Eosinophil Granule Proteins Research Articles

Assessment of eosinophil granule proteins in various body fluids: is there a relation to clinical variables in childhood asthma?

The eosinophil plays a central role in the inflammatory process in bronchial asthma. Recent studies have indicated that the assessment of eosinophil-derived proteins in various body fluids could be used for monitoring disease activity of childhood asthma. Till now, no study exists which compared the levels of eosinophil-derived proteins in various body fluids such as serum, nasal lavage fluid (NALF) and urine. To investigate whether eosinophil granule proteins in different compartments were correlated and whether there is a relationship between disease activity, pulmonary function and bronchial hyperreactivity. Twenty-eight children with atopic bronchial asthma were recruited. Serum, NALF and urine samples were obtained and assessed for eosinophil cationic protein (ECP) and eosinophil protein X (EPX). The levels of eosinophil proteins were analysed for a relationship with lung function variables, bronchial hyperreactivity and disease activity. Eleven healthy control subjects were used as controls. Median ECP and EPX concentrations in serum (31.4 and 74.8 microg/L vs 15.8 and 24.3 microg/L, respectively), NALF (9.9 and 44. 9 microg/L vs 0 and 2.5 microg/L, respectively) and urine (49.4 vs 16.5 microg/mmol creatinine) were significantly raised in children with bronchial asthma compared with healthy control subjects. In addition, ECP and EPX levels in serum and urine samples were significantly higher in symptomatic patients compared with asymptomatic subjects with asthma. Although no relationship between eosinophil-derived proteins in serum, NALF or urine and the level of nonspecific bronchial hyperreactivity could be detected, the concentrations of EPX in serum and urine were correlated with variables of pulmonary function. Our findings demonstrate increased eosinophil activity in serum, NALF and urine derived from children with bronchial asthma. Due to the relationship between levels of eosinophil proteins in serum/urine samples and lung function, as well as significant concentration differences between symptomatic and asymptomatic asthmatic children, the assessment of eosinophil proteins in serum or urine samples appear to be more appropriate in monitoring disease activity than measurement of ECP or EPX in NALF. Thus, the determination of serum ECP/EPX or urinary EPX may be preferentially used in monitoring eosinophilic inflammation in childhood asthma.

Two eosinophil granule proteins, eosinophil peroxidase and major basic protein, inhibit mucin release from hamster tracheal surface epithelial cells in primary culture.

Effects of eosinophil proteins on airway goblet cell mucin release were investigated using primary hamster tracheal surface epithelial (HTSE) cell cultures. HTSE cells were metabolically labeled using 3H-glucosamine and chased in the presence of varying concentrations of eosinophil proteins. The amount of 3H-mucin in the spent media was measured by Sepharose CL-4B gel-filtration column chromatography. Possible cytotoxicity by the eosinophil proteins was assessed by measurements of both lactic dehydrogenase and mucin release during as well as after the treatment period. (1) Neither eosinophil cationic protein nor eosinophil-derived neurotoxin affected mucin release at concentrations up to 10(-6) M; (2) both major basic protein (MBP) and eosinophil peroxidase (EPO) inhibited mucin release in a dose-dependent fashion, and the inhibitory effect by these proteins appeared to be reversible; (3) neither MBP nor EPO caused any apparent cytotoxicity at concentrations up to 10(-6) M. Eosinophil proteins such as MBP and EPO inhibit mucin release from primary HTSE cells without causing any apparent cytotoxicity.

Degranulation of eosinophils mediated by intercellular adhesion molecule-1 and its ligands is involved in adhesion molecule expression on endothelial cells–selective induction of VCAM-1

