Eosinophils in innate immunity: an evolving story

Eosinophils are innate immune leukocytes found in relatively low numbers within the blood. Terminal effector functions of eosinophils, deriving from their capacity to release their content of tissue-destructive cationic proteins, have historically been considered primary effector mechanisms against specific parasites, and are likewise implicated in tissue damage accompanying allergic responses such as asthma. However, the past decade has seen dramatic advancements in the field of eosinophil immunobiology, revealing eosinophils to also be key participants in many other facets of innate immunity, from bridging innate and adaptive immune responses to orchestrating tissue remodeling events. Here, we review the multifaceted functions of eosinophils in innate immunity that are currently known, and discuss new avenues in this evolving story.     

α-Galactosylceramide-induced airway eosinophilia is mediated through the activation of NKT cells

Invariant NKT (iNKT) cells bridge innate and adaptive immune responses, resulting in the expansion of Ag-specific B and T cell responses. α-Galactosylceramide (α-GalCer), the most studied glycolipid that activates iNKT cells, has been proposed to be an effective adjuvant against infections and tumors. We found that the activation of iNKT cells by intranasal injection of α-GalCer induced airway eosinophilia in naive mice. Eosinophils, which mediate tissue damage and dysfunction by secreting mediators, play important roles in the pathogenesis of allergic diseases. In this study, we investigated the mechanism of how eosinophils are recruited to the lung by α-GalCer. Our results demonstrated that α-GalCer-induced eosinophil inflammation was mediated through iNKT cells. These cells secreted IL-5 to recruit eosinophils directly to the lung and/or secreted IL-4 and IL-13 to recruit eosinophils indirectly by inducing lung epithelial cells, endothelial cells, and fibroblast to secrete the eosinophil chemoattractant eotaxin. In addition, in the OVA-alum murine model of allergic asthma, α-GalCer administration in OVA-immunized mice also increased airway eosinophilia after challenge. Given our findings, intranasal administration of α-GalCer induced airway eosinophilic inflammation in both naive and allergic mice. Hence, it remains to be determined whether the activation of iNKT cells would be applicable in therapeutics for human diseases.     

CD48 is an allergen and IL-3-induced activation molecule on eosinophils

Eosinophils are involved in a variety of allergic, parasitic, malignant, and idiopathic disorders by releasing a variety of factors including specific granule proteins, lipid mediators, and proinflammatory and immunoregulatory cytokines and chemokines. In addition, they interact with various cell types in the inflamed tissue. Yet, the mechanism of eosinophil activation is still poorly understood. Recently, we described the expression and function of the CD2-subfamily of receptors and especially 2B4 on human eosinophils. In this study we focus on CD48, the high-affinity ligand of 2B4. CD48 is a GPI-anchored protein involved in cellular activation, costimulation, and adhesion, but has not been studied on eosinophils. We demonstrate that human eosinophils from atopic asthmatics display enhanced levels of CD48 expression and that IL-3 up-regulates CD48 expression. Furthermore, cross-linking CD48 on human eosinophils triggers release of eosinophil granule proteins. Assessment of CD48 expression in a murine model of experimental asthma revealed that CD48 is induced by allergen challenge and partially regulated by IL-3. Additionally, anti-IL-3 reduces CD48 expression and the degree of airway inflammation. Thus, CD48 is an IL-3-induced activating receptor on eosinophils, likely involved in promoting allergic inflammation.     

Innate antifungal immunity of human eosinophils mediated by a beta 2 integrin, CD11b

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 beta(2) integrin molecule, CD11b, to adhere to a major cell wall component, beta-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 beta-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.     

Nonpathogenic, environmental fungi induce activation and degranulation of human eosinophils

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 M(r) of approximately 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.     

Surfactant protein D attenuates sub-epithelial fibrosis in allergic airways disease through TGF-β

Background

Surfactant protein D (SP-D) can regulate both innate and adaptive immunity. Recently, SP-D has been shown to contribute to the pathogenesis of airway allergic inflammation and bleomycin-induced pulmonary fibrosis. However, in allergic airways disease, the role of SP-D in airway remodeling remains unknown. The objective of this study was to determine the contribution of functional SP-D in regulating sub-epithelial fibrosis in a mouse chronic house dust mite model of allergic airways disease.

Methods

C57BL/6 wild-type (WT) and SP-D−/− mice (C57BL/6 background) were chronically challenged with house dust mite antigen (Dermatophagoides pteronyssinus, Dp). Studies with SP-D rescue and neutralization of TGF-β were conducted. Lung histopathology and the concentrations of collagen, growth factors, and cytokines present in the airspace and lung tissue were determined. Cultured eosinophils were stimulated by Dp in presence or absence of SP-D.

