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Eosinophils are major effector cells against parasites, fungi, bacteria, and viruses. However, these cells also take part in local and systemic inflammation, which are central to eczema, atopy, rhinitis, asthma, and autoimmune diseases. A role for eosinophils has been also shown in vascular thrombotic disorders and in cancer. Many, if not all, above-mentioned conditions involve the release of intracellular nucleotides (ATP, ADP, UTP, etc.) and nucleosides (adenosine) in the extracellular environment. Simultaneously, eosinophils further release ATP, which in autocrine and paracrine manners, stimulates P2 receptors. Purinergic signaling in eosinophils mediates a variety of responses including CD11b induction, ROI production, release of granule contents and enzymes, as well as cytokines. Exposure to extracellular ATP also modulates the expression of endothelial adhesion molecules, thereby favoring eosinophil extravasation and accumulation. In addition, eosinophils express the immunosuppressive adenosine P1 receptors, which regulate degranulation and migration. However, pro-inflammatory responses induced by extracellular ATP predominate. Due to their important role in innate immunity and tissue damage, pharmacological targeting of nucleotide- and nucleoside-mediated signaling in eosinophils could represent a novel approach to alleviate eosinophilic acute and chronic inflammatory diseases. These innovative approaches might also have salutary effects, particularly in host defense against parasites and in cancer.

Previous research in our laboratory has demonstrated that repeated intranasal exposure to Aspergillus fumigatus conidia in C57BL/6 mice results in a chronic pulmonary inflammatory response that reaches its maximal level after four challenges. The inflammatory response is characterized by eosinophilia, goblet cell metaplasia, and T helper T H 2 cytokine production, which is accompanied by sustained interleukin-17 (IL-17) expression that persists even after the T H 2 response has begun to resolve. T H 17 cells could develop in mice deficient in gamma interferon (IFN-γ), IL-4, or IL-10. In the lungs of IL-17 knockout mice repeatedly challenged with A. fumigatus conidia, inflammation was attenuated (with the most significant decrease occurring in eosinophils), conidial clearance was enhanced, and the early transient peak of CD4 + CD25 + FoxP3 + cells blunted. IL-17 appeared to play only a minor role in eosinophil differentiation in the bone marrow but a central role in eosinophil extravasation from the blood into the lungs. These observations point to an expanded role for IL-17 in driving T H 2-type inflammation to repeated inhalation of fungal conidia.

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.

Eosinophils are blood and tissue immune cells that participate in a diverse range of activities normally beneficial for the host defense, but in circumstances of untoward inflammatory conditions these cells can be responsible for pathological responses. Accordingly the transit of eosinophils from the blood to tissues is a subject of considerable importance in immunology. In this article we review how the complement anaphylatoxins, C3a and C5a bring about eosinophil extravasation. These mediators do not merely provide a chemotactic or haptotactic gradient but are responsible for orchestrating innumerable responses by other cells types, including of endothelial cells, mast cells, and basophils in order to create an environment that is conducive for eosinophil infiltration. C5a has the capacity to prime the endothelium directly to present P-selectin, and C5a stimulated generation of eosinophil hydrogen peroxide and other oxidants can cause additional upregulation of endothelial P-selectin and ICAM-1. Moreover, the anaphylatoxins have the ability to recruit mast cells and basophils and can stimulate these cells to release IL-4 and IL-13, which by augmenting endothelial VCAM-1, convey some selectivity for eosinophils. The anaphylatoxins also have the capability to evoke the release and activation of eosinophil MMP-9, which is employed by this cell type to digest its way past the subendothelial matrix. Finally, because C3a and C5a can stimulate the generation of nitric oxide along with the secretion of histamine and LTC4 from several cell types, the anaphylatoxins can bring about an increase in vascular permeability that facilitates eosinophil accumulation at sites of allergic inflammation.

Extracts from the plant Synadenium carinatum latex are widely and indiscriminately used in popular medicine to treat a great number of inflammatory disorders and although the mechanisms underlying these effects remain undefined, the lectin isolated from S. carinatum latex (ScLL) is thought to be in part responsible for these anti-inflammatory effects. In order to elucidate possible immunoregulatory activities of ScLL, we investigated the effects of ScLL administration in models of acute and chronic inflammation. Oral administration of ScLL significantly inhibited neutrophil and eosinophil extravasation in models of acute and chronic inflammation and reduced eosinophil and mononuclear blood counts during chronic inflammation. ScLL administration reduced IL(interleukin)-4 and IL-5 levels but increased interferon-gamma and IL-10 in an asthma inflammatory model, which suggested that it might induce a TH2 to TH1 shift in the adaptive immune response. ScLL also inhibited IkappaBalpha degradation, a negative regulator of proinflammatory NF-kappaB. Taken together, these results provide the first description of a single factor isolated from S. carinatum latex extract with immunoregulatory functions and suggest that ScLL may be useful in the treatment of allergic inflammatory disorders.