Background: Adhesion molecules and eosinophils may play an important role in the pathogenesis of allergic inflammatory reactions. Objective: We attempted to clarify eosinophil activation, such as degranulation, by signaling through adhesion molecule and to determine whether degranulation is involved in adhesion molecule expression on endothelial cells. Methods: Eosinophils were cultured with or without recombinant soluble intercellular adhesion molecule-1 (ICAM-1), and the levels of eosinophil cationic protein and eosinophil-derived neurotoxin were determined. The influence of these eosinophil granule proteins or supernatant from eosinophil cultured with ICAM-1 on the expression of ICAM-1 or vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells was also examined by flow-cytometric analysis. Results: Supernatant levels of eosinophil granule protein were significantly increased by culture for 4 hourss or 16 hours with recombinant soluble ICAM-1, suggesting degranulation by adherence to ICAM-1. Both granule proteins and the supernatants of eosinophils cultured with recombinant soluble ICAM-1 induced expression of ICAM-1 and VCAM-1 on endothelial cells, with the latter showing a more prominant increase. Conclusion: Degranulation mediated through adherence to endothelial cells by ICAM-1 and its ligands may be involved in the expression of adhesion molecules, such as ICAM-1 or VCAM-1, on these cells. Our finding of the selective induction of VCAM-1 expression suggests that eosinophil adherence to endothelial cells, even if it is because of ICAM-1, may be involved in selective eosinophil recruitment and accumulation at sites of allergic inflammation. (J Allergy Clin Immunol 1999;103:S452-6.)

Effects of eosinophil granule major basic protein on phosphatidylcholine secretion in rat type II pneumocytes.

Eosinophils are involved in inflammatory diseases such as asthma. We previously reported that activated eosinophils increased the phosphatidylcholine (PC) secretion in primary cultures of rat type II pneumocytes. Increased PC secretion was confirmed to be partly mediated by superoxide anions released from activated eosinophils. However, the influence of eosinophil granule proteins on PC secretion is unknown at present. In this study, we determined whether eosinophil major basic protein (MBP) influences PC secretion. MBP dose dependently increased the PC secretion in rat type II pneumocytes without producing any cell damage. The MBP-induced increase in PC secretion was significantly reduced by preadministration of either H-7, a protein kinase inhibitor, or 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, a chelator of intracellular Ca2+, but not by H-89, a protein kinase inhibitor. Our results suggest that the MBP-induced increase in PC secretion may provide mechanical stability and protect against lung atelectasis.

Eosinophils, eosinophil cationic protein and eosinophil-derived neurotoxin in serum and urine of patients with onchocerciasis coinfected with intestinal nematodes and in urinary schistosomiasis

Eosinophils, eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN/EPX), myeloperoxidase (MPO) and IgE were measured in blood, serum and/or urine in Schistosoma haematobium- and Onchocerca volvulus-infected Guineans and O. volvulus- and S. haematobium-negative Guineans coinfected or infected with intestinal nematodes. The number of eosinophils and levels of eosinophil granule proteins but not of MPO were found to be strongly elevated in all Africans as compared to European controls. The highest serum ECP and serum and urinary EDN/EPX levels were observed in the hyperreactive form of onchocerciasis (sowda). Onchocerciasis patients and O. volvulus-negative Africans coinfected or infected with intestinal nematodes (hookworm and/or Ascaris lumbricoides) revealed higher serum granule protein concentrations and/or absolute eosinophil counts and urinary ECP than those without nematode infections. Statistical differences between both sections were found for the absolute eosinophil counts and for serum EDN/EPX and IgE in generalized onchocerciasis, and for urinary ECP in sowda, indicating stimulation of the eosinophil potential of O. volvulus-positive patients by coexistent hookworm infection. This worm species, in contrast to A. lumbricoides, causes especially high eosinophil counts and EDN/EPX and IgE levels. From these results it is concluded that in nematode diseases, ECP and EDN/EPX levels reflect the degree of antigenic stimulation, eosinophil activation and eosinophil turnover rates. Serum ECP and serum and urinary EDN/EPX may, therefore, serve as parameters to monitor helminth infection. Urinary ECP may be a marker of eosinophiluria secondary to urogenital manifestation of S. haematobium. It is elevated in hyperreactive onchocerciasis activated by intestinal nematodes.