Results

Dp-challenged SP-D−/− mice demonstrate increased sub-epithelial fibrosis, collagen production, eosinophil infiltration, TGF-β1, and IL-13 production, when compared to Dp-challenged WT mice. By immunohistology, we detected an increase in TGF-β1 and IL-13 positive eosinophils in SP-D−/− mice. Purified eosinophils stimulated with Dp produced TGF-β1 and IL-13, which was prevented by co-incubation with SP-D. Additionally, treatment of Dp challenged SP-D−/− mice with exogenous SP-D was able to rescue the phenotypes observed in SP-D−/− mice and neutralization of TGF-β1 reduced sub-epithelial fibrosis in Dp-challenged SP-D−/− mice.

Conclusion

These data support a protective role for SP-D in the pathogenesis of sub-epithelial fibrosis in a mouse model of allergic inflammation through regulation of eosinophil-derived TGF-β.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0143-9) contains supplementary material, which is available to authorized users.     

Surfactant protein-A suppresses eosinophil-mediated killing of Mycoplasma pneumoniae in allergic lungs

Surfactant protein-A (SP-A) has well-established functions in reducing bacterial and viral infections but its role in chronic lung diseases such as asthma is unclear. Mycoplasma pneumoniae (Mp) frequently colonizes the airways of chronic asthmatics and is thought to contribute to exacerbations of asthma. Our lab has previously reported that during Mp infection of non-allergic airways, SP-A aides in maintaining airway homeostasis by inhibiting an overzealous TNF-alpha mediated response and, in allergic mice, SP-A regulates eosinophilic infiltration and inflammation of the airway. In the current study, we used an in vivo model with wild type (WT) and SP-A(-/-) allergic mice challenged with the model antigen ovalbumin (Ova) that were concurrently infected with Mp (Ova+Mp) to test the hypothesis that SP-A ameliorates Mp-induced stimulation of eosinophils. Thus, SP-A could protect allergic airways from injury due to release of eosinophil inflammatory products. SP-A deficient mice exhibit significant increases in inflammatory cells, mucus production and lung damage during concurrent allergic airway disease and infection (Ova+Mp) as compared to the WT mice of the same treatment group. In contrast, SP-A deficient mice have significantly decreased Mp burden compared to WT mice. The eosinophil specific factor, eosinophil peroxidase (EPO), which has been implicated in pathogen killing and also in epithelial dysfunction due to oxidative damage of resident lung proteins, is enhanced in samples from allergic/infected SP-A(-/-) mice as compared to WT mice. In vitro experiments using purified eosinophils and human SP-A suggest that SP-A limits the release of EPO from Mp-stimulated eosinophils thereby reducing their killing capacity. These findings are the first to demonstrate that although SP-A interferes with eosinophil-mediated biologic clearance of Mp by mediating the interaction of Mp with eosinophils, SP-A simultaneously benefits the airway by limiting inflammation and damage.     

Coexpression of IL-5 and eotaxin-2 in mice creates an eosinophil-dependent model of respiratory inflammation with characteristics of severe asthma

Mouse models of allergen provocation and/or transgenic gene expression have provided significant insights regarding the cellular, molecular, and immune responses linked to the pathologies occurring as a result of allergic respiratory inflammation. Nonetheless, the inability to replicate the eosinophil activities occurring in patients with asthma has limited their usefulness to understand the larger role(s) of eosinophils in disease pathologies. These limitations have led us to develop an allergen-naive double transgenic mouse model that expresses IL-5 systemically from mature T cells and eotaxin-2 locally from lung epithelial cells. We show that these mice develop several pulmonary pathologies representative of severe asthma, including structural remodeling events such as epithelial desquamation and mucus hypersecretion leading to airway obstruction, subepithelial fibrosis, airway smooth muscle hyperplasia, and pathophysiological changes exemplified by exacerbated methacholine-induced airway hyperresponsiveness. More importantly, and similar to human patients, the pulmonary pathologies observed are accompanied by extensive eosinophil degranulation. Genetic ablation of all eosinophils from this double transgenic model abolished the induced pulmonary pathologies, demonstrating that these pathologies are a consequence of one or more eosinophil effector functions.     