Asthma is characterized by airway inflammation rich in eosinophils. Airway eosinophilia is associated with exacerbations and has been suggested to play a role in airway remodelling. Recruitment of eosinophils from the circulation requires that blood eosinophils become activated, leading to their arrest on the endothelium and extravasation. Circulating eosinophils can be envisioned as potentially being in different activation states, including non-activated, pre-activated or 'primed', or fully activated. In addition, the circulation can potentially be deficient of pre-activated or activated eosinophils, because such cells have marginated on activated endothelium or extravasated into the tissue. A number of eosinophil surface proteins, including CD69, L-selectin, intercellular adhesion molecule-1 (ICAM-1, CD54), CD44, P-selectin glycoprotein ligand-1 (PSGL-1, CD162), cytokine receptors, Fc receptors, integrins including αM integrin (CD11b), and activated conformations of Fc receptors and integrins, have been proposed to report cell activation. Variation in eosinophil activation states may be associated with asthma activity. Eosinophil surface proteins proposed to be activation markers, with a particular focus on integrins, and evidence for associations between activation states of blood eosinophils and features of asthma are reviewed here. Partial activation of β1 and β2 integrins on blood eosinophils, reported by monoclonal antibodies (mAbs) N29 and KIM-127, is associated with impaired pulmonary function and airway eosinophilia, respectively, in non-severe asthma. The association with lung function does not occur in severe asthma, presumably due to greater eosinophil extravasation, specifically of activated or pre-activated cells, in severe disease.

Airway eosinophilia is the hallmark of asthma exacerbation. Coordination of cytokines such as interleukin (IL)-5, eotaxin and regulated on activation, normal T-cell expressed and secreted (RANTES) seem to be necessary for eosinophil extravasation including adhesion, chemotaxis, and activation. The purpose of this study was to characterize both the kinetics of allergen-induced inflammatory cell recruitment to the airways and cytokines selective for eosinophil chemotaxis, activation, or resolution. Eight atopic asthmatic individuals demonstrating a dual response to inhaled allergen completed a diluent-controlled crossover study. The subjects showed significant allergen-induced early and late airway asthmatic responses (p < 0.001), and an increase in the number of sputum eosinophils and metachromatic cells (p < 0.05). The number of eosinophils immunopositive for IL-5, eotaxin, and RANTES increased 7 h after allergen inhalation (p < 0.05), coincident with the peak number of activated eosinophils. Sputum cells immunopositive for IL-10 decreased significantly following allergen challenge (p = 0. 04), and correlated negatively with sputum eosinophils (r = -0.34, p = 0.02). This study shows that allergen-induced increases in sputum eosinophils are associated with the presence of cytokines specific for the activation and chemotaxis of eosinophils, and suggests that cooperation of eosinophilic cytokines may be important for the accumulation and regulation of activated eosinophils at the site of allergic inflammation.

1. We have developed a novel model of allergen-induced eosinophil into mouse air-pouches following sensitization and challenge with ovalbumin (Ova). This model was used to investigate the mechanism(s) underlying the anti-inflammatory action of the glucocorticoid hormone dexamethasone (Dex). 2. Injection of 10 micrograms Ova into 6-day-old dorsal air-pouches of mice sensitized to the same antigen provoked an intense cell accumulation as early as 6 h post-challenge (0.08 +/- 0.03 and 4.0 +/- 1.0 x 10(5) leucocytes in saline and Ova-treated air-pouches, respectively), maximal at 24 h (0.02 +/- 0.01 and 6.0 +/- 0.8 x 10(5) leucocytes in saline and Ova-treated air-pouches, respectively) and persisted up to 48 h. At the 24 h time-point, the cellular infiltrate consisted of 37% eosinophils, 18% neutrophils and 45% mononuclear cells, as assessed by histological examination. The same ratio of eosinophil/neutrophil was obtained by fluorescence-activated cell sorting (FACS) analysis, since 72% of the polymorphonuclear (PMN) population was positive for very-late antigen-4 (VLA-4) expression. 3. Subcutaneous (s.c.) administration of Dex (50 or 100 micrograms per mouse, -1 h) inhibited eosinophil accumulation into Ova challenged air-pouches by about 70% (P < 0.05) and 75% (P < 0.05), respectively, when compared to controls. Cell accumulation measured at 48 h after Ova injection was also significantly reduced (-75%) by Dex administration at the 24 h time-point (n = 12, P < 0.05). 4. Eosinophil numbers in the bone marrow and blood were quantitated. We found that the sensitization protocol induced a 3 fold increase in eosinophil numbers in the bone marrow (naive mice: 2.7 +/- 0.3 x 10(5); sensitized mice: 8.7 +/- 1.7 x 10(5), P < 0.05) and blood (naive mice: 0.5 +/- 0.2 x 10(5); sensitized mice: 1.5 +/- 0.3 x 10(5), P < 0.01). However, 24 h following Ova challenge, the eosinophil numbers in the bone marrow had dropped (3.7 +/- 0.8 x 10(5) with no change in the circulating pool, suggesting an equilibrium within the eosinophil pools had been reached. 5. Dex administration provoked a profound eosinopaenia in the blood of naive (5.2 +/- 1.5 to 0.9 +/- 0.6 x 10(4)) and sensitized mice (1.5 +/- 0.3 to 0.08 +/- 0.02 x 10(5)) at 4 h. This effect was reversed within 24 h. Dex also inhibited the release of eosinophils from the bone marrow in response to Ova challenge. 6. We show for the first time that express the steroid-inducible protein lipocortin 1 (LC1). FACS analysis of eosinophils emigrated into the Ova challenged air-pouches revealed detectable LC1-like immunoreactivity (373 x 10(3)). These data were also substantiated by LC1 detection in circulating eosinophils of interleukin-5 transgenic mice (strain: CBA/Ca). However, s.c. injection of Dex (50 micrograms) did not alter LC1 levels in blood eosinophils, such that 235 +/- 21 x 10(3) LC1-like molecules per cell were measured after vehicle treatment (n = 5), and 224 +/- 8 x 10(3) LC1-like molecules per cell were associated with this cell type 1 h after steroid treatment (n = 5, not significant). Finally, resident eosinophils (in the pleural cavity) were found to have much higher LC1 levels than that found in the blood circulation (2 fold increase, P < 0.05). 7. Passive immunization of mice against LC1 with a validated antiserum (termed LCS3) and protocol failed to modify the anti-migratory activity exerted by Dex towards eosinophil extravasation into Ova-challenged air-pouches. The steroid (50 micrograms s.c., -1 h) produced a similar degree of inhibition of eosinophil accumulation both in control animals (treated with a non-immune sheep serum) the LCS3-treated mice (-56% and 59%, respectively, n = 15-21, not significant). 8. In conclusion, the air-pouch provides a novel and convenient cavity to study allergen-induced cell recruitment which is sensitive to glucocorticoid hormone treatment. The effect of Dex on eosinophil distribution in these experimental conditions has been studied in detail and

Asthma is a chronic inflammatory condition of the airways characterized by reversible airway obstruction, airway hyperresponsiveness (AHR) to normally harmless stimuli and airway inflammation. Eosinophilic asthma is a phenotype of the condition characterized by increased blood or sputum eosinophils whose numbers correlate with disease severity. Release of their potent pro-inflammatory arsenal by infiltrating tissue eosinophils, including granule-derived basic proteins, mediators, cytokines and chemokines, contributes to airway inflammation, and lung tissue remodeling that includes airway thickening and fibrosis. More recent evidence suggests that in addition to their role as degranulating effector cells, eosinophils have the capacity to act as antigen presenting cells resulting in T cell proliferation and activation (Blanchard and Rothenberg, 2009). Eosinophil extravasation from the post-capillary venules, migration within the interstitium, cellular activation and tissue retention are controlled by cell adhesion molecules, i.e., selections, integrins and members of the immunoglobulin superfamily (Barthel et al., 2008; Barnes, 2011). The expression and function of these adhesion molecules and the subsequent chemotactic attraction and activation of infiltrating pro-inflammatory cells are controlled by a myriad of cytokines, chemokines and mediators with the Th2 cytokines IL-4, IL-5, and IL-13 representing essential and central coordinators of asthmatic inflammation (Barrett and Austen, 2009; Walsh, 2010). Thus, modulating the cytokine network in asthma with biological therapy targeted to patients with particular eosinophilic phenotypes represents a plausible paradigm for treatment of this important condition (Petsky et al., 2007; Barnes, 2008; Desai and Brightling, 2009; Walsh, 2011).

The establishment of animal models of asthma is critical to elucidate the mechanisms involved in the pathogenesis of the disease. In the present study, we have used a parasite antigen from Schistosoma mansoni eggs, which induces a TH2 response, to elicit a pulmonary inflammatory reaction that resolves after 3 to 4 days. Histologic examination of the lungs of soluble egg antigens (SEA) or saline vehicle-challenged mice demonstrated a large influx of cells in the antigen- but not vehicle-challenged mice, thus demonstrating an antigen-specific reaction. A characteristic influx of eosinophils could be detected as early as 8 h, with significant increases at 24 to 72 h after challenge. An assessment of the bronchial alveolar lavage (BAL) fluid demonstrated dominant neutrophil infiltration at 8 h, with a subsequent decrease to background by 48 h. In addition, peak monocyte infiltration occurred at 24 h, and peak eosinophil extravasation into the airway was shown at 48 h. The examination of leukocyte infiltrates in the interstitium in dispersed lung preparations again demonstrated early neutrophil and monocyte infiltration at 8 h after challenge, with increases in lymphocyte and eosinophil infiltrates at 24 h. Examination of interleukin-4 (IL-4) production in the BAL fluid demonstrated the presence of IL-4 early in the response, with levels peaking between 8 and 24 h after antigen challenge, with no detectable IL-4 in the saline vehicle-challenged mice. Mice treated with anti-IL-4 antibodies demonstrated a tenfold decrease in BAL eosinophil influx at 48 h after challenge and a reduction in total pulmonary leukocyte cellularity.(ABSTRACT TRUNCATED AT 250 WORDS)

Background: Skin eosinophilia is a common feature of allergic skin diseases, but it is unclear how epidermal and dermal eosinophil infiltration is controlled. To investigate regulation of localization of eosinophils in skin, we examined the regulatory mechanisms of expression of eosinophil-specific chemoattractants in dermal fibroblasts and epidermal keratinocytes. Methods: We analyzed production of eotaxin, eotaxin-2, eotaxin-3 and galectin-9 by dermal fibroblasts and epidermal keratinocytes in response to several stimuli in vitro. Results: Dermal fibroblasts produced eotaxin and eotaxin-3 in response to stimulation by interleukin (IL)-4 and/or tumor necrosis factor-α. Similarly, IL-4 stimulated epidermal keratinocytes to secrete eotaxin-3. However, we did not detect eotaxin mRNA expression or protein secretion by keratinocytes stimulated in vitro. Interferon (IFN)-γ induced galectin-9 expression on dermal fibroblasts. Conversely, expression of galectin-9 on epidermal keratinocytes was dose-dependently inhibited by IFN-γ. The immunohistochemical assays revealed that dermal fibroblasts (but not epidermal keratinocytes) in the lesional skin of psoriasis vulgaris (a Th1-polarized disease) express significant levels of galectin-9. Conclusion: Eotaxin-3 contributes to dermal and epidermal eosinophil infiltration in Th2-polarized skin inflammation in which IL-4 is produced. In contrast, IFN-γ-dominated inflammation appears to mediate eosinophil extravasation into the dermis and eosinophil adhesion to dermal fibroblasts via galectin-9 in association with decreased chemoattractant activity of epidermal galectin-9. The present results reveal a novel mechanism of dermal eosinophilia in IFN-γ-mediated skin inflammation, and reflect concerted chemoattractant production involving dermal and/or epidermal eosinophilia during changes in the local cytokine profile.

Eotaxin is a potent eosinophil-specific CC-chemokine, which has been shown to play a role in the selective induction of eosinophil accumulation in a number of allergic models of inflammation. Many aspects of the mechanism by which eotaxin induces eosinophil accumulation in vivo remain unresolved. In the present study, we investigated the direct effect of synthetic human eotaxin on leucocyte/endothelial cell interactions within rat mesenteric venules, as quantified by intravital microscopy. Topical eotaxin (30 pmol) induced rapid firm adhesion and extravasation of leucocytes within the rat mesentery, the extravasated leucocytes all being eosinophils, as determined by histological analysis. Whilst eotaxin was unable to stimulate the interaction of rat eosinophils with vascular cell adhesion molecule-1 (VCAM-1) under static conditions in vitro, eotaxin-induced responses in vivo were significantly suppressed by anti-alpha4 integrin and anti-VCAM-1 monoclonal antibodies (mAbs). The anti-alpha4 integrin mAb, HP2/1 (3.5 mg/kg), inhibited the eotaxin-induced firm adhesion and extravasation, 60 min postapplication of the chemokine, by 89% and 84%, respectively. In the same set of experiments, the anti-VCAM-1 mAb, 5F10 (3.5 mg/kg), inhibited leucocyte adhesion and extravasation by 61% and 63%, respectively. These results demonstrate that eotaxin-induced migration of eosinophils through rat mesenteric venules in vivo is dependent on an alpha4 integrin/VCAM-1 adhesion pathway, the significance of which may only be evident under flow conditions and/or following the ligation of other adhesion molecules expressed on eosinophils.