Norm values for eosinophil cationic protein in nasal secretions: influence of specimen collection

Eosinophil granulocytes play an important role in allergic inflammation of the nasal mucosa. Eosinophil cationic protein (ECP) is a specific eosinophil granule protein released upon activation of these cells. ECP concentration in nasal secretions has been demonstrated to be a good marker for the activity of eosinophilic nasal mucosal inflammation. The clinical use of such a marker requires defined values which are regarded as pathological or within normal range. In analyses of nasal secretion samples, the sampling method has an important influence on the data obtained. We investigated ECP levels in nasal secretions (NS) of healthy volunteers obtained by seven different methods of sample collection to define norm values and to evaluate the clinical use of the different methods. A total of 839 healthy individuals were evaluated using blowing the nose (Bl: n = 82), suction (Suc: n = 69), Okuda microsuction technique (MSuc: n = 93), absorbent cotton wool samplers (CWS: n = 156), rubber-foam samplers (RFS: n = 193), nasal lavage (Lav: n = 112) and nasal spray washing (NSW: n = 134). Missing values occurred in more than 60% in Bl, Suc and MSuc, so that no norm range was defined for these methods. Norm range for ECP in NS was 5-46 ng/mL for CWS, 7-41 ng/mL for RFS, 4-51 ng/mL for NSW, and 3-31 ng/mL for Lav. When comparing seven different methods used in this study to collect nasal secretions and determine ECP levels, the method based upon absorption or nasal washing was the best.

Human Eotaxin Induces Eosinophil-Derived Neurotoxin Release from Normal Human Eosinophils

Background: Eosinophil granule proteins deposition at the site of allergic inflammation contributes to the late-phase reaction of hypersensitivity diseases. In the present communication, we describe the effect of human eotaxin on normal human eosinophil exocytosis measured as degranulation of eosinophil-derived neurotoxin (EDN). Methods: Purified eosinophils were obtained from normal healthy volunteers with the CD16-negative procedure. Purified eosinophils were stimulated with various concentrations of eotaxin and the amount of EDN released was analysed by radioimmunoassay. Flow cytometry was used to examine the surface expression of adhesion molecules on eosinophils. Results: Eotaxin significantly induced EDN release in a dose-dependent manner. The potency of eotaxin in this effect was equal to that of RANTES, and comparable to that of platelet-activating factor. Eotaxin-induced EDN release was blocked by cytochalasin B in a dose-dependent manner. The surface expression of CD11a, CD11b, CD18 and VLA-4 adhesion molecules on normal human eosinophils were not modulated by eotaxin stimulation. Conclusions: These results indicate that eotaxin may play an important role not only as a selective chemotaxin for the cell type but also as a secretagogue. Furthermore, they demonstrate a degranulation mechanism(s) involving cytoskeletal changes which is probably independent of the quantitative expression of adhesion molecules.

CYTOKINES IN ALLERGIC BRONCHOPULMONARY ASPERGILLOSIS

Asthma is clinically characterized as a disease manifested as reversible or mostly reversible bronchial obstruction with cough and wheeze accompanied by hyperresponsiveness of the airways to a variety of irritant triggers, including allergens. The current concept of asthma is that it is a unique cellular immune response responsible for the characteristic pathophysiologic and clinical manifestations. The recruitment and activation of helper T cells and production of their unique family of cytokines network in concert to increase the production of immunoglobulin E (IgE) and enhance the recruitment and activation of eosinophils and differentiation of mast cells, leading to the pathologic expression of asthma. The concerted effect of vasoactive and spasmogenic mediators, along with chemoattractant molecules, active enzymes, and the unique granule proteins of eosinophils and mast cells, leads to the development of the characteristic physiologic picture associated with asthma. This article focuses on the role of cytokines in precipitating the allergic cascade in allergic asthma with particular emphasis on allergic bronchopulmonary aspergillosis (ABPA). Very few reports are available on the role of cytokines in human ABPA. In the past few years, however, a number of animal models of ABPA have been described. Here we have compiled the salient findings of these studies. The sequence of the inflammatory cascade has been elucidated in studies of allergen bronchoprovocation in atopic individuals. Allergic individuals, when challenged with specific allergenic proteins, develop an acute bronchoconstrictive event and recover spontaneously, only to manifest a second, more prolonged and persistent bronchoconstriction. 5 During the early phase, histamine, prostaglandin D2, leukotriene C4, and platelet-activating factor release are accompanied by increased mucous secretion; a brisk infiltration of eosinophils is added and an influx of CD4+ T lymphocytes can be demonstrated at 24 to 48 hours. 4,20 ABPA occurs primarily in patients with asthma or with cystic fibrosis (CF) and is characterized by a variety of clinical and immunologic responses to antigens of Aspergillus fumigatus , a ubiquitous fungus that colonizes the bronchial tree of patients. 3,27 Some immunologic manifestations are (1) peripheral blood and pulmonary eosinophilia, (2) immediate cutaneous reactivity to Aspergillus antigen, (3) elevated total serum IgE, (4) precipitating antibody to A. fumigatus , (5) elevated serum IgE and IgG antibodies to Aspergillus compared with sera from patients with asthma alone without ABPA, and (6) increased serum interleukin (IL)-2 receptor concentrations. 7 Frequently, hyphae of A. fumigatus grow saprophytically in the bronchial lumen. Bronchiectasis in patients with asthma is proximal, occurring in the central two thirds of lung field on high-resolution examinations.