Staphylococcus aureus induces eosinophil cell death mediated by α-hemolysin

Staphylococcus aureus, a major human pathogen, exacerbates allergic disorders, including atopic dermatitis, nasal polyps and asthma, which are characterized by tissue eosinophilia. Eosinophils, via their destructive granule contents, can cause significant tissue damage, resulting in inflammation and further recruitment of inflammatory cells. We hypothesised that the relationship between S. aureus and eosinophils may contribute to disease pathology. We found that supernatants from S. aureus (SH1000 strain) cultures cause rapid and profound eosinophil necrosis, resulting in dramatic cell loss within 2 hours. This is in marked contrast to neutrophil granulocytes where no significant cell death was observed (at equivalent dilutions). Supernatants prepared from a strain deficient in the accessory gene regulator (agr) that produces reduced levels of many important virulence factors, including the abundantly produced α-hemolysin (Hla), failed to induce eosinophil death. The role of Hla in mediating eosinophil death was investigated using both an Hla deficient SH1000-modified strain, which did not induce eosinophil death, and purified Hla, which induced concentration-dependent eosinophil death via both apoptosis and necrosis. We conclude that S. aureus Hla induces aberrant eosinophil cell death in vitro and that this may increase tissue injury in allergic disease.     

IgA-induced eosinophil degranulation

Eosinophils play an important role as effector cells in allergic, parasitic, and other conditions. The mechanism(s) by which eosinophils mediate their effector functions was studied by incubation of human normodense eosinophils with Sepharose beads coupled to various Ig isotypes as targets. Controls included eosinophils incubated alone or incubated with uncoated beads, human serum albumin-, or OVA-coated beads. An eosinophil granule protein, the eosinophil-derived neurotoxin (EDN), was measured as an indicator of eosinophil degranulation. Eosinophils released eosinophil-derived neurotoxin when incubated with Sepharose beads coupled to Ig of the IgG or IgA isotypes, as well as IgA-Fc fragments. Mixtures of IgG and IgA on beads did not act synergistically. Secretory IgA (sIgA) provided the most potent signal for eosinophil degranulation and was two to three times more potent than IgG. Furthermore, 2 to 17% of the normodense eosinophils bound to IgG- or IgA-coated beads, whereas 24 to 27% of the eosinophils bound to sIgA-coated beads. Thus, sIgA may be the principal Ig mediating eosinophil effector function at mucosal surfaces in helminth infections and hypersensitivity diseases, especially bronchial asthma.     

Computational and experimental analysis reveals a requirement for eosinophil-derived IL-13 for the development of allergic airway responses in C57BL/6 mice

Eosinophils are found in the lungs of humans with allergic asthma, as well as in the lungs of animals in models of this disease. Increasing evidence suggests that these cells are integral to the development of allergic asthma in C57BL/6 mice. However, the specific function of eosinophils that is required for this event is not known. In this study, we experimentally validate a dynamic computational model and perform follow-up experimental observations to determine the mechanism of eosinophil modulation of T cell recruitment to the lung during development of allergic asthma. We find that eosinophils deficient in IL-13 were unable to rescue airway hyperresponsiveness, T cell recruitment to the lungs, and Th2 cytokine/chemokine production in ΔdblGATA eosinophil-deficient mice, even if Th2 cells were present. However, eosinophil-derived IL-13 alone was unable to rescue allergic asthma responses in the absence of competence of other IL-13-producing cells. We further computationally investigate the role of other cell types in the production of IL-13, which led to the various predictions including early and late pulses of IL-13 during airway hyperresponsiveness. These experiments suggest that eosinophils and T cells have an interdependent relationship, centered on IL-13, which regulates T cell recruitment to the lung and development of allergic asthma.     

Treatment with Pyranopyran-1, 8-Dione Attenuates Airway Responses in Cockroach Allergen Sensitized Asthma in Mice

Chronic allergic asthma is characterized by Th2-typed inflammation, and contributes to airway remodeling and the deterioration of lung function. Viticis Fructus (VF) has long been used in China and Korea as a traditional herbal remedy for treating various inflammatory diseases. Previously, we have isolated a novel phytochemical, pyranopyran-1, 8-dione (PPY), from VF. This study was conducted to evaluate the ability of PPY to prevent airway inflammation and to attenuate airway responses in a cockroach allergen-induced asthma model in mice. The mice sensitized to and challenged with cockroach allergen were treated with oral administration of PPY. The infiltration of total cells, eosinophils and lymphocytes into the BAL fluid was significantly inhibited in cockroach allergen-induced asthma mice treated with PPY (1, 2, or 10 mg/kg). Th2 cytokines and chemokine, such as IL-4, IL-5, IL-13 and eotaxin in BAL fluid were also reduced to normal levels following treatment with PPY. In addition, the levels of IgE were also markedly suppressed after PPY treatment. Histopathological examination demonstrated that PPY substantially inhibited eosinophil infiltration into the airway, goblet cell hyperplasia and smooth muscle hypertrophy. Taken together, these results demonstrate that PPY possesses a potent efficacy on controlling allergic asthma response such as airway inflammation and remodeling.     

