Citations

Abstract IL-5 is a key cytokine for eosinophil maturation, recruitment, activation, and possibly the development of inflammation in asthma. High concentrations of IL-5 are present in the airway after Ag challenge, but the responsiveness of airway eosinophils to IL-5 is not well characterized. The objectives of this study were to establish, following airway Ag challenge: 1) the expression of membrane (m)IL-5Rα on bronchoalveolar lavage (BAL) eosinophils; 2) the responsiveness of these cells to exogenous IL-5; and 3) the presence of soluble (s)IL-5Rα in BAL fluid. To accomplish these goals, blood and BAL eosinophils were obtained from atopic subjects 48 h after segmental bronchoprovocation with Ag. There was a striking reduction in mIL-5Rα on airway eosinophils compared with circulating cells. Furthermore, sIL-5Rα concentrations were elevated in BAL fluid, but steady state levels of sIL-5Rα mRNA were not increased in BAL compared with blood eosinophils. Finally, BAL eosinophils were refractory to IL-5 for ex vivo degranulation, suggesting that the reduction in mIL-5Rα on BAL eosinophils may regulate IL-5-mediated eosinophil functions. Together, the loss of mIL-5Rα, the presence of sIL-5Rα, and the blunted functional response (degranulation) of eosinophils to IL-5 suggest that when eosinophils are recruited to the airway, regulation of their functions becomes IL-5 independent. These observations provide a potential explanation for the inability of anti-IL-5 therapy to suppress airway hyperresponsiveness to inhaled Ag, despite a reduction in eosinophil recruitment.

Abstract Eosinophils and their products are probably important in the pathophysiology of allergic diseases, such as bronchial asthma, and in host immunity to certain organisms. An association between environmental fungal exposure and asthma has been long recognized clinically. Although products of microorganisms (e.g., lipopolysaccharides) directly activate certain inflammatory cells (e.g., macrophages), the mechanism(s) that triggers eosinophil degranulation is unknown. In this study we investigated whether human eosinophils have an innate immune response to certain fungal organisms. We incubated human eosinophils with extracts from seven environmental airborne fungi (Alternaria alternata, Aspergillus versicolor, Bipolaris sorokiniana, Candida albicans, Cladosporium herbarum, Curvularia spicifera, and Penicillium notatum). Alternaria and Penicillium induced calcium-dependent exocytosis (e.g., eosinophil-derived neurotoxin release) in eosinophils from normal individuals. Alternaria also strongly induced other activation events in eosinophils, including increases in intracellular calcium concentration, cell surface expression of CD63 and CD11b, and production of IL-8. Other fungi did not induce eosinophil degranulation, and Alternaria did not induce neutrophil activation, suggesting specificity for fungal species and cell type. The Alternaria-induced eosinophil degranulation was pertussis toxin sensitive and desensitized by preincubating cells with G protein-coupled receptor agonists, platelet-activating factor, or FMLP. The eosinophil-stimulating activity in Alternaria extract was highly heat labile and had an Mr of ∼60 kDa. Thus, eosinophils, but not neutrophils, possess G protein-dependent cellular activation machinery that directly responds to an Alternaria protein product(s). This innate response by eosinophils to certain environmental fungi may be important in host defense and in the exacerbation of inflammation in asthma and allergic diseases.

Abstract Ecalectin, produced by Ag-stimulated T lymphocytes, is a potent eosinophil-specific chemoattractant in vitro as well as in vivo and thus is implicated in allergic responses. Ecalectin differs structurally from other known eosinophil chemoattractants (ECAs); ecalectin belongs to the galectin family defined by their affinity for β-galactosides and by their conserved carbohydrate recognition domains. These characteristic features suggest that ecalectin has unique activities associated with allergic inflammation besides ECA activity. Conversely, ecalectin may mediate ECA activity by binding to a receptor of a known ECA via affinity for the β-galactosides present on this receptor. In this study, we have tested whether ecalectin mediates ECA activity by binding to a receptor of a known ECA, and we have assessed its effects on eosinophils. Ecalectin did not mediate ECA activity by binding to the IL-5R or to CCR3. Also, the ECA activity of ecalectin was mainly chemokinetic. In addition, ecalectin induced concentration-dependent eosinophil aggregation, a marker for eosinophil activation. Ecalectin induced concentration-dependent superoxide production from eosinophils but did not induce degranulation; usually these two events are coupled in eosinophil activation. Moreover, ecalectin directly prolonged eosinophil survival in vitro and did not trigger eosinophils to secrete cytokines that prolong eosinophil survival. These results demonstrate that ecalectin has several unique effects on eosinophils. Therefore, we conclude that ecalectin is a novel eosinophil-activating factor. Presumably, these effects allow ecalectin to play a distinctive role in allergic inflammation.

Abstract In granulocytes, platelet-activating factor (PAF) shares many of its biological effects with other chemotactic factors, such as FMLP, complement fragments, and lipid mediators. Two unique effects are that PAF is relatively resistant to pertussis toxin (PTX) and that PAF activates the inflammatory functions of eosinophils more strongly than it activates those of neutrophils. To investigate the molecular mechanisms of the responses of eosinophils to PAF, we analyzed superoxide anion production by a chemiluminescence method that provides real-time kinetic data for the cellular responses. We found that PAF induced bimodal superoxide anion production in human eosinophils, consisting of an intense, but transient, first phase and a larger and sustained second phase. In contrast, PAF induced essentially a transient unimodal response in human neutrophils. The two phases of eosinophil response were mediated by distinct cellular mechanisms: the second phase was highly dependent on cellular adhesion and β2 integrins, but the first phase was independent of both adhesion and β2 integrins. The upstream signaling mechanisms were also different: the second phase was mediated by PTX-resistant G-protein(s) and through activation of phosphatidylinositol 3-kinase, while the first phase was mediated by PTX-sensitive G-protein(s). Furthermore, the second-phase response was ∼100-fold more resistant to inhibition by a competitive PAF receptor antagonist than the first phase. Thus, eosinophils and neutrophils react differently to PAF, and PAF activates two separate and distinct effector pathways in human eosinophils. These two activation pathways may explain the eosinophils’ strong and diverse biological responses to PAF.

Abstract Tissue eosinophilia probably plays important roles in the pathophysiology of bronchial asthma and allergic disorders; however, this concept was challenged recently by controversial results in mouse models of bronchial asthma treated with anti-IL-5 Ab and the failure of anti-IL-5 therapy in humans. We have now used a unique model, IL-5 transgenic (TG) mice, to address a fundamental question: is airway eosinophilia beneficial or detrimental in the allergic response? After sensitization and challenge with OVA, IL-5 TG mice showed a marked airway eosinophilia. Surprisingly, these IL-5 TG mice showed lower airway reactivity to methacholine. Immunohistochemical analysis of the lungs revealed a marked peribronchial infiltration of eosinophils, but no eosinophil degranulation. In vitro, mouse eosinophils from peritoneal lavage fluids did not produce superoxide anion, but did produce an anti-inflammatory and fibrotic cytokine, TGF-β1. Indeed, the TGF-β1 levels in bronchoalveolar lavage fluid specimens from IL-5 TG mice directly correlated with airway eosinophilia (r = 0.755). Furthermore, anti-IL-5 treatment of IL-5 TG mice decreased both airway eosinophilia and TGF-β1 levels in bronchoalveolar lavage fluids and increased airway reactivity. Thus, in mice, marked eosinophilia prevents the development of airway hyper-reactivity during an allergic response. Overall, the roles of eosinophils in asthma and in animal models need to be addressed carefully for their potentially detrimental and beneficial effects.

Abstract Protease-activated receptors (PARs) are a unique class of G protein-coupled receptors, which are activated by proteolytic cleavage of the amino terminus of the receptor itself. PARs are most likely involved in various biological responses, such as hemostasis and regulation of muscle tone; however, the roles of PARs in the functions of inflammatory and immune cells are poorly understood. Because eosinophils are most likely involved in allergic inflammation and are exposed to a variety of proteases derived from allergens and other inflammatory cells, we investigated whether PARs regulate effector functions of eosinophils. Human eosinophils constitutively transcribe mRNA for PAR2 and PAR3, but not those for PAR1 and PAR4. The expression of PAR2 protein was confirmed by flow cytometry. When trypsin, an agonist for PAR2, was incubated with eosinophils, it potently induced superoxide anion production and degranulation; 5 nM trypsin induced responses that were 50∼70% of those induced by 100 nM platelet-activating factor, a positive control. In contrast, thrombin, an activator for PAR1, PAR3, and PAR4, showed minimal effects. The stimulatory effect of trypsin was dependent on its serine protease activity and was blocked 59% by anti-PAR2 Ab. Furthermore, a specific tethered peptide ligand for PAR2 potently induced superoxide production and degranulation; the effects of peptide ligands for PAR1, PAR3, and PAR4 were negligible. These findings suggest that human eosinophils express functional PAR2, and serine proteases at the inflammation site may play important roles in regulating effector functions of human eosinophils. The expression and functional relevance of other PARs still need to be determined.

Abstract We present a novel immunotherapeutic strategy using a human B7-DC cross-linking Ab that prevents lung inflammation, airway obstruction, and hyperreactivity to allergen in a mouse model of allergic inflammatory airway disease. Dendritic cells (DC) have the ability to skew the immune response toward a Th1 or Th2 polarity. The sHIgM12 Ab functions in vitro by cross-linking the costimulatory family molecule B7-DC (PD-L2) on DC up-regulating IL-12 production, homing to lymph nodes, and T cell-activating potential of these APCs. Using chicken OVA as a model Ag, the administration of sHIgM12 Ab to BALB/c mice blocked lung inflammation, airway pathology, and responsiveness to methacholine, even after animals were presensitized and a Th2-polarized immune response was established. This therapeutic strategy was ineffective in STAT4-deficient animals, indicating that IL-12 production is critical in this system. Moreover, the polarity of the immune response upon in vitro restimulation with Ag is changed in wild-type mice, with a resulting decrease in Th2 cytokines IL-4 and IL-5 and an increase in the immunoregulatory cytokine IL-10. These studies demonstrate that the immune response of hypersensitized responders can be modulated using B7-DC cross-linking Abs, preventing allergic airway disease upon re-exposure to allergen.

Abstract IL-12p70, a heterodimer composed of p35 and p40 subunits, is a key polarizing cytokine produced by maturing dendritic cells (DCs). We report that cigarette smoke extract (CSE), an extract of soluble cigarette smoke components, suppresses both p35 and p40 production by LPS or CD40L-matured DCs. Suppression of IL-12p70 production from maturing DCs was not observed in the presence of nicotine concentrations achievable in CSE or in the circulation of smokers. The suppressed IL-12p70 protein production by CSE-conditioned DCs was restored by pretreatment of DCs or CSE with the antioxidants N-acetylcysteine and catalase. Inhibition of DC IL-12p70 by CSE required activation of ERK-dependent pathways, since inhibition of ERK abrogated the suppressive effect of CSE on IL-12 secretion. Oxidative stress and sustained ERK phosphorylation by CSE enhanced nuclear levels of the p40 transcriptional repressor c-fos in both immature and maturing DCs. Suppression of the p40 subunit by CSE also resulted in diminished production of IL-23 protein by maturing DCs. Using a murine model of chronic cigarette smoke exposure, we observed that systemic and lung DCs from mice “smokers” produced significantly less IL-12p70 and p40 protein upon maturation. This inhibitory effect was selective, since production of TNF-α during DC maturation was enhanced in the smokers. These data imply that oxidative stress generated by cigarette smoke exposure suppresses the generation of key cytokines by maturing DCs through the activation of ERK-dependent pathways. Some of the cigarette smoke-induced inhibitory effects on DC function may be mitigated by antioxidants.

Abstract Eosinophils and their products are likely important in the pathophysiology of allergic diseases, such as bronchial asthma, and in host immunity to parasitic organisms. However, the mechanisms for proinflammatory mediator release by eosinophils are poorly understood. CD66b (CEACAM8, CGM6, NCA-95) is a single chain, GPI-anchored, highly glycosylated protein belonging to the carcinoembryonic Ag supergene family. CD66b is an activation marker for human granulocytes; however, its biological functions are largely unknown in eosinophils. We found that CD66b is highly expressed on the surface of human peripheral blood eosinophils isolated from healthy individuals. Engagement of CD66b, but not CD66a, by mAb or a natural ligand, galectin-3, activated a Src kinase family molecule, hemopoietic cell kinase (Hck), and induced cellular adhesion, superoxide production, and degranulation of eosinophils. CD66b molecules were localized in lipid rafts, and disruption of lipid rafts or removal of the GPI anchor inhibited the adhesion and activation of eosinophils. Importantly, CD66b was constitutively and physically associated with a β2 integrin, CD11b, and cross-linking of CD66b induced a striking clustering of CD11b molecules. Thus, CD66b molecules are involved in regulating adhesion and activation of eosinophils, possibly through their localization in lipid rafts and interaction with other cell surface molecules, such as CD11b. Binding of exogenous or endogenous carbohydrate ligands(s) to CD66b may be important in the release of proinflammatory mediators by human eosinophils.

Abstract Eosinophils produce and release various proinflammatory mediators and also show immunomodulatory and tissue remodeling functions; thus, eosinophils may be involved in the pathophysiology of asthma and other eosinophilic disorders as well as host defense. Several major questions still remain. For example, how do human eosinophils become activated in diseased tissues or at the site of an immune response? What types of host immunity might potentially involve eosinophils? Herein, we found that human eosinophils react vigorously to a common environmental fungus, Alternaria alternata, which is implicated in the development and/or exacerbation of human asthma. Eosinophils release their cytotoxic granule proteins, such as eosinophil-derived neurotoxin and major basic protein, into the extracellular milieu and onto the surface of fungal organisms and kill the fungus in a contact-dependent manner. Eosinophils use their versatile β2 integrin molecule, CD11b, to adhere to a major cell wall component, β-glucan, but eosinophils do not express other common fungal receptors, such as dectin-1 and lactosylceramide. The I-domain of CD11b is distinctively involved in the eosinophils’ interaction with β-glucan. Eosinophils do not react with another fungal cell wall component, chitin. Because human eosinophils respond to and kill certain fungal organisms, our findings identify a previously unrecognized innate immune function for eosinophils. This immune response by eosinophils may benefit the host, but, in turn, it may also play a role in the development and/or exacerbation of eosinophil-related allergic human diseases, such as asthma.