathered together, in front of the nucleus. On 10 μg/ml periostin or TGFBI, more eosinophils adopted a flattened pancake morphology with dispersed granules and nuclear lobes, and slower migration. Conversion between acorn and pancake morphologies were observed. We conclude that TGFBI or periostin supports two modes of migration by IL-5 family cytokine-activated eosinophils. The rapid mode is favored by intermediate protein coatings and the slower by higher coating concentrations. We speculate that eosinophils move by haptotaxis up a gradient of adhesive ECM protein and then slow down to surveil the tissue.

FABP4 regulates eosinophil recruitment and activation in allergic airway inflammation.

Fatty acid binding protein 4 (FABP4), a member of a family of lipid-binding proteins, is known to play a role in inflammation by virtue of its ability to regulate intracellular events such as lipid fluxes and signaling. Studies have indicated a proinflammatory role for FABP4 in allergic asthma although its expression and function in eosinophils, the predominant inflammatory cells recruited to allergic airways, were not investigated. We examined expression of FABP4 in murine eosinophils and its role in regulating cell recruitment in vitro as well as in cockroach antigen (CRA)-induced allergic airway inflammation. CRA exposure led to airway recruitment of FABP4-expressing inflammatory cells, specifically eosinophils, in wild-type (WT) mice. FABP4 expression in eosinophils was induced by TNF-α as well as IL-4 and IL-13. FABP4-deficient eosinophils exhibited markedly decreased cell spreading/formation of leading edges on vascular cell adhesion molecule-1 and significantly decreased adhesion to intercellular adhesion molecule-1 associated with reduced β2-integrin expression relative to WT cells. Furthermore, FABP4-deficient eosinophils exhibited decreased migration, F-actin polymerization, calcium flux, and ERK(1/2) phosphorylation in response to eotaxin-1. In vivo, CRA-challenged FABP4-deficient mice exhibited attenuated eosinophilia and significantly reduced airway inflammation (improved airway reactivity, lower IL-5, IL-13, TNF-α, and cysteinyl leukotriene C4 levels, decreased airway structural changes) compared with WT mice. In conclusion, expression of FABP4 in eosinophils is induced during conditions of inflammation and plays a proinflammatory role in the development of allergic asthma by promoting eosinophil adhesion and migration and contributing to the development of various aspects of airway inflammation.

Activation of IL5R and CRTH2 on Human Eosinophils Elicit a Similar Molecular Response and Reveal a Synergistic Effect

Eosinophils represent a key cell driving inflammatory processes in diseases such as asthma and eosinophilic esophagitis. The entire process of migration and activation of eosinophils is orchestrated by a myriad of molecules, among which interleukin 5 (IL5) plays a crucial role. Another receptor expressed on eosinophils is chemo-attractant receptor like expressed on Th2 cells (CRTH2). CRTH2 activation has a prominent role in stimulating Th2 and type 2 innate lymphoid cells to produce IL5 in disease tissue, but also induces eosinophils migration and adhesion. Prostaglandin D2 is the endogenous ligand of CRTH2, predominately released by activated mast cells, another key cell involved in eosinophilic diseases. Consequently, the IL5 and CRTH2 pathway are likely triggered simultaneously in eosinophils in disease tissue. Understanding the molecular effects of CRTH2 and IL5 receptor (IL5R) activated eosinophils might provide new insights for efficient eosinophils blocking. We performed gene expression profiling with isolated human eosinophils to compare the molecular signatures of CRTH2 and IL5R activation. The identified genes were confirmed using an orthogonal technology, and simultaneous IL5R and CRTH2 activation was investigated on the protein level. This study reveals that IL5R and CRTH2 activation trigger a similar molecular response in eosinophils with stronger IL5R effects. C-C chemokine ligand 2 was produced synergistically upon IL5R and CRTH2 stimulation and might contribute to monocyte and macrophage recruitment to disease tissue. The effect on C-C chemokine receptor 1 suggests that eosinophils maturation in the tissue might even occur in absence of IL5 via the CRTH2 pathway.