Extensive eosinophil degranulation and peroxidase-mediated oxidation of airway proteins do not occur in a mouse ovalbumin-challenge model of pulmonary inflammation

Paradigms of eosinophil effector function in the lungs of asthma patients invariably depend on activities mediated by cationic proteins released from secondary granules during a process collectively referred to as degranulation. In this study, we generated knockout mice deficient for eosinophil peroxidase (EPO) to assess the role(s) of this abundant secondary granule protein in an OVA-challenge model. The loss of EPO had no effect on the development of OVA-induced pathologies in the mouse. The absence of phenotypic consequences in these knockout animals extended beyond pulmonary histopathologies and airway changes, as EPO-deficient animals also displayed OVA-induced airway hyperresponsiveness after provocation with methacholine. In addition, EPO-mediated oxidative damage of proteins (e.g., bromination of tyrosine residues) recovered in bronchoalveolar lavage from OVA-treated wild-type mice was <10% of the levels observed in bronchoalveolar lavage recovered from asthma patients. These data demonstrate that EPO activities are inconsequential to the development of allergic pulmonary pathologies in the mouse and suggest that degranulation of eosinophils recruited to the lung in this model does not occur at levels comparable to those observed in humans with asthma.     

The roles of autotaxin/lysophosphatidic acid in immune regulation and asthma

Lysophosphatidic acid (LPA) species are present in almost all organ systems and play diverse roles through its receptors. Asthma is an airway disease characterized by chronic allergic inflammation where various innate and adaptive immune cells participate in establishing Th2 immune response. Here, we will review the contribution of LPA and its receptors to the functions of immune cells that play a key role in establishing allergic airway inflammation and aggravation of allergic asthma.     

In vitro and in vivo characterization of a novel CCR3 antagonist, YM-344031

Eosinophils play a prominent proinflammatory role in a broad range of diseases, including atopic dermatitis and asthma. Eotaxin-1 and its receptor CCR3 are implicated in the recruitment of eosinophils from blood into inflammatory tissues, therefore inhibition of Eotaxin-1/CCR3 interaction may have therapeutic potential for allergic inflammation with eosinophil infiltration. YM-344031, a novel and selective small molecule CCR3 antagonist, potently inhibited ligand binding (IC(50)=3.0nM), ligand-induced Ca(2+) flux (IC(50)=5.4nM), and the chemotaxis of human CCR3-expressing cells (IC(50)=19.9nM). YM-344031 (1-10mg/kg) orally administered to cynomolgus monkeys significantly inhibited Eotaxin-1-induced eosinophil shape change in whole blood. Additionally, orally administered YM-344031 (100mg/kg) prevented both immediate- and late-phase allergic skin reactions in a mouse allergy model. YM-344031 therefore has potential as a novel and orally available compound for the treatment of allergic inflammation, such as atopic dermatitis and asthma.     

Differential proteomic analysis of bronchoalveolar lavage fluid in asthmatics following segmental antigen challenge

Allergic asthma is characterized by persistent airway inflammation and remodeling. Bronchoalveolar lavage conducted with fiberoptic bronchoscopy has been widely used for investigating the pathogenesis of asthma and other lung disorders. Identification of proteins in the bronchoalveolar lavage fluid (BALF) and their expression changes at different stages of asthma could provide further insights into the complex molecular mechanisms involved in this disease. In this report, we describe the first comprehensive differential proteomic analysis of BALF from both asthmatic patients and healthy subjects before and 24 h after segmental allergen challenge. Our proteomic analysis involves affinity depletion of six abundant BALF proteins, SDS-PAGE fractionation, protein in-gel digestion, and subsequent nano-LC-MS/MS analysis in conjunction with database searching for protein identification and semiquantitation. More than 1,500 distinct proteins were identified of which about 10% displayed significant up-regulation specific to the asthmatic patients after segmental allergen challenge. The differentially expressed proteins represent a wide spectrum of functional classes such as chemokines, cytokines, proteases, complement factors, acute phase proteins, monocyte-specific granule proteins, and local matrix proteins, etc. The majority of these protein expression changes are closely associated with many aspects of the pathophysiology of asthma, including inflammation, eosinophilia, airway remodeling, tissue damage and repair, mucus production, and plasma infiltration. Importantly a large portion of these proteins and their expression changes were identified for the first time from BALF, thus providing new insights for finding novel pathological mediators and biomarkers of asthma.     

