Mouse eosinophil associated ribonucleases: Mechanism of cytotoxic, antibacterial and antiparasitic activities.

Ribonuclease A family is a group of proteins having similar structures and catalytic mechanism but different functions. Human eosinophil granules contain two ribonucleases belonging to the RNase A family, eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN). In mouse, 15 orthologs of EDN and ECP, called mouse eosinophil associated ribonucleases (mEARs) have been reported which are expressed under different pathophysiological conditions. In this study, we have characterized mEAR2, mEAR5, mEAR7 and mEAR11, and compared them with ECP for their catalytic, cytotoxic, antibacterial and antiparasitic activities. All four mEARs had cytotoxic, antibacterial and antiparasitic activities. Generally, mEAR5 and mEAR2 were more cytotoxic than mEAR7, mEAR11 and ECP. The antimicrobial activities of mEAR7 and mEAR5 were higher than those of mEAR11 and mEAR2. The cytotoxic activity appeared to be associated with the basicity and RNase activity of mEARs, whereas no such correlation was observed for antimicrobial activities. The differential selective expression of mEARs under various pathophysiological conditions may be associated with the different biological activities of various mEARs.

Enhanced expression of neuropeptide S (NPS) receptor in eosinophils from severe asthmatics and subjects with total IgE above 100IU/ml.

Eosinophils are inflammatory cells of particular relevance to asthma exacerbations. Neuropeptide S (NPS) receptor was identified in a search for asthma susceptibility genes, where the risk haplotypes of the NPS receptor gene associated with total serum IgE above 100IU/ml and asthma. The aim of the present study was to investigate and compare expression of NPS receptor in human peripheral blood eosinophils derived from subjects with total serum IgE above and below 100IU/ml and patients with different phenotypes of asthma. Additionally, we aimed to study the function of NPS receptor in human eosinophils. We found higher NPS receptor protein expression in eosinophils derived from subjects with high IgE when compared to those from subjects with low IgE and the level of NPS receptor positively correlated with serum IgE. NPS receptor expression was also higher in eosinophils from patients with severe asthma than in cells from mild asthmatics or healthy controls. The receptor agonist NPS was a chemotactic agent for eosinophils. NPS also increased N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated CD11b integrin levels in eosinophils from subjects with high IgE. Furthermore, eosinophils from those subjects exhibited Ca(2+) mobilization but not cAMP rise in response to NPS. Altogether, NPS receptor may have a pathological role in individuals with severe asthma and/or elevated serum IgE levels as eosinophils from these patients express higher levels of NPS receptor protein and respond to NPS by enhanced migration and adhesion molecule expression.

Eosinophil-derived neurotoxin / RNase 2: connecting the past, the present and the future.

The eosinophil-derived neurotoxin (EDN, also known as eosinophil protein-X) is best-known as one of the four major proteins found in the large specific granules of human eosinophilic leukocytes. Although it was named for its discovery and initial characterization as a neurotoxin, it is also expressed constitutively in human liver tissue and its expression can be induced in macrophages by proinflammatory stimuli. EDN and its divergent orthologs in rodents have ribonuclease activity, and are members of the extensive RNase A superfamily, although the relationship between the characterized physiologic functions and enzymatic activity remains poorly understood. Recent explorations into potential physiologic functions for EDN have provided us with some insights into its role in antiviral host defense, as a chemoattractant for human dendritic cells, and most recently, as an endogenous ligand for toll-like receptor (TLR)2.

Eosinophil-derived neurotoxin acts as an alarmin to activate the TLR2-MyD88 signal pathway in dendritic cells and enhances Th2 immune responses.

Eosinophil-derived neurotoxin (EDN) is an eosinophil granule-derived secretory protein with ribonuclease and antiviral activity. We have previously shown that EDN can induce the migration and maturation of dendritic cells (DCs). Here, we report that EDN can activate myeloid DCs by triggering the Toll-like receptor (TLR)2-myeloid differentiation factor 88 signaling pathway, thus establishing EDN as an endogenous ligand of TLR2. EDN activates TLR2 independently of TLR1 or TLR6. When mice were immunized with ovalbumin (OVA) together with EDN or with EDN-treated OVA-loaded DCs, EDN enhanced OVA-specific T helper (Th)2-biased immune responses as indicated by predominant production of OVA-specific interleukin (IL)-5, IL-6, IL-10, and IL-13, as well as higher levels of immunoglobulin (Ig)G1 than IgG2a. Based on its ability to serve as a chemoattractant and activator of DCs, as well as the capacity to enhance antigen-specific immune responses, we consider EDN to have the properties of an endogenous alarmin that alerts the adaptive immune system for preferential enhancement of antigen-specific Th2 immune responses.

Serum eosinophil-derived neurotoxin (EDN) in diagnosis and evaluation of severity and bronchial hyperresponsiveness in childhood asthma.

This study sought to evaluate the use of serum eosinophil-derived neurotoxin (EDN), which has been proposed as a marker of airway inflammation in asthma in the diagnosis and evaluation of the severity and bronchial hyperresponsiveness in childhood asthma. We studied 72 children with atopic asthma, 36 children with nonatopic asthma, and 43 healthy controls. Skin prick tests, pulmonary function tests, and methacholine challenge tests were performed, in addition to total eosinophil count, serum ECP, and EDN being measured in all subjects. EDN levels were significantly higher in the atopic asthma group than those in the nonatopic asthma group or control group (p < 0.001), as were ECP levels (p < 0.001). EDN levels differed more significantly among groups divided by asthma severity (p < 0.001) than did ECP levels for these groups (p < 0.05). For the groups divided according to bronchial hyperresponsiveness, both EDN and ECP levels were significantly different (p < 0.005 and p < 0.01, respectively). Significant correlations were found between EDN and PC(20) (gamma = -0.281; p < 0.001), between ECP and PC(20) (gamma = -0.274; p < 0.005), and between EDN and ECP (gamma = 0.443; p < 0.001). In conclusion, serum EDN, as another marker of eosinophilic inflammation together with ECP, may aid in the diagnosis of asthma, especially atopic asthma, and in the evaluation of the severity and bronchial hyperresponsiveness in childhood asthma.

Human ribonuclease A superfamily members, eosinophil-derived neurotoxin and pancreatic ribonuclease, induce dendritic cell maturation and activation.

A number of mammalian antimicrobial proteins produced by neutrophils and cells of epithelial origin have chemotactic and activating effects on host cells, including cells of the immune system. Eosinophil granules contain an antimicrobial protein known as eosinophil-derived neurotoxin (EDN), which belongs to the RNase A superfamily. EDN has antiviral and chemotactic activities in vitro. In this study, we show that EDN, and to a lesser extent human pancreatic RNase (hPR), another RNase A superfamily member, activates human dendritic cells (DCs), leading to the production of a variety of inflammatory cytokines, chemokines, growth factors, and soluble receptors. Human angiogenin, a RNase evolutionarily more distant to EDN and hPR, did not display such activating effects. Additionally, EDN and hPR also induced phenotypic and functional maturation DCs. These RNases were as efficacious as TNF-alpha, but induced a different set of cytokine mediators. Furthermore, EDN production by human macrophages could be induced by proinflammatory stimuli. The results reveal the DC-activating activity of EDN and hPR and suggest that they are likely participants of inflammatory and immune responses. A number of endogenous mediators in addition to EDN have been reported to have both chemotactic and activating effects on APCs, and can thus amplify innate and Ag-specific immune responses to danger signals. We therefore propose these mediators be considered as endogenous multifunctional immune alarmins.