Inhibiting focal adhesion kinase (FAK) blocks IL-4 induced VCAM-1 expression and eosinophil recruitment in vitro and in vivo.

Leukocyte recruitment plays a critical role during both normal inflammation and chronic inflammatory diseases, and ongoing studies endeavor to better understand the complexities of this process. Focal adhesion kinase (FAK) is well known for its role in cancer, yet it also has been shown to regulate aspects of neutrophil and B16 melanoma cell recruitment by rapidly influencing endothelial cell focal adhesion dynamics and junctional opening. Recently, we found that FAK related non-kinase (FRNK), a protein that is often used as a FAK dominant negative, blocked eosinophil transmigration by preventing the transcription of vascular cell adhesion molecule-1 (VCAM-1) and eotaxin-3 (CCL26). Surprisingly, the blocking occurred even in the absence of endogenous FAK. To better understand the role of FAK in leukocyte recruitment, we used a FAK-specific inhibitor (PF-573228) and determined the effect on IL-4 induced eosinophil recruitment in vitro and in vivo. PF-573228 prevented the expression of VCAM-1 and CCL26 expression in IL-4-stimulated human endothelial cells in vitro. As a result, eosinophil adhesion and transmigration were blocked. PF-572338 also prevented IL-4-induced VCAM-1 expression in vivo. Using brightfield intravital microscopy, we found that PF-573228 decreased leukocyte rolling flux, adhesion, and emigration. We specifically examined eosinophil recruitment in vivo by using an eosinophil-GFP reporter mouse and found PF-573228 attenuated eosinophil emigration. This study reveals that a FAK inhibitor influences inflammation through its action on eosinophil recruitment.

Fasudil, a ROCK inhibitor, attenuates endotelial‐leukocyte interaction in sickle cell transgenic mice

IntroductionSickle cell disease (SCD) is one of the most common genetic disorder worldwide, caused by point mutations in the gene encoding the hemoglobin β chain. The most common form of SCD is homozygous sickle cell anemia (SCA; HbSS), where the presence of two βs globin alleles results in an almost exclusive production of hemoglobin S (HbS) instead of HbA in erythrocytes. In SCD, leukocyte plays an important role in the pathophysiology of disease, and leukocyte adhesion may be, in fact, the initiating event in vaso‐occlusive episodes. The neutrophil appears to be the leukocyte that most participates in the vaso‐occlusive process. Changes in the Rho‐A/Rho‐kinase signaling pathway can modulate some pathophysiological aspects of the sickle cell disease, such as priapism, increased ROS production and altered sickle cell cytoskeletal phosphorylation. In addition, Rho kinase inhibitors were able to reduce endothelial activation and consequent eosinophil adhesion in vitro and reduced the allergic lung inflammation in SCA mice.ObjectiveThe aim of this study was to investigate the effect of acute treatment with Fasudil, a specific inhibitor of Rho‐kinase, in the initial steps of the leukocytes transmigration processes in an in vivo model of inflammation‐induced vaso‐occlusion in SCA mice model.MethodsInflammation was induced by the administration of TNF‐α (0.5 μg, i.p.) in male HbSS knock‐in mouse model of SCA (Townes; Wu et al., Blood 2006) and C57BL/6JuniB control mice. Fasudil (10 mg/kg) was intraperitoneally administrated in the animals 1 h before TNF‐α. Four hours later, the animals were surgically prepared for intravital microscopy of the microvasculature of the cremaster muscle.ResultsIntravital microscopy demonstrated that TNF‐α induced an increase in the number of rolling and adhesion leukocytes to endothelium and accumulation of leukocytes outside the vessels. However, this augment is significantly higher in SCA mice compared to control animals (Rolling: 15.8±2.3 and 4.5±0.7 leukocyte min−1; Adhesion: 15.3±0.7 and 10.1±0.6 leukocyte adhered 100μm−1; Extravasated: 5.7±0.5 and 3.5±0.5 leukocyte ×100×50 μm2, p&lt;0.05, respectively). Pre‐treatment with Fasudil had no effect on leukocyte rolling (10.5±1.8 leukocyte min−1), however, significantly reduced firm adhesion by 30% (11.1±0.8 leukocyte adhered 100μm−1) and decreased leukocyte extravasation by 46% (3.1±0.4 leukocyte ×100×50 μm2) in SCA mice.ConclusionIn summary, our data demonstrated that Rho‐kinase signaling regulates inflammation‐induced vaso‐occlusion in SCA mice model in vivo and that Rho‐kinase inhibition may be useful in the treatment of vaso‐occlusion process in SCA, limiting the infiltration of leukocytes, and reducing vascular inflammation.Support or Funding InformationFAPESPThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MHC Class II Activation and Interferon-γ Mediate the Inhibition of Neutrophils and Eosinophils by Staphylococcal Enterotoxin Type A (SEA).

Staphylococcal enterotoxins are classified as superantigens that act by linking T-cell receptor with MHC class II molecules, which are expressed on classical antigen-presenting cells (APC). Evidence shows that MHC class II is also expressed in neutrophils and eosinophils. This study aimed to investigate the role of MHC class II and IFN-γ on chemotactic and adhesion properties of neutrophils and eosinophils after incubation with SEA. Bone marrow (BM) cells obtained from BALB/c mice were resuspended in culture medium, and incubated with SEA (3–30 ng/ml; 1–4 h), after which chemotaxis and adhesion were evaluated. Incubation with SEA significantly reduced the chemotactic and adhesive responses in BM neutrophils activated with IL-8 (200 ng/ml). Likewise, SEA significantly reduced the chemotactic and adhesive responses of BM eosinophils activated with eotaxin (300 ng/ml). The inhibitory effects of SEA on cell chemotaxis and adhesion were fully prevented by prior incubation with an anti-MHC class II blocking antibody (2 μg/ml). SEA also significantly reduced the intracellular Ca2+ levels in IL-8- and eotaxin-activated BM cells. No alterations of MAC-1, VLA4, and LFA-1α expressions were observed after SEA incubation. In addition, SEA elevated by 3.5-fold (P < 0.05) the INF-γ levels in BM cells. Incubation of BM leukocytes with IFN-γ (10 ng/ml, 2 h) reduced both neutrophil and eosinophil chemotaxis and adhesion, which were prevented by prior incubation with anti-MHC class II antibody (2 μg/ml). In conclusion, SEA inhibits neutrophil and eosinophil by MHC class II-dependent mechanism, which may be modulated by concomitant release of IFN-γ.

Activation of human eosinophils with palladium nanoparticles (Pd NPs): importance of the actin cytoskeleton in Pd NPs-induced cellular adhesion

Palladium (Pd) is known to be released into the environment in the fine and ultrafine (at the nanoscale) airborne particle fractions mainly from automobile catalytic converters leading to an increase human exposure to this noble metal. It was reported that Pd can induce allergic reactions in individuals exposed to it via different ways. Some studies reported an increased number of eosinophils into airways following NP exposure in vivo in rodent models of allergies and inflammation. Knowing the importance of eosinophils in allergies, asthma and other lung diseases, it is surprising to observe that the direct effect of Pd at the nanoscale in eosinophils has been poorly documented. The aim of this study was to determine how Pd NPs will affect the biology of human eosinophils. Characterization of Pd NPs by dynamic light scattering indicates the presence of some aggregates when suspended in diverse solutions used here for the different experiments. Pd NPs did not significantly induce cell necrosis and apoptosis in eosinophils (0.5–150μg/ml) as assessed by trypan blue exclusion assay, flow cytometry after staining with FITC-annexin V and propidium iodide and by morphological observations by optical microscopy. PD NPs, unlike the positive controls, did not induce reactive oxygen species (ROS) but were found to target the actin cytoskeleton, since actin was differently re-located intracellularly when compared to untreated cells as determined by fluorescence microscopy. Clearly, Pd NPs were found to increase adhesion of eosinophils onto human endothelial EA.hy926 cells. Using cytochalasin D, a cell-permeable and potent inhibitor of actin polymerization, this ability to increase adhesion was drastically reversed. Our results indicate that Pd NPs can target the cytoskeleton and increase the adhesion of human eosinophils by an actin-dependent mechanism. These findings show that human eosinophils can be activated by Pd NPs emphasizing the importance of fully investigating how these NPs could alter the biology of human cells involved in allergies, asthma and other lung diseases as well as in various other inflammatory disorders.