Inhibition of nonspecific binding of fluorescent-labelled antibodies to human eosinophils

Eosinophils and their products play a major role in inflammatory reactions associated with asthma and allergic diseases. There is a growing body of evidence that eosinophils synthesize, store, and release bioactive cytokines and chemokines with the potential to contribute to local inflammatory changes. Fluorescein isothiocyanate (FITC) has been widely used as an immunofluorescent conjugate for antibodies specific for detection of these molecules. However, FITC is an ionic fluorochrome (negatively charged) which binds strongly to positively charged eosinophil granule proteins. We developed new methods to prevent charge-based interactions of ionic fluorochromes with granule proteins, and optimised immunofluorescent staining techniques for eosinophils. An antibody to interleukin-6 (IL-6) was used to optimise this procedure for eosinophil-derived granule proteins. We attempted to block nonspecific binding of FITC-labelled anti-IL-6 using normal human IgG, foetal calf serum (FCS), bovine serum albumin (BSA), and goat, horse, and normal human sera at concentrations ranging between 1–10%. Only human IgG (2%; 20 mg/ml) was able to reduce background fluorescence. These results were confirmed using Texas Red conjugates. We also used antibodies conjugated to a nonionic fluorochrome, BODIPY FL, to detect IL-6 in eosinophils. Unlike FITC, BODIPY FL-conjugated antibodies did not require strong blocking conditions (2% BSA). We recommend that a neutral fluorochrome (BODIPY FL) should be used for immunofluorescence studies in eosinophils. Alternatively, strong blocking conditions may be used to decrease background binding of FITC-conjugated antibodies.

Cytolysis and piecemeal degranulation as distinct modes of activation of airway mucosal eosinophils

Background: Cytotoxic eosinophil granule proteins are considered important in the pathogenesis of inflammatory airway diseases, including asthma, rhinitis, and polyposis. However, little is known about the mechanisms involved in the deposition of these tissue-damaging granular products in vivo. Objective: We sought to determine the occurrence of degranulating eosinophils, those with morphologic evidence of cytolysis with associated clusters of free eosinophil granules (Cfegs), and to identify the frequency of apoptotic eosinophils in inflamed upper airway tissue. Methods: Eosinophil-rich nasal polyps were processed for transmission electron microscopy and for light microscopic evaluation of whole-mount preparations subjected to deep tissue staining for eosinophil peroxidase. Results: The mean proportion of eosinophil subtypes were intact and resting (6.8%), intact but degranulating (83%), cytolytic or Cfegs (9.9%), and apoptotic (0.0%). All degranulating eosinophils exhibited piecemeal degranulation. The occurrence of Cfegs was confirmed in nonsectioned whole-mount preparations. Depending on the appearance of their core and matrix, the specific granules were divided into four subtypes, and a degranulation index (altered per total granules) was calculated for each eosinophil. Cytolytic eosinophils had a much lower degranulation index than intact eosinophils present in the same tissue ( P < .001). Conclusions: These data indicate that eosinophil cytolysis is present in human airway mucosa, that its occurