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Clinically relevant and reliable reports derived from in vitro research are dependent on the choice of cell isolation protocols adopted between different laboratories. Peripheral blood eosinophils are conventionally isolated using density-gradient centrifugation followed by immunomagnetic selection (positive/negative) while neutrophils follow a more simplified dextran-sedimentation methodology. With the increasing sophistication of molecular techniques, methods are now available that promise protocols with reduced user-manipulations, improved efficiency, and better yield without compromising the purity of enriched cell populations. These recent techniques utilize immunomagnetic particles with multiple specificities against differential cell surface markers to negatively select non-target cells from whole blood, greatly reducing the cost/time taken to isolate granulocytes. Herein, we compare the yield efficiencies, purity and baseline activation states of eosinophils/neutrophils isolated using one of these newer protocols that use immunomagnetic beads (MACSxpress isolation) vs. the standard isolation procedures. The study shows that the MACSxpress method consistently allowed higher yields per mL of peripheral blood compared to conventional methods (P

Background: Asthma severity and eosinophilia correlate with a deficiency in vitamin D and its active metabolite calcitriol. Calcitriol modulates numerous leukocyte functions, but its effect on eosinophils is not fully understood. We postulated that calcitriol exerts a direct effect on eosinophil biology by modulating cell survival. Methods: Purified peripheral blood eosinophils from atopic donors were incubated in the presence of calcitriol for up to 14 days with or without IL-5. The effect of calcitriol on eosinophil viability was measured using the annexin-V/propidium iodide flow cytometry assay. We also examined the release of eosinophil peroxidase (EPX) in media using a flow cytometry assay with anti-EPX antibodies, and the enzymatic activity of EPX was measured by an OPD-based colorimetric assay. Results: We observed that calcitriol sustained cell viability in eosinophils with a concurrent reduction of necrotic cells. This effect was amplified by the addition of IL-5. In parallel, we observed that a physiological dose of calcitriol (10 nM) significantly reduced eosinophil necrosis and cytolytic release of EPX in media when coincubated with IL-5. Conclusion: These results suggest that calcitriol may exert a direct effect on eosinophils by reducing necrosis and the cytolytic release of inflammatory mediators like EPX.

Platelet-activating factor (PAF [1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine]) is a phospholipid mediator released from activated macrophages, mast cells, and basophils that promotes pathophysiologic inflammation. Eosinophil responses to PAF are complex and incompletely elucidated. We show in this article that PAF and its 2-deacetylated metabolite (lysoPAF) promote degranulation (release of eosinophil peroxidase) via a mechanism that is independent of the characterized PAFR. Specifically, we demonstrate that receptor antagonists CV-3988 and WEB-2086 and pertussis toxin have no impact on PAF- or lysoPAF-mediated degranulation. Furthermore, cultured mouse eosinophils from PAFR−/− bone marrow progenitors degranulate in response to PAF and lysoPAF in a manner indistinguishable from their wild-type counterparts. In addition to PAF and lysoPAF, human eosinophils degranulate in response to lysophosphatidylcholine, but not phosphatidylcholine, lysophosphatidylethanolamine, or phosphatidylethanolamine, demonstrating selective responses to phospholipids with a choline head-group and minimal substitution at the sn-2 hydroxyl. Human eosinophils release preformed cytokines in response to PAF, but not lysoPAF, also via a PAFR-independent mechanism. Mouse eosinophils do not release cytokines in response to PAF or lysoPAF, but they are capable of doing so in response to IL-6. Overall, our work provides the first direct evidence for a role for PAF in activating and inducing degranulation of mouse eosinophils, a crucial feature for the interpretation of mouse models of PAF-mediated asthma and anaphylaxis. Likewise, we document and define PAF and lysoPAF-mediated activities that are not dependent on signaling via PAFR, suggesting the existence of other unexplored molecular signaling pathways mediating responses from PAF, lysoPAF, and closely related phospholipid mediators.

Eosinophils expressing indoleamine 2, 3-dioxygenase (IDO) may contribute to T-helper cell (Th)2 predominance. To characterize human thymus IDO+ eosinophil ontogeny relative to Th2 regulatory gene expression, we processed surgically obtained thymi from 22 children (age: 7 days to 12 years) for immunohistochemistry and molecular analysis, and measured cytokine and kynurenine levels in tissue homogenates. Luna+ eosinophils (∼2% of total thymic cells) decreased in number with age (P = 0.02) and were IDO+. Thymic IDO immunoreactivity (P = 0.01) and kynurenine concentration (P = 0.01) decreased with age as well. In addition, constitutively-expressed interleukin (IL)-5 and IL-13 in thymus supernatants was highest in youngest children. Eosinophil numbers correlated positively with expression of the Th2 cytokines IL-5, IL-13 (r = 0.44, P = 0.002), and IL-4 (r = 0.46, P = 0.005), transcription factor signal transducer and activator of transcription-6 (r = 0.68, P = 0.001), and the chemokine receptor, CCR3 (r = 0.17, P = 0.04), but negatively with IL-17 mRNA (r = −0.57, P = 0.02) and toll-like receptor 4 expression (r = −0.74, P = 0.002). Taken together, these results suggest that functional thymic IDO+ eosinophils during human infant life may have an immunomodulatory role in Th2 immune responses.

Degranulation from eosinophils in response to secretagogue stimulation is a regulated process that involves exocytosis of granule proteins through specific signalling pathways. One potential pathway is dependent on cyclin-dependent kinase 5 (Cdk5) and its effector molecules, p35 and p39, which play a central role in neuronal cell exocytosis by phosphorylating Munc18, a regulator of SNARE binding. Emerging evidence suggests a role for Cdk5 in exocytosis in immune cells, although its role in eosinophils is not known. We sought to examine the expression of Cdk5 and its activators in human eosinophils, and to assess the role of Cdk5 in eosinophil degranulation. We used freshly isolated human eosinophils and analysed the expression of Cdk5, p35, p39 and Munc18c by Western blot, RT-PCR, flow cytometry and immunoprecipitation. Cdk5 kinase activity was determined following eosinophil activation. Cdk5 inhibitors were used (roscovitine, AT7519 and small interfering RNA) to determine its role in eosinophil peroxidase (EPX) secretion. Cdk5 was expressed in association with Munc18c, p35 and p39, and phosphorylated following human eosinophil activation with eotaxin/CCL11, platelet-activating factor, and secretory IgA-Sepharose. Cdk5 inhibitors (roscovitine, AT7519) reduced EPX release when cells were stimulated by PMA or secretory IgA. In assays using small interfering RNA knock-down of Cdk5 expression in human eosinophils, we observed inhibition of EPX release. Our findings suggest that in activated eosinophils, Cdk5 is phosphorylated and binds to Munc18c, resulting in Munc18c release from syntaxin-4, allowing SNARE binding and vesicle fusion, with subsequent eosinophil degranulation. Our work identifies a novel role for Cdk5 in eosinophil mediator release by agonist-induced degranulation.

Background: Animal and human studies demonstrated that interleukin (IL)-12, a Th1 cytokine, reduces blood and bronchial eosinophilia, and airway hyperreactivity. According to current concepts, these effects are mediated through the release of cytokines promoting eosinophil recruitment and activation. However, the presence of IL-12 receptors on eosinophils suggests that IL-12 also acts directly on eosinophils. We postulated that IL-12 directly modulates eosinophil functions and has the capacity to regulate eosinophil degranulation, migration and survival, in vitro. Method: Effects of IL- 12 on purified human blood eosinophils were evaluated for peroxidase (EPO) release, eotaxin-induced migration through a model of basement membrane (Matrigel), and survival. Results: IL-12 inhibited 50% of PAF and secretory IgA-induced EPO release (n = 8, p < 0.001). IL-12 also reduced eotaxin-induced migration through Matrigel by 54 ±6% (n = 6, p < 0.01). These effects were not explained by an IL-12-induced impaired viability or apoptosis. Conclusion: Our results demonstrate that IL-12 directly modulates eosinophil functions without promoting apoptosis and explain, at least in part, the effects of IL-12 on eosinophils observed in in vivo studies.

Background: Blood eosinophils have mRNA for FcγRIIIB (CD16) but no or minimal spontaneous CD16 expression. Because IFN-γ and chemotactic factors induce eosinophil CD16 expression in vitro, we postulated that blood eosinophils could express CD16. Objective: Blood of nonallergic controls and subjects with allergic rhinitis, allergic and nonallergic asthma, or hypereosinophilia of various etiologies were analyzed for leukocyte CD16 surface expression. Methods: CD16 + eosinophils were identified on the basis of physico-optic characteristics, major basic protein, CD49b expression, and sorting by flow cytometry and microscope examination. Results: Subjects with allergic rhinitis and subjects with asthma had higher median percentages of CD16 + eosinophils (8.1% [1% to 48.6%] and 7.3% [1.4% to 31.1%], respectively) than nonallergic controls and nonallergic asthmatics (3% [0% to 11%] and 4.6% [2.9% to 5.1%], respectively). In subjects with hypereosinophilia, CD16 + eosinophils were increased only in a case of drug allergy. When subjects with mild allergic asthma were challenged with a relevant aeroallergen, blood CD16 + eosinophils further increased during or after the late-phase response (6 to 48 hours after challenge; mean ± SEM, 9.4% ± 2.5% to 20.0% ± 3.0%). CD16 + eosinophils expressed more IL-5 receptor but less CD11b and IL-12p35 than did CD16 − eosinophils. Conclusion: Upregulation of blood CD16 + eosinophils in allergic conditions and its association with a modified phenotype suggest that CD16 receptor could play a role in eosinophil activation in allergy. (J Allergy Clin Immunol 2002;109:463-9.)

Eosinophils derive from the bone marrow and circulate at low levels in the blood in healthy individuals. These granulated cells preferentially leave the circulation and marginate to tissues, where they are implicated in the regulation of innate and adaptive immunity. In diseases such as allergic inflammation, eosinophil numbers escalate markedly in the blood and tissues where inflammatory foci are located. Eosinophils possess a range of immunomodulatory factors that are released upon cell activation, including over 35 cytokines, growth factors, and chemokines. Unlike T and B cells, eosinophils can rapidly release cytokines within minutes in response to stimulation. While some cytokines are stored as pre-formed mediators in crystalloid granules and secretory vesicles, eosinophils are also capable of undergoing de novo synthesis and secretion of these immunological factors. Some of the molecular mechanisms that coordinate the final steps of cytokine secretion are hypothesized to involve binding of membrane fusion complexes comprised of soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs). These intracellular receptors regulate the release of granules and vesicles containing a range of secreted proteins, among which are cytokines and chemokines. Emerging evidence from both human and animal model-based research has suggested an active participation of eosinophils in several physiological/pathological processes such as immunomodulation and tissue remodeling. The observed eosinophil effector functions in health and disease implicate eosinophil cytokine secretion as a fundamental immunoregulatory process. The focus of this review is to describe the cytokines, growth factors, and chemokines that are elaborated by eosinophils, and to illustrate some of the intracellular events leading to the release of eosinophil-derived cytokines.

Identification of eosinophils in whole blood samples by flow cytometry is often problematic. There are usually only a low number and percentage of cells that may be detected, and it may be difficult to discriminate eosinophils from other granulocytes. Here, we propose a simple approach using the eosinophil's intrinsic autofluorescence properties, combined with detection of CCR3 expression, to reliably identify eosinophils in a mixed leukocyte population.

Identification of eosinophils in whole blood samples by flow cytometry is often problematic. There are usually only a low number and percentage of cells that may be detected, and it may be difficult to discriminate eosinophils from other granulocytes. Here, we propose a simple approach using the eosinophil's intrinsic autofluorescence properties, combined with detection of CCR3 expression, to reliably identify eosinophils in a mixed leukocyte population.

IL-17 is a cytokine that has been reported to be produced by T lymphocytes. In vitro, IL-17 activates fibro-blasts and macrophages for the secretion of GM-CSF, TNF-alpha, IL-1beta, and IL-6. A number of these cytokines are involved in the airway remodeling that is observed within the lungs of asthmatic individuals.In this study, we investigated the expression of IL-17 in sputum and bronchoalveolar lavage specimens obtained from asthmatic subjects and from nonasthmatic control subjects.IL-17 was detected through use of immunocytochemistry, in situ hybridization, and Western blot. Bronchial fibroblasts were stimulated with IL-17, and cytokine production and chemokine production were detected through use of ELISA and RT-PCR.Using immunocytochemistry, we demonstrated that the numbers of cells positive for IL-17 are significantly increased in sputum and bronchoalveolar lavage fluids of subjects with asthma in comparison with control subjects (P.001 and P.005, respectively). We demonstrated that in addition to T cells, eosinophils in sputum and bronchoalveolar lavage fluids expressed IL-17. Peripheral blood eosinophils were also positive for IL-17, and the level of IL-17 in eosinophils purified from peripheral blood was significantly higher in subjects with asthma than in controls (P.01). To further investigate the mechanism of action of IL-17 in vivo, we examined the effect of this cytokine on fibroblasts isolated from bronchial biopsies of asthmatic and nonasthmatic subjects. IL-17 did enhance the production of pro-fibrotic cytokines (IL-6 and IL-11) by fibroblasts, and this was inhibited by dexamethasone. Similarly, IL-17 increased the level of other fibroblast-derived inflammatory mediators, such as the alpha-chemokines, IL-8, and growth-related oncogene-alpha.Our results, which demonstrate for the first time that eosinophils are a potential source of IL-17 within asthmatic airways, suggest that IL-17 might have the potential to amplify inflammatory responses through the release of proinflammatory mediators such as alpha-chemokines.

Introduction Eosinophils are continually targeted in allergic airway inflammatory disease therapies. The presence of their granules in airway tissues remains unexplained, yet implies the involvement of necrosis. The latter’s definition has evolved into a highly regulated and distinct signalling pathway leading to physiological inflammation, known as necroptosis. Even though necroptosis is a recently introduced concept, we currently recognize its role as an alternative mechanism in the absence of apoptosis. Furthermore, necroptosis seems to act as a host-defence strategy against viral infections, which consequently associates itself with eosinophils’ role in airway viral clearance. The aim of this review is to discuss if necroptosis of eosinophils is involved in allergic airway inflammation. Conclusion The investigation of necroptosis in eosinophil is currently an area which lacks research, yet harbours great promise for drug development.

Objectives There is interest in developing new compounds to enhance the immune response to airway virus infections. CVT-E002 is a patented ginseng extract shown to decrease symptoms of virus infection in clinical trials. We hypothesized that the mechanism for this antiviral effect could be through modulation of dendritic cells leading to enhanced T-cell activation. Methods Human monocyte-derived dendritic cells (moDC) exposed to CVT-E002 (or not) were co-cultured with autologous T cells, with or without virus (respiratory syncytial virus or parainfluenza virus). Effects of CVT-E002 on cell function were determined through flow cytometry, 5-bromo-2′-deoxyuridine (BrdU) incorporation and ELISA. Key findings moDC cultured with CVT-E002 or virus induced greater activation of T cells, as measured by CD25 expression and BrdU incorporation, compared with untreated moDC. Responding T cells were CD4+CD45RO+. Co-cultures of CVT-E002 treated moDC with T cells responded with increased release of Th1-type cytokines (interferon-gamma, tumour necrosis factor and interleukin-12). CVT-E002-treated moDC showed increased expression of CD83, CD80 and CD86. Lipopolysaccharide levels were not detected in CVT-E002 and antagonists for Toll-like receptor-4 did not inhibit CVT-E002-induced moDC maturation. Conclusions CVT-E002 induced moDC maturation, which caused increased memory T-cell activation and Th1-type cytokine response.