Rock Inhibitor Fasudil Reduces Leukocyte-Endothelium Interactions in the Microvasculature of a Sickle Cell Mouse Model of Allergic Inflammation

Introduction: Sickle cell disease (SCD) vaso-occlusion process involves different cell types, such as red blood cells,activated endothelial cells, platelets and leukocytes. Endothelialdysfunction contributes to the vaso-occlusion process and leadsto inflammation. Data suggest that Rho-kinase signaling may regulate numerous aspects of the inflammatory process. Alterations in the Rho-A/Rho-kinase signaling pathway modulate pathophysiological aspects of the sickle cell disease such as priapism, and these enzymes are involved in increased reactive oxygen species generation and altered sickle cell cytoskeletal phosphorylation. In addition, Rho kinase inhibitors were able to reduce endothelial activation and consequent eosinophil adhesion in vitro and reduced allergic inflammation in the lungs of SCD mice. However, the involvement of Rho/Rho-kinase signaling pathways in the mechanism underlying systemic vascular occlusion in SCD remains unclear. This study aimed to determine whether Rho/Rho-kinase pathways are involved in the mechanism of microvascular SCD vaso-occlusion. We investigated the effect of fasudil, a specific inhibitor of Rho-kinase, in the initial steps of the leukocyte transmigration process in a model of allergic inflammation using intravital microscopy of the cremaster muscle in SCD mice. Methods: Experimental groups consisted of male homozygous Tim Townes transgenic sickle cell mice and C57BL/6JuniB control mice. SCD and control mice were actively sensitized with a subcutaneous injection of 100 μg of ovalbumin (OVA) mixed with 1.6 mg Al(OH)3 in 0.9% NaCl (Day zero). On day 7, mice received a second injection of 100 μg of OVA. On day 14, mice were subcutaneously challenged with eotaxin (100 ng/per dose) with or without pre-treatment with intraperitoneal fasudil (10 mg/kg) 1 hour before eotaxin. Four hours later, the animals were surgically prepared for intravital microscopy of the microvasculature of the cremaster muscle. Results: Intravital microscopy showed that eotaxin challenge in OVA-sensitized animals increased rolling and adhesion of leukocytes to the endothelium, and accumulation of leukocytes outside the vessels was observed. However, this increase is significantly higher in SCD mice compared to control animals (Rolling: 23±1.9 and 15±1.2 leukocyte min-1; Adhesion: 16.3±0.9 and 11.7±0.5 leukocyte adhered 100µm-1; Extravasation: 3.88±0.3 and 2.4±0.2 leukocyte perx100x50 µm2, p<0.05, respectively). The leukocyte rolling flow was similarly inhibited by fasudil treatment in control animals by 50% and in SCD mice by 42%. Eotaxin-induced firm adhesion after fasudil was also reduced by 57% in control animals and 63% in SCD mice. Notably, pre-treatment with fasudil caused a greater decrease in leukocyte extravasation in SCD mice (44%) than in control animals (16%), p<0.0001. Conclusion: Our data show that inhibition of Rho-kinase decreased endothelial-leukocyte interaction. These findings suggest that Rho-kinase inhibitors may have therapeutic benefits in the vaso-occlusive process in SCD, limiting the extravasation of leukocytes, and reducing vascular inflammation. DisclosuresConran:Bayer AG: Research Funding. Fertrin:Alexion Pharmaceuticals: Consultancy.

Titanium dioxide nanoparticles induce human eosinophil adhesion onto endothelial EA.hy926 cells via activation of phosphoinositide 3-kinase/Akt cell signalling pathway

The use of nanoparticles (NPs) for developing new therapeutic strategies in a variety of diseases is gaining increasing attention. However, NPs could possess undesired effects, including pro-inflammatory activities. Despite the fact that several studies reported that NPs may induce or exacerbate eosinophilic inflammation in vivo in rodents, the information regarding the direct interaction between NPs and human eosinophils is lacking. In the present study, we test the possibility that NPs could alter the capacity of human eosinophils to adhere onto a cellular substratum. Using a panel of NPs, we found that several were able to increase the adhesion of human eosinophil onto endothelial EA.hy926 cells. Among them, TiO2 NPs were the most potent and we therefore pursue this study with these NPs. TiO2 NPs were found to increase the adhesion of eosinophils in a concentration dependent fashion. TiO2 NPs did not alter the cell surface expression of a panel of cellular adhesion molecules, but CD29. Indeed, a weak to moderate, but significant, decrease of CD29 was observed after 30min but returned to normal levels after 90min. TiO2 NPs were found to activate Akt, one important target of phosphoinositide 3-kinase (PI3K). However, despite the fact that cells were fully responsive to the cytokine GM-CSF activating both Akt and Erk-1/2, TiO2 NPs did not activate Erk-1/2. Using a pharmacological approach with the PI3K/Akt inhibitor, wortmannin, the ability of TiO2 NPs to activate Akt was drastically inhibited and, further, their capacity to increase adhesion of eosinophils was reversed. This study provides insights into the effects of NPs on the biology of human eosinophils indicating that as other agents, NPs, namely TiO2 NPs, can induce intracellular events associated with a cellular function, adhesion.

Sialic acid–binding immunoglobulin-like lectin 8 (Siglec-8) is an activating receptor mediating β2-integrin–dependent function in human eosinophils

Siglec-8 is a CD33 subfamily cell-surface receptor selectively expressed on human eosinophils. After cytokine priming, Siglec-8 mAb or glycan ligand binding causes eosinophil apoptosis associated with reactive oxygen species (ROS) production. Most CD33-related Siglecs function as inhibitory receptors, but the ability of Siglec-8 to stimulate eosinophil ROS production and apoptosis suggests that Siglec-8 might instead function as an activating receptor. We sought to determine the role of IL-5 priming and identify the signaling molecules involved in Siglec-8 function for human eosinophils. We used an mAb and/or a multimeric synthetic sulfated sialoglycan ligand recognizing Siglec-8 in combination with integrin blocking antibodies, pharmacologic inhibitors, phosphoproteomics, and Western blot analysis to define the necessity of various proteins involved in Siglec-8 function for human eosinophils. Cytokine priming was required to elicit the unanticipated finding that Siglec-8 engagement promotes rapid β2-integrin-dependent eosinophil adhesion. Also novel was the finding that this adhesion was necessary for subsequent ROS production and apoptosis. Siglec-8-mediated ROS was generated through reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation because pretreatment of eosinophils with catalase (an extracellular superoxide scavenger) or NSC 23766 (a Rac GTPase inhibitor) completely inhibited Siglec-8-mediated eosinophil apoptosis. Finally, engagement of Siglec-8 on IL-5-primed eosinophils resulted in increased phosphorylation of Akt, p38, and c-Jun N-terminal kinase 1 that was also β2-integrin dependent; pharmacologic inhibition of these kinases completely prevented Siglec-8-mediated eosinophil apoptosis. These data demonstrate that Siglec-8 functions uniquely as an activating receptor on IL-5-primed eosinophils through a novel pathway involving regulation of β2-integrin-dependent adhesion, NADPH oxidase, and a subset of protein kinases.

