C­C chemokine receptor type 3 gene knockout alleviates inflammatory responses in allergic rhinitis model mice by regulating the expression of eosinophil granule proteins and immune factors.

The present study aimed to investigate the effects of C­C chemokine receptor type 3 (CCR3) gene knockout on allergic rhinitis (AR) in mice, as well as the underlying molecular mechanisms. Ovalbumin was administrated to CCR3+/+ and CCR3­/­ BALB/c mice to establish an AR model. The mice were divided into four groups: i) Normal control (CG), ii) AR model (AR), iii) CCR3 knockout CG (CCR3­/­CG) and iv) AR model with CCR3 knockout (CCR3­/­AR). Histological sections of nasal mucosae were examined by hematoxylin and eosin staining, which revealed that CCR3 knockout suppressed the invasion of inflammatory cells and relieved the damage of nasal mucosae. Peripheral blood smear and nasal­washing smears were evaluated by Wright's staining. Eosinophil (EOS) numbers in nasal mucosae, peripheral blood, and nasal washings of the various groups were ranked in the order: AR>CCR3­/­AR>CG>CCR3­/­. mRNA expression levels of CCR3, EOS peroxidase (EPO), EOS cationic protein (ECP), and major basic protein (MBP) in the peripheral serum and nasal washings were detected by reverse transcription­polymerase chain reaction. Interferon­Î³ (IFN­Î³), interleukin (IL)­4, IL­10, and immunoglobulin E (IgE) protein levels in the peripheral serum and nasal washings were investigated by ELISA. CCR3 mRNA expression was not detected in the CCR3­/­ and CCR3­/­AR groups, whereas expression levels in the AR group were markedly higher compared with expression in the CG group. Compared with the CG­associated groups (i.e., the CG and CCR3­/­CG groups), the levels of EPO, ECP, MBP, IL­4, and IgE were significantly increased in the AR­associated groups (that is, R and CCR3­/­AR). In addition, the CCR3­/­AR group mice produced significantly lower levels of EPO, ECP, MBP, IL­4 and IgE compared with the AR group, whereas the expression levels of IFN­Î³ and IL­10 were increased. CCR3 gene knockout may alleviate EOS invasion and the inflammatory response in AR model mice by reducing the expression levels of EPO, ECP, MBP, IL­4, and IgE, and increasing the expression of IL­10 and IFN­Î³.

Elevated Systemic Levels of Eosinophil, Neutrophil, and Mast Cell Granular Proteins in Strongyloides Stercoralis Infection that Diminish following Treatment.

Infection with the helminth parasite Strongyloides stercoralis (Ss) is commonly clinically asymptomatic that is often accompanied by peripheral eosinophilia. Granulocytes are activated during helminth infection and can act as immune effector cells. Plasma levels of eosinophil and neutrophil granular proteins convey an indirect measure of granulocyte degranulation and are prominently augmented in numerous helminth-infected patients. In this study, we sought to examine the levels of eosinophil, neutrophil, and mast cell activation-associated granule proteins in asymptomatic Ss infection and to understand their kinetics following anthelmintic therapy. To this end, we measured the plasma levels of eosinophil cationic protein, eosinophil-derived neurotoxin, eosinophil peroxidase, eosinophil major basic protein, neutrophil elastase, myeloperoxidase, neutrophil proteinase-3, mast cell tryptase, leukotriene C4, and mast cell carboxypeptidase-A3 in individuals with asymptomatic Ss infection or without Ss infection [uninfected (UN)]. We also estimated the levels of all of these analytes in infected individuals following definitive treatment of Ss infection. We demonstrated that those infected individuals have significantly enhanced plasma levels of eosinophil cationic protein, eosinophil-derived neurotoxin, eosinophil peroxidase, eosinophil major basic protein, elastase, myeloperoxidase, mast cell tryptase, leukotriene C4, and carboxypeptidase-A3 compared to UN individuals. Following the treatment of Ss infection, each of these granulocyte-associated proteins drops significantly. Our data suggest that eosinophil, neutrophil, and mast cell activation may play a role in the response to Ss infection.

Development of a suspension array assay in multiplex for the simultaneous measurement of serum levels of four eosinophil granule proteins.

The concentrations of major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil derived neurotoxin (EDN) and eosinophil peroxidase (EPO) have been associated with eosinophilic disease severity. Whereas a variety of techniques have been used to measure individual eosinophil granule protein concentration, none of these methods efficiently measures MBP, ECP, EDN and EPO simultaneously. A multiplex suspension array system was developed to simultaneously measure the concentrations of MBP, ECP, EDN and EPO in serum. The assay showed excellent inter- and intra-assay reliability, and serum levels of MBP, ECP and EDN from eosinophilic subjects analyzed by ELISA and multiplex were highly correlated (r=0.8579; P<0.0001, r=0.6356; P=0.0006 and r=0.8600; P<0.0001, respectively, Spearman rank correlation). Moreover, the multiplex assay required 500-fold less serum than a single ELISA to achieve comparable sensitivity. Absolute eosinophil count and eosinophil surface expression of the activation marker, CD69, were significantly correlated with concentrations of MBP, EDN and EPO, but not ECP, in serum from eosinophilic subjects. Furthermore, subjects with eosinophilic gastrointestinal disorder and normal peripheral absolute eosinophil counts (<0.5×10(9)/l) had significantly increased concentrations of MBP (P<0.0001), ECP (P<0.0001), EDN (P=0.0001) and EPO (P<0.0001) compared to normal donors. In summary, the eosinophil granule protein multiplex assay provides a rapid and reliable way to measure eosinophil granule protein levels and should prove useful in assessing patterns of degranulation in patients with eosinophilic disorders.

Morphological changes in eosinophils are reliable markers of the severity of an acute asthma exacerbation in children.

BACKGROUND: Early identification of the severity of asthma exacerbation would be helpful for the management of patients. We aimed to evaluate the correlation of morphological change in activated eosinophils and the severity of an asthma exacerbation. METHODS: Blood was collected from 55 asthmatic children: 40 of whom were having an exacerbation, 15 symptom-free, and 15 healthy controls. The percentage of eosinophils with morphological changes (emission of single or multiple pseudopods, presence of cytoplasmic vacuoles, releasing a small, moderate, or large quantity of granules, spreading, eosinophil death, and presence of cluster of free eosinophil granules) was quantified after the adherence to a slide and compared using the Mann-Whitney test. The correlation between the severity of the asthma exacerbation and the percentage changed eosinophils was tested with Spearman's correlation. RESULTS: The proportion of activated eosinophils was higher in asthmatic symptom-free children than in the control group, and acute asthma exacerbation produced an additional increase in eosinophil activation (P < 0.01). More significantly increased morphological changes were emissions of multiple pseudopods, presence of cytoplasmic vacuoles, spreading, and presence of a cluster of free eosinophil granules (P < 0.001). The following were correlated with the severity of an asthma exacerbation: ≥14% of eosinophils emitting single pseudopod, 8% emitting multiple pseudopods, 17% with vacuoles, 28% eosinophils releasing a large quantity of granules, and 66% of spread eosinophils. CONCLUSIONS: Quantifying the morphological changes in eosinophils is a feasible, easy, and reliable manner to identify the severity of an asthma exacerbation and therefore might improve the clinical management of asthmatic children.

Eosinophil granular proteins damage bronchial epithelial cells infected with respiratory syncytial virus.

BACKGROUND: Respiratory syncytial (RS) virus infection is an important exacerbating factor in acute bronchial asthma. However, the precise mechanisms responsible for viral infection-induced exacerbations of asthma are uncertain. To elucidate the role of eosinophilic inflammation in the pathogenesis of virus-induced asthma, we investigated the effects of eosinophil granule proteins on bronchial epithelial cell infected with RS virus. METHODS: Morphological changes and cytopathic effects in human type II pulmonary alveolar epithelial cells (A549) infected with RS virus and/or eosinophil granule proteins such as major basic protein (MBP), eosinophil peroxidase (EPO), eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) were observed by microscopy. Apoptosis/necrosis was evaluated by trypan blue exclusion test. We also measured 8 types of phosphorylated proteins in MBP-treated A549 cells infected with RS virus. RESULTS: Although RS virus alone did not affect the cytopathic effects of A549 cells, high concentrations of MBP or a combination of 4 granule proteins resulted in cytopathic effects. MBP or EPO, but not ECP or EDN, induced cytotoxicity and necrosis of the infected A549 cells. Furthermore, MBP induced the phosphorylation of the extracellular signal-regulated kinase (ERK) 1/2, p38 mitogen-activated protein kinase (MAPK), Jun-N-terminal protein kinase (JNK), and signal transducer and activator of transcription (STAT)3 in the infected cells. CONCLUSIONS: These results suggest that eosinophil granule proteins, specifically MBP, damage bronchial epithelial cells infected with RS virus and that the MAPK family are involved in these responses, indicating that eosinophilic inflammation might be associated with the pathophysiology of RS virus-induced acute exacerbations of asthma.

Antimicrobial activity of human eosinophil granule proteins: involvement in host defence against pathogens.

Eosinophils have been associated with the pathophysiology of various allergic diseases and asthma. Eosinophils secrete a number of granule proteins that have been identified as effector molecules responsible for many of the actions of eosinophils. The four major eosinophil granule proteins, major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil derived neurotoxin (EDN) and eosinophil peroxidase have been shown to be involved in a number of eosinophil associated functions. EDN possesses antiviral activity against single stranded RNA viruses like respiratory syncytial virus, Hepatitis and HIV, whereas ECP and MBP have antibacterial and antiparasitic properties. This review summarizes the studies on antipathogenic activities of eosinophil granule proteins against bacteria, viruses, protozoans and helminths.

The development of a sensitive and specific ELISA for mouse eosinophil peroxidase: assessment of eosinophil degranulation ex vivo and in models of human disease.

Mouse models of eosinophilic disorders are often part of preclinical studies investigating the underlying biological mechanisms of disease pathology. The presence of extracellular eosinophil granule proteins in affected tissues is a well established and specific marker of eosinophil activation in both patients and mouse models of human disease. Unfortunately, assessments of granule proteins in the mouse have been limited by the availability of specific antibodies and a reliance on assays of released enzymatic activities that are often neither sensitive nor eosinophil specific. The ability to detect immunologically and quantify the presence of a mouse eosinophil granule protein in biological fluids and/or tissue extracts was achieved by the generation of monoclonal antibodies specific for eosinophil peroxidase (EPX). This strategy identified unique pairs of antibodies with high avidity to the target protein and led to the development of a unique sandwich ELISA for the detection of EPX. Full factorial design was used to develop this ELISA, generating an assay that is eosinophil-specific and nearly 10 times more sensitive than traditional OPD-based detection methods of peroxidase activity. The added sensitivity afforded by this novel assay was used to detect and quantify eosinophil degranulation in several settings, including bronchoalveolar fluid from OVA sensitized/challenged mice (an animal model of asthma), serum samples derived from peripheral blood recovered from the tail vasculature, and from purified mouse eosinophils stimulated ex vivo with platelet activating factor (PAF) and PAF + ionomycin. This ability to assess mouse eosinophil degranulation represents a specific, sensitive, and reproducible assay that fulfills a critical need in studies of eosinophil-associated pathologies in mice.

Eosinophils: multifaceted biological properties and roles in health and disease.

Eosinophils are leukocytes resident in mucosal tissues. During T-helper 2 (Th2)-type inflammation, eosinophils are recruited from bone marrow and blood to the sites of immune response. While eosinophils have been considered end-stage cells involved in host protection against parasite infection and immunopathology in hypersensitivity disease, recent studies changed this perspective. Eosinophils are now considered multifunctional leukocytes involved in tissue homeostasis, modulation of adaptive immune responses, and innate immunity to certain microbes. Eosinophils are capable of producing immunoregulatory cytokines and are actively involved in regulation of Th2-type immune responses. However, such new information does not preclude earlier observations showing that eosinophils, in particular human eosinophils, are also effector cells with proinflammatory and destructive capabilities. Eosinophils with activation phenotypes are observed in biological specimens from patients with disease, and deposition of eosinophil products is readily seen in the affected tissues from these patients. Therefore, it would be reasonable to consider the eosinophil a multifaceted leukocyte that contributes to various physiological and pathological processes depending on their location and activation status. This review summarizes the emerging concept of the multifaceted immunobiology of eosinophils and discusses the roles of eosinophils in health and disease and the challenges and perspectives in the field.

Functional role of eosinophils in gastrointestinal inflammation.

Eosinophilic gastrointestinal (GI) diseases (EGIDs) are characterized by a rich eosinophilic inflammation of the GI tract. Clinical and experimental studies suggest that eosinophils have a pathogenic role in EGIDs; however, the function of eosinophils in these diseases remains an enigma. This article describes eosinophil immunoregulatory and effector function and discusses the possible involvement of these pathways in EGIDs.

The role of eosinophils in airway tissue remodelling in asthma.

There is an increasing evidence that airway structural change (termed remodelling) is associated with the severity and chronicity of asthma. Recent studies support an important role for eosinophils in the remodelling process. In particular eosinophil depletion studies have demonstrated that several aspects of remodelling are attenuated. Eosinophils have been confirmed as an important source of TGF-beta(1) as well as other important cytokines that can lead to the direct activation of epithelium and mesenchymal cells that are considered to drive airway remodelling. The current studies that support a role for eosinophils in airway remodelling are reviewed in article.

Mechanisms of eosinophilia in the pathogenesis of hypereosinophilic disorders.

The increased numbers of activated eosinophils in the blood and tissues that typically accompany hypereosinophilic disorders result from a variety of mechanisms. Exciting advances in translating discoveries achieved from mouse models and molecular strategies to the clinic have led to a flurry of new therapeutics specifically designed to target eosinophil-associated diseases. So far, this form of hypothesis testing in humans in vivo through pharmacology generally has supported the paradigms generated in vitro and in animal models, raising hopes that a spectrum of novel therapies soon may become available to help those who have eosinophil-associated diseases.

Selected eosinophil proteins as markers of inflammation in atopic dermatitis patients.

Atopic dermatitis (AD) is an inflammatory skin disease characterized by chronic and recurrent course, beginning primarily in early childhood. The etiopathogenesis of AD has not yet been fully understood, although various types of inflammatory cells including eosinophils may be involved in its pathomechanism. The basic aim of the study was to evaluate the usefulness of selected eosinophil proteins in serum and urine of AD patients, as markers of disease severity. The study also aimed to analyze correlations between the level of examined proteins and parameters such as skin prick test (SPT) results, serum concentration of total IgE, and coexistence of symptoms of other atopic diseases. The study included 30 AD patients and two control groups: 30 patients suffering from chronic urticaria and 30 healthy individuals. The mean level of eosinophil proteins measured in serum and urine of AD patients was higher than that in controls, although a significant difference was only recorded for serum and urine level of eosinophil protein X (EPX). Patients with very severe/severe AD presented higher levels of eosinophil proteins than patients presenting with mild/moderate AD, although no significant difference was found between these two groups. AD patients with positive SPT results and detectable specific IgE in serum, and with coexisting symptoms of other atopic diseases presented with higher mean levels of serum and urine eosinophil proteins than AD cases with negative SPT results and without any symptoms of other atopic diseases. In children suffering from AD, serum eosinophil cationic protein level, EPX level and urine EPX level were higher than those in healthy children, however, without statistical significance. Study results suggested a significant role of eosinophils in the etiopathogenesis of AD. Serum and urine levels of selected eosinophil proteins may serve as an important part of diagnostic approach to AD patients, especially in differentiation of allergic and non-allergic forms of AD. The results are also promising for the usefulness of selected eosinophil proteins in the diagnosis of AD in children, however, thorough analysis on a larger group of patients is needed.

The eosinophil.

Eosinophils have been considered end-stage cells involved in host protection against parasites. However, numerous lines of evidence have now changed this perspective by showing that eosinophils are pleiotropic multifunctional leukocytes involved in initiation and propagation of diverse inflammatory responses, as well as modulators of innate and adaptive immunity. In this review, we summarize the biology of eosinophils, focusing on the growing properties of eosinophil-derived products, including the constituents of their granules as well as the mechanisms by which they release their pleiotropic mediators. We examine new views on the role of eosinophils in homeostatic function, including developmental biology and innate and adaptive immunity (as well as interaction with mast cells and T cells). The molecular steps involved in eosinophil development and trafficking are described, with special attention to the important role of the transcription factor GATA-1, the eosinophil-selective cytokine IL-5, and the eotaxin subfamily of chemokines. We also review the role of eosinophils in disease processes, including infections, asthma, and gastrointestinal disorders, and new data concerning genetically engineered eosinophil-deficient mice. Finally, strategies for targeted therapeutic intervention in eosinophil-mediated mucosal diseases are conceptualized.

Th1 and Th2 cell population in chronic ethmoidal rhinosinusitis: a chemokine receptor assay.

BACKGROUND: It is necessary for the immune system in the nasal and paranasal sinus mucosa to possess appropriate responses to infective agents as well as allergic responses. Recently, the T helper (Th)1/Th2 paradigm has been proposed as a new concept to explain various immunologic phenomena. OBJECTIVE: Toward the goal of better understanding chronic rhinosinusitis, we have tried to define the predominating helper T-cell subsets among patients with chronic rhinosinusitis through characterizing the expressed chemokine receptors by these cells to find out the relation between these chemokine receptors' expression and the underlying pathogenesis of chronic rhinosinusitis. METHODS: Thirty patients with ethmoidal chronic rhinosinusitis were used in our study. Patients were divided into atopic and nonatopic groups according to their serum immunoglobulin (Ig)E levels. Samples from ethmoidal sinus mucosa were processed as follows: frozen sections were examined immunohistochemically for detection of CCR4, CCR5, and EG2 positive cells and the mRNA of CCR4, and CCR5 transcripts were then examined by real-time quantitative polymerase chain reaction (PCR). RESULTS: By immunohistochemistry, atopic patients showed high expression of CCR4 and EG2 positive cells, whereas nonatopic patients showed high expression of CCR5 positive cells. The expression of CCR4 and CCR5 mRNA, detected by real-time quantitative PCR, supported the data obtained by immunohistochemistry. CONCLUSION: The present study suggests that eosinophil recruitment associated with Th2 cell infiltration is the main factor responsible for the pathology of atopic rhinosinusitis.

Correlation of serum total IgE, eosinophil granule cationic proteins, sensitized allergens and family aggregation in atopic dermatitis patients with or without rhinitis.

Atopic dermatitis (AD) is a complex disease with both a genetic background and environmental interactions. Although multiple linkage-analyses about AD have been studied, there have been only a few family aggregation tests of AD or perennial allergic rhinitis (AR) to date. The association of allergen-specific IgE in AD and atopic dermatitis with allergic perennial rhinitis (ADR) have also been seldom discussed. The purpose of this study was to evaluate family aggregation and assess allergen-specific IgE in patients with AD and ADR. We also planned to investigate the effect of family history of AD on the prevalence of allergen-specific antibodies. The serum levels of IgE, eosinophil cationic protein (ECP) and major basic protein (MBP) were measured and compared in patients with AD and those with ADR. Proportional analysis compared allergen-specific IgE between AD and ADR. The family aggregation was conducted to estimate the odds ratio for various atopic diseases in different family members. Total IgE and allergen-specific antibodies in serum were compared between those patients who had AD with AR and those without. The result revealed that allergic rhinitis is the most common concomitant atopic disease associated with AD. The ADR group was more likely to have serum mite-, cockroach-, and feather-specific IgE. The positive rates for wheat, peanut and soybean were higher in those AD without rhinitis. In the family aggregation of AD, the odds ratio for siblings was higher than for parents, the ratios for brother and sister were 9.91 and 8.75, respectively. However, the odds ratio for parents of ADR was higher than siblings; the ratios for father and mother of ADR were 8.22 and 2.94, respectively. AD patients with family histories of AD were more likely to have mite-, soybean-, and peanut-specific antibodies in their serum. We concluded that aeroallergens are the most important allergens aggravating atopic diseases in Taiwan. Food plays an important role in the pathogenesis of AD. Measurement of serum total IgE combined with the MAST-CLA test could be helpful in the diagnosis of atopic diseases. The differential aggregation tendency for AD and ADR implicated the complexity of the gene-environment interaction in these atopic diseases.

[Eosynophils in parasitic infections--clinical and functional significance]. / Eozynofile w chorobach pasozytniczych--znaczenie kliniczne i funkcjonalne.

Eosinophils are multifunctional cells which contain and produce many biologically active substances. Their presence is mainly associated with parasitic infections or allergic manifestations. Eosinophilia is a common feature of helminth infections occurring both in the blood and at local sites of infection. The highly toxic basic proteins in eosinophil granules exert a range of biological effects not only against helminth parasites but also in host tissue being responsible for their damage. Eosinophils have been shown to be strong effectors killing helminth parasites in vitro, especially the larval stages. However, this function in vivo was established only for the very small number of parasites. In the last years an opportunity has appeared to perform experiments with parasites on genetically modified mice: over expressing the gene encoding Il-5 as well as lacking some receptors on eosinophils. These new studies in mice demonstrated that Il-5 and eosinophils had a different impact on different helminth infections. Eosinophilia in human patients has only a limited predictive value for the presence of helminth infections. However, the likelihood of the presence of helminth infections increases considerably with the extent of eosinophilia.