Fungal chitin from asthma-associated home environments induces eosinophilic lung infiltration

Development of asthma and allergic inflammation involves innate immunity, but the environmental contributions remain incompletely defined. Analysis of dust collected from the homes of asthmatic individuals revealed that the polysaccharide chitin is environmentally widespread and associated with β-glucans, possibly from ubiquitous fungi. Cell wall preparations of Aspergillus isolated from house dust induced robust recruitment of eosinophils into mouse lung, an effect that was attenuated by enzymatic degradation of cell wall chitin and β-glucans. Mice expressing constitutively active acidic mammalian chitinase in the lungs demonstrated a significant reduction in eosinophil infiltration after fungal challenge. Conversely, chitinase inhibition prolonged the duration of tissue eosinophilia. Thus, fungal chitin derived from home environments associated with asthma induces eosinophilic allergic inflammation in the lung, and mammalian chitinases, including acidic mammalian chitinase, limit this process.     

Human eosinophils exert TNF-α and granzyme A-mediated tumoricidal activity toward colon carcinoma cells

Peripheral blood and tissue eosinophilia is a prominent feature in allergic diseases and helminth infections. In cancer patients, tumor-associated tissue eosinophilia is frequently observed. Tumor-associated tissue eosinophilia can be associated with a favorable prognosis, notably in colorectal carcinoma. However, underlying mechanisms of eosinophil contribution to antitumor responses are poorly understood. We have in this study investigated the direct interactions of human eosinophils with Colo-205, a colorectal carcinoma cell line, and show that eosinophils induce apoptosis and directly kill tumor cells. Using blocking Abs, we found that CD11a/CD18 complex is involved in the tumoricidal activity. Coculture of eosinophils with Colo-205 led to the release of eosinophil cationic protein and eosinophil-derived neurotoxin as well as TNF-α secretion. Moreover, eosinophils expressed granzyme A, which was released upon interaction with Colo-205, whereas cytotoxicity was partially inhibited by FUT-175, an inhibitor of trypsin-like enzymatic activity. Our data present the first demonstration, to our knowledge, that granzyme A is a cytotoxic mediator of the eosinophil protein arsenal, exerting eosinophil tumoricidal activity toward Colo-205, and provide mechanistic evidence for innate responses of eosinophil against tumor cells.     

Eosinophil granule cationic proteins regulate complement. I. Activity on the alternative pathway.

Eosinophil granules contain several cationic proteins that mediate tissue damage in allergic disease. The present study examined the capacity and mechanisms by which these cationic proteins regulate activity of the alternative pathway of C. Eosinophil peroxidase and eosinophil cationic protein inhibited formation of cell-bound alternative pathway C3 convertase, causing 50% inhibition of lysis at about 0.19 and 0.75 microgram/10(7) cellular intermediates, respectively. Major basic protein inhibited alternative pathway C3 activity by only 19% at 1.5 micrograms/10(7) cellular intermediates. Eosinophil-derived neurotoxin had no activity on the alternative pathway. The eosinophil granule proteins were examined for the mechanism by which they inhibited alternative pathway activity. Eosinophil peroxidase and major basic protein inhibited fluid phase factor B consumption in a reaction mixture that also contained factors D and C3b, eosinophil-derived neurotoxin had no activity on factor B consumption, and eosinophil cationic protein consumed factor B in the absence of C3b and factor D. Both eosinophil cationic protein and eosinophil peroxidase enhanced the decay of preformed alternative pathway convertase. Lysis of EAC4b,3b cellular intermediates formed to contain a low surface amount of C3b was more inhibited than was lysis of cells formed with a standard amount of C3b on the surface. This suggests that these eosinophil proteins acted predominantly on C3b to regulate alternative pathway activity. We also found that none of the eosinophil granule cationic proteins had any effect on later events after the formation of the C3 convertase. We conclude that although eosinophil-derived neurotoxin (isoelectric pH value (pI) = 8.9) does not regulate alternative pathway activity, the more highly charged eosinophil granule cationic proteins--major basic protein (pI = 10.9), eosinophil cationic protein (pI = 10.8), and eosinophil peroxidase (pI = 10.8)--do share the capacity to regulate C activity and may exert this activity in vivo.