Platelet-Eosinophil Interactions As a Potential Therapeutic Target in Allergic Inflammation and Asthma.

The importance of platelet activation during hemostasis is well understood. An understanding of these mechanisms has led to the use of several classes of anti-platelet drugs to inhibit aggregation for the prevention of thrombi during cardiovascular disease. It is now also recognized that platelets can function very differently during inflammation, as part of their role in the innate immune response against pathogens. This dichotomy in platelet function occurs through distinct physiological processes and alternative signaling pathways compared to that of hemostasis (leading to platelet aggregation) and is manifested as increased rheological interactions with leukocytes, the ability to undergo chemotaxis, communication with antigen-presenting cells, and direct anti-pathogen responses. Mounting evidence suggests platelets are also critical in the pathogenesis of allergic diseases such as asthma, where they have been associated with antigen presentation, bronchoconstriction, bronchial hyperresponsiveness, airway inflammation, and airway remodeling in both clinical and experimental studies. In particular, platelets have been reported bound to eosinophils in the blood of patients with asthma and the incidence of these events increases after both spontaneous asthma attacks in a biphasic manner, or after allergen challenge in the clinic. Platelet depletion in animal models of allergic airway inflammation causes a profound reduction in eosinophil recruitment to the lung, suggesting that the association of platelets with eosinophils is indeed an important event during eosinophil activation. Furthermore, in cases of severe asthma, and in animal models of allergic airways inflammation, platelet–eosinophil complexes move into the lung through a platelet P-selectin-mediated, eosinophil β1-integrin activation-dependent process, while platelets increase adherence of eosinophils to the vascular endothelium in vitro, demonstrating a clear interaction between these cell types in allergic inflammatory diseases. This review will explore non-thrombotic platelet activation in the context of allergy and the association of platelets with eosinophils, to reveal how these phenomena may lead to the discovery of novel therapeutic targets.

IL-5-stimulated eosinophils adherent to periostin undergo stereotypic morphological changes and ADAM8-dependent migration.

IL-5 causes suspended eosinophils to polarize with filamentous (F)-actin and granules at one pole and the nucleus in a specialized uropod, the "nucleopod," which is capped with P-selectin glycoprotein ligand-1 (PSGL-1). IL-5 enhances eosinophil adhesion and migration on periostin, an extracellular matrix protein upregulated in asthma by type 2 immunity mediators. Determine how the polarized morphology evolves to foster migration of IL-5-stimulated eosinophils on a surface coated with periostin. Blood eosinophils adhering to adsorbed periostin were imaged at different time points by fluorescent microscopy, and migration of eosinophils on periostin was assayed. After 10minutes in the presence of IL-5, adherent eosinophils were polarized with PSGL-1 at the nucleopod tip and F-actin distributed diffusely at the opposite end. After 30-60minutes, the nucleopod had dissipated such that PSGL-1 was localized in a crescent or ring away from the cell periphery, and F-actin was found in podosome-like structures. The periostin layer, detected with monoclonal antibody Stiny-1, shown here to recognize the FAS1 4 module, was cleared in wide areas around adherent eosinophils. Clearance was attenuated by metalloproteinase inhibitors or antibodies to disintegrin metalloproteinase 8 (ADAM8), a major eosinophil metalloproteinase previously implicated in asthma pathogenesis. ADAM8 was not found in podosome-like structures, which are associated with proteolytic activity in other cell types. Instead, immunoblotting demonstrated proteoforms of ADAM8 that lack the cytoplasmic tail in the supernatant. Anti-ADAM8 inhibited migration of IL-5-stimulated eosinophils on periostin. Migrating IL-5-activated eosinophils on periostin exhibit loss of nucleopodal features and appearance of prominent podosomes along with clearance of the Stiny-1 periostin epitope. Migration and epitope clearance are both attenuated by inhibitors of ADAM8. We propose, therefore, that eosinophils remodel and migrate on periostin-rich extracellular matrix in the asthmatic airway in an ADAM8-dependent manner, making ADAM8 a possible therapeutic target.

Adhesion-induced eosinophil cytolysis requires the receptor-interacting protein kinase 3 (RIPK3)–mixed lineage kinase-like (MLKL) signaling pathway, which is counterregulated by autophagy

Eosinophils are a subset of granulocytes that can be involved in the pathogenesis of different diseases, including allergy. Their effector functions are closely linked to their cytotoxic granule proteins. Release takes place through several different mechanisms, one of which is cytolysis, which is associated with release of intact granules, so-called clusters of free eosinophil granules. The mechanism underlying this activation-induced form of cell death in eosinophils has remained unclear. We aimed to elucidate the molecular mechanism of eosinophil cytolysis. Isolated blood eosinophils were incubated on glass coverslips coated with intravenous immunoglobulin and inactive complement component 3b. Amorphologic characterization of the distinct stages of the proposed cascade was addressed by means of time-lapse automated fluorescence microscopy, electron microscopy, and immunohistochemistry. Experiments with pharmacologic inhibitors were performed to elucidate the sequence of events within the cascade. Tissue samples of patients with eosinophilic skin diseases or eosinophilic esophagitis were used for invivo analyses. After eosinophil adhesion, we observed reactive oxygen species production, early degranulation, and granule fusion processes, leading to a distinct morphology exhibiting cytoplasmic vacuolization and, finally, cytolysis. Using a pharmacologic approach, we demonstrate the presence of a receptor-interacting protein kinase 3 (RIPK3)-mixed lineage kinase-like (MLKL) signaling pathway in eosinophils, which, after its activation, leads to the production of high levels of reactive oxygen species in a p38 mitogen-activated protein kinase and phosphatidylinositol 3'-kinase-dependent manner. All these steps are required for cytoplasmic vacuolization and subsequent cytolysis to occur. Interestingly, triggering cytolysis is associated with an induction of autophagy in eosinophils, and additional stimulation of autophagy by means of pharmacologic inhibition of the mechanistic target of rapamycin counterregulates cell death. Moreover, MLKL phosphorylation, cytoplasmic vacuolization, and cytolysis were observed in eosinophils under invivo inflammatory conditions. We report that adhesion-induced eosinophil cytolysis takes place through RIPK3-MLKL-dependent necroptosis, which can be counterregulated by autophagy.

Evidence for a role of eosinophils in blister formation in bullous pemphigoid.

Bullous pemphigoid (BP) is an autoimmune bullous disease of the skin characterized by subepidermal blister formation due to tissue-bound and circulating autoantibodies to the hemidesmosomal antigens BP180 and BP230. Although eosinophils and their toxic mediators are found abundantly in BP lesions, their role in blister formation has remained unclear. To investigate the role of eosinophils in the pathogenesis of BP with a specific focus on blister formation and to define conditions inducing dermal-epidermal separation (DES). In an ex vivo human model of BP, normal human skin cryosections were incubated with purified human peripheral blood eosinophils with or without activation in the presence or absence of BP autoantibodies, brefeldin A, diphenyleneiodonium, DNase or blocking F(ab')2 fragments to CD16, CD18, CD32 and CD64. Dermal-epidermal separation was assessed by light microscopy studies and quantified using Fiji software. Following activation with IL-5 and in the presence of BP autoantibodies, eosinophils induced separation along the dermal-epidermal junction of ex vivo skin. Dermal-epidermal separation was significantly reduced by blocking any of the following: Fcγ receptor binding (P = 0.048), eosinophil adhesion (P = 0.046), reactive oxygen species (ROS) production (P = 0.002), degranulation (P < 0.0001) or eosinophil extracellular trap (EET) formation (P = 0.048). Our results provide evidence that IL-5-activated eosinophils directly contribute to BP blister formation in the presence of BP autoantibodies. Dermal-epidermal separation by IL-5-activated eosinophils depends on adhesion and Fcγ receptor activation, requires elevated ROS production and degranulation and involves EET formation. Thus, targeting eosinophils may be a promising therapeutic approach for BP.

Periostin up-Regulates the Effector Functions of Eosinophils

Periostin, an extracellular matrix protein, is up-regulated in asthmatic airways, mainly by T helper (Th) 2 cytokines. Periostin functions as a matricellular protein in cell activation by binding to cell surface receptors. However, the role of periostin in the development of eosinophilic airway inflammation has not been fully clarified. Here, we examined whether periostin could modify eosinophil functions such as superoxide anion (O2−) generation, degranulation, and production of cytokine/chemokines. Eosinophils were isolated from the blood of healthy volunteers or allergic subjects, and their adhesion to recombinant human periostin was measured using eosinophil peroxidase assays. Eosinophil O2−generation was examined based on the superoxide dismutase-inhibitable reduction of cytochrome C. Eosinophil-derived neurotoxin (EDN) concentrations in cell media were measured by ELISA as a marker of degranulation, and concentrations of cytokine/chemokines were also measured. Periostin directly induces eosinophil adhesion, which was enhanced by IL-5. Periostin also activated other functions of eosinophils such as O2− generation and EDN release. Anti-αM or anti-β2 integrin monoclonal antibody suppressed the eosinophil adhesion, O2−generation, and EDN release induced by periostin. Finally, periostin increased the production of transforming growth factor (TGF-β)1, TGF-β2 and cysteinyl leukotrienes (cysLTs) from eosinophils. These findings suggested that periostin up-regulates eosinophil functions through αMβ2 integrin. These effects may be involved in the activation of eosinophils and in the development of remodeling in the airway of Th2-dominant asthma, and could possibly aggravate the disease.

Suppression of Eosinophil Integrins Prevents Remodeling of Airway Smooth Muscle in Asthma.

Background: Airway smooth muscle (ASM) remodeling is an important component of the structural changes to airways seen in asthma. Eosinophils are the prominent inflammatory cells in asthma, and there is some evidence that they contribute to ASM remodeling via released mediators and direct contact through integrin–ligand interactions. Eosinophils express several types of outer membrane integrin, which are responsible for cell–cell and cell–extracellular matrix interactions. In our previous study we demonstrated that asthmatic eosinophils show increased adhesion to ASM cells and it may be important factor contributing to ASM remodeling in asthma. According to these findings, in the present study we investigated the effects of suppression of eosinophil integrin on eosinophil-induced ASM remodeling in asthma.Materials and Methods: Individual combined cell cultures of immortalized human ASM cells and eosinophils from peripheral blood of 22 asthmatic patients and 17 healthy controls were prepared. Eosinophil adhesion was evaluated using eosinophil peroxidase activity assay. Genes expression levels in ASM cells and eosinophils were measured using quantitative real-time PCR. ASM cell proliferation was measured using alamarBlue® solution. Eosinophil integrins were blocked by incubating with Arg-Gly-Asp-Ser peptide.Results: Eosinophils from the asthma group showed increased outer membrane α4β1 and αMβ2 integrin expression, increased adhesion to ASM cells, and overexpression of TGF-β1 compared with eosinophils from the healthy control group. Blockade of eosinophil RGD-binding integrins by Arg-Gly-Asp-Ser peptide significantly reduced adhesion of eosinophils to ASM cells in both groups. Integrin-blocking decreased the effects of eosinophils on TGF-β1, WNT-5a, and extracellular matrix protein gene expression in ASM cells and ASM cell proliferation in both groups. These effects were more pronounced in the asthma group compared with the control group.Conclusion: Suppression of eosinophil-ASM interaction via RGD-binding integrins attenuates eosinophil-induced ASM remodeling in asthma.Trial Registration: ClinicalTrials.gov Identifier: NCT02648074.