Eosinophils expressing indoleamine 2, 3-dioxygenase (IDO) may contribute to T-helper cell (Th)2 predominance. To characterize human thymus IDO+ eosinophil ontogeny relative to Th2 regulatory gene expression, we processed surgically obtained thymi from 22 children (age: 7 days to 12 years) for immunohistochemistry and molecular analysis, and measured cytokine and kynurenine levels in tissue homogenates. Luna+ eosinophils ( approximately 2% of total thymic cells) decreased in number with age (P = 0.02) and were IDO+. Thymic IDO immunoreactivity (P = 0.01) and kynurenine concentration (P = 0.01) decreased with age as well. In addition, constitutively-expressed interleukin (IL)-5 and IL-13 in thymus supernatants was highest in youngest children. Eosinophil numbers correlated positively with expression of the Th2 cytokines IL-5, IL-13 (r = 0.44, P = 0.002), and IL-4 (r = 0.46, P = 0.005), transcription factor signal transducer and activator of transcription-6 (r = 0.68, P = 0.001), and the chemokine receptor, CCR3 (r = 0.17, P = 0.04), but negatively with IL-17 mRNA (r = -0.57, P = 0.02) and toll-like receptor 4 expression (r = -0.74, P = 0.002). Taken together, these results suggest that functional thymic IDO+ eosinophils during human infant life may have an immunomodulatory role in Th2 immune responses.

The inhibitory roles of nitric oxide (NO) in T cell proliferation have been observed and studied extensively over the last two decades. Despite efforts, the fundamental pathway by which NO exerts its inhibitory actions remains to be elucidated although recent evidence suggests that the transcription factor Wilms tumor 1 (WT1) may be important. WT1 has been linked to numerous developmental pathways in particular nephrogenesis. Due to its roles in development and cell proliferation, polymorphisms within the WT1 gene can result in malignancies such as leukemia and Wilms tumor. WT1 functions as a transcriptional regulator and its activity is controlled through phosphorylation by protein kinase A (PKA). PKA-dependent WT1 phosphorylation results in translocation of WT1 from the nucleus to the cytosol, a process that interferes with WT1 transcriptional activities. In the current study we demonstrate that WT1 is expressed in human lymphocytes. Using the proliferative compound PHA we induced T cell proliferation and growth correlated with an increase in the expression of WT1 measured by RT-PCR, flow cytometry and immunoblot. Co-stimulation with the NO donor SNOG at concentrations of 0, 100, 300 and 600 μM reduced in a concentration dependent way the PHA-induced upregulation of WT1 that correlated with a reduction in T cell proliferation. We conclude that WT1 might be an important component of the NO-dependent regulation of T lymphocyte proliferation and potential function.

Increased eosinophil counts are a major feature of asthmatic airways. Eosinophil recruitment requires migration through epithelium and tissue extracellular matrix by activation of proteases. We assessed the capacity of IL-16, a CD4+ cell chemotactic factor, to induce migration of eosinophils through a reconstituted basement membrane and evaluated the proteases, mediators, and receptors involved in this migration. IL-16 added to lower chambers of Invasion Chambers elicited eosinophil migration through Matrigel. This effect was decreased by inhibition of the plasminogen-plasmin system (Abs against urokinase plasminogen activator receptor or plasminogen depletion), but not by anti-matrix metalloproteinase-9 Abs. Abs against CD4 also inhibited IL-16-induced eosinophil migration. At the baseline level, few eosinophils (4.6% positive cells with a mean fluorescence of 0.9) expressed surface membrane CD4, while most permeabilized eosinophils (68% positive cells with a mean fluorescence of 18) express the CD4 Ag. TNF-pretreatment increased surface membrane CD4+ expression by 6-fold as previously described, and increased IL-16-induced cell migration by 2.2-fold. Incubation of eosinophils with IL-16 also increased surface membrane CD4 expression by 5.4-fold, supporting the role of CD4 as receptor for IL-16. Abs against CCR3, eotaxin, or RANTES blocked IL-16-induced migration. In conclusion, IL-16 promotes eosinophil migration in vitro, by activating the plasminogen-plasmin system and increasing the membrane expression of its receptor. This effect is initiated via CD4 and mediated via the release of CCR3 ligand chemokines. Interestingly, most eosinophils express intracellular CD4. Hence, IL-16 may play an important role in the recruitment of blood eosinophils to the bronchial mucosa of asthmatics.

Background: Blood eosinophils express CD16 on their surface when stimulated in vitro with platelet-activating factor or IFNγ. Transient expression of CD16 is also observed in vivo following aeroallergen challenge of asthmatic subjects. The present work is aimed at evaluating the possible mechanisms modulating eosinophil expression of CD16 and the biological functions of this receptor. Methods: First, purified blood eosinophils were incubated with IL-1β, IL-2, IL-4, IL-5, IL-9 or IL-16, GM-CSF, IFNγ, eotaxin or 5-oxo-ETE and CD16 expression was measured. Second, the capacity of CD16 to mediate degranulation induced by IgG immune complexes (IC) was evaluated in eosinophils with low and high CD16 expression. Finally, serum allergen-specific IgE and IgG, and total IgE levels were measured at baseline in allergic asthmatics and correlated with changes observed in blood eosinophil CD16 expression (ΔCD16) following allergen challenge. Results: Only IFNγ and IL-2 significantly increased the number of CD16+ eosinophils, respectively, 37 ± 10% (p = 0.0038) and 38 ± 8% (p = 0.0006), compared to control, 7 ± 2%. IgG IC induced degranulation in eosinophils with low and high CD16 expression and monoclonal anti-CD16 and anti-CD32 antibodies inhibited this. IgG IC increased eosinophil CD16 expression (14 ± 6%, p = 0.0008) and this effect was blocked by pretreatment with anti-CD32 antibodies. ΔCD16 following allergen challenge correlated with the specific IgG/total IgE ratio (r2 = 0.41, p = 0.036). Conclusion: These data suggest that formation of IgG IC is associated with surface eosinophil CD16 expression in asthma and that CD16 in cooperation with CD32 mediates IC-induced degranulation.

Migration from blood to tissue modulates eosinophil function, possibly through interactions with endothelial cells. The effects of contact with and migration through endothelial cells on eosinophil expression of surface markers and release of leukotriene C4 were evaluated. A small proportion (2.6%) of eosinophils spontaneously migrated through endothelial cell monolayers. Activation of endothelial cells by interleukin (IL)-4 or IL-1beta slightly increased this migration (to 12.4%), which became much greater when a chemoattractant was placed in the lower chamber (84.3%). However, the chemotactic effect was downregulated by pretreating endothelial cells with interferon gamma (IFN-gamma; 63.1%). At baseline, 5% of eosinophils expressed CD69; this increased to 30.7% in culture on untreated endothelial cells and to 50.9% on IL-1beta-pretreated endothelial cells. This effect was mediated through intercellular adhesion molecule-1/CD11b interaction. Eosinophil migration through endothelial cells further increased CD69 expression to 63.9% and also increased CD35 expression from 83.3 to 91.3%. Upon stimulation, eosinophils that had migrated through endothelial cells produced more leukotriene C4 than control cells (872.4 and 103.9 pg x mL(-1), respectively). Endothelial cell pretreatment with IL-4 or IL-1beta further increased leukotriene C4 release (1,789.1 and 2,895.1 pg x mL(-1), respectively), whereas pretreatment with IFN-gamma decreased it (293.7 pg x mL(-1)). These data show that in vitro interactions with endothelial cells upregulate eosinophil membrane receptor expression and mediator release and that these effects are differently modulated by T-helper cell type 1 and 2 cytokines. These eosinophil modulations may play an important role in asthma pathogenesis.

Background Epidemiological studies show a strong correlation between vitamin D deficiency and asthma severity. Genetic studies have identified vitamin D receptor polymorphisms as a risk factor for asthma in diverse human populations. Calcitriol (1,25-dihydroxyvitamin D3), the physiologically active metabolite of mammalian vitamin D, is able to modulate receptor and cytokine expression as well as cell differentiation and maturation of various leukocytes in an anti-inflammatory manner. Despite the role of eosinophils in allergic asthma pathology, little is known about the effects of calcitriol on eosinophil biology. Calcitriol has a direct modulatory effect on eosinophil survival and effector functions in vitro.

Eosinophils are often predominant inflammatory leukocytes infiltrating oral squamous carcinoma (OSC) sites. Prostaglandins are secreted by oral carcinomas and may be involved in eosinophil infiltration. The objective of this study was to determine the factors contributing to eosinophil migration and potential anti-neoplastic effects on OSC. Eosinophil degranulation was evaluated by measuring release of eosinophil peroxidase (EPO). Eosinophil chemotaxis towards OSC cells was assessed using artificial basement membrane. Eosinophil infiltration was prominent within the tissue surrounding the OSC tumor mass. We observed growth inhibition of the OSC cell line, SCC-9, during co-culture with human eosinophils, in vitro, which correlated with EPO activity that possesses growth inhibitory activity. The PGD2 synthase inhibitor, HQL-79, abrogated migration towards SCC-9. Our data suggest that OSC-derived PGD2 may play an important role via CRTH2 (the PGD2 receptor on eosinophils) in eosinophil recruitment and subsequent anti-tumor activity through the action of eosinophil cationic proteins.

Background The most frequent trigger of asthma exacerbation is infection with common airway viruses; however, the precise mechanism regulating such severe reactions is not understood. The presence of airway eosinophil products is a unique feature detected in asthmatic airways. Using an animal model, we previously demonstrated that T cells play an important role in regulating an eosinophil-dependant pathway of virus-induced airway hyperreactivity. We hypothesize that human eosinophils respond to viruses, although only after interaction with T cells. Objectives We sought to determine whether eosinophils can respond to airway viruses in vitro and determine the mechanism of response. Methods An in vitro coculture model of human eosinophils, antigen-presenting cells, and T cells was used with parainfluenza virus, respiratory syncytial virus, or rhinovirus. We measured release of eosinophil peroxidase (EPO) in concert with T-cell proliferation, cytokine release, and changes in T-cell phenotype. Results The viruses induced release of EPO when coincubated in the presence of antigen-presenting cells (dendritic cells or macrophages) and T cells. Virus-mediated release was associated with proliferation of CD3 + CD4 + T cells and release of cytokines. UV inactivation of the virus did not prevent virus-induced EPO release or T-cell proliferation. Proliferating CD4 + T cells show increased expression of CD25 and CD45RO. CD8 + T cells were not activated. Conclusion Virus-induced EPO release can occur in the context of antigen presentation to CD4 + T cells.

Rationale Exocytosis is dependent on interaction of vesicle (v-) and plasma membrane (t-) SNAREs to form SNARE complexes. SNARE assembly is a prerequisite for secretion, which is specifically blocked by clostridial neurotoxins. We showed that the v-SNARE, VAMP-2, was localized to cytoplasmic vesicles in eosinophils, as shown in neutrophils. The t-SNAREs, SNAP-23 and syntaxin-4 are conserved in hematopoeitic cells and localized to eosinophil plasma membranes. We hypothesized that SNAREs are essential in granule-derived mediator release in eosinophils and neutrophils. Methods Purified human peripheral blood eosinophils and neutrophils were preincubated with or without inhibitory antibodies in the presence of the permeabilization agent, streptolysin-O, then stimulated with Ca2+ and GTPγS and supernatants examined for mediator release. Syntaxin-4 immunoprecipitates were examined for binding with SNARE isoforms. Expression of SNARE isoforms was determined using RT-PCR analysis. Protein expression was determined by Western blot and confirmed by FACS analysis of whole cells and granule fractions. Results Anti-syntaxin-4 impaired peroxidase secretion from both granulocytes. In contrast, anti-VAMP-2 blocked only eosinophil peroxidase release. Release of lactoferrin and MMP-9 from secondary and tertiary neutrophil granules, respectively, was modestly reduced by anti-VAMP-2. VAMP-7, -8 and syntaxin-6 mRNA was detected in both granulocyte types. Negligible expression of syntaxin-6 protein was found in both cell types. Analysis of purified CD63+ granules indicated that VAMP-7 is weakly expressed, while significant levels of VAMP-8 expression were detected in both cell types. Conclusions Our observations suggest that mediator release from granulocytes is SNARE-dependent and that subsets of v-SNAREs may be implicated in exocytosis of specific granules.

The effect of eotaxin, a potent eosinophil chemotactic factor, on eosinophil transmigration through a reconstituted basal membrane (Matrigel®) was evaluated. Eotaxin induced significant eosinophil transmigration in the presence of 10% fetal bovine serum (FBS) and interleukin-5. Its effect was optimal at 0.01 μM, and it plateaued at 18 h. Eotaxin's effect was greater with eosinophils from asthmatic subjects (61.1 ± 3.4%) than with eosinophils from normal subjects (38.7 ± 4.2%) (P < 0.001). Inhibition of metalloproteinases decreased eotaxin-induced transmigration by ≤10.4%, whereas inhibition of the plasminogen-plasmin system decreased eotaxin's effect by ≤44.4% (P = 0.0002). Moreover, eotaxin-induced transmigration was largely diminished in medium with low concentrations of serum [0.5% FBS: 6.1 ± 2.4%; 10% FBS: 40.2 ± 5.8% (P = 0.0001)] but returned to its initial level with the addition of plasminogen (2 U/mL) to 0.5% FBS (43.1 ± 6.5%). These data show that eotaxin is an efficient promoter of eosinophil transmigration in vitro, that it is more potent with cells from asthmatics than with normal cells, and that its effect depends predominantly on the activation of the plasminogen-plasmin system.

Objective/purpose Induction of IDO in dendritic cells (DCs) depletes the essential amino acid, tryptophan, and generates a family of catabolites known as kynurenines (KYN). IDO activity is reported to have immunomodulatory effects, including the selective induction of apoptosis in T-helper 1 (Th1) lymphocytes, an effect not seen with Th2 cells that are dominant in allergic asthma. Infants hospitalized for RSV-related bronchiolitis have increased risk of developing asthma (48% vs. 8% in control). Induction of IDO activity by RSV may explain the link between RSV bronchiolitis and asthma pathogenesis. IDO is induced by various cytokines and a number of non-airway viruses; however, RSV has not yet been studied. We hypothesize that RSV induce IDO activation in human dendritic cells (DCs). Methods Primary human dendritic cells (DCs) were infected with sucrose gradient purified RSV with a multiplicity of infection (MOI) rate of 1.0. Flow cytometry and confocal microscopy were used to confirm infection. We measured KYN in culture media by a spectrophotometric method using Ehrlich reagent. We blocked RSV infection with the RSV-mAb, Palivizumab, and UV-inactivation to determine a role for infection. The potent competitive inhibitor of viral RNA polymerase, Ribavirin, was used to block RSV replication and protein synthesis. To evaluate dependency of RSV-induced IDO induction on different cell signaling pathways, we used a variety of specific inhibitors including MEK inhibitor I (120 nM), MEK inhibitor II (4 μM), SB202190 (p38MAPK, 3 μM), JNK inhibitor II (1 μM), IKK inhibitor II (Wedelolactone, 30 μM), IKK inhibitor III (BMS345541, 3 μM) and relevant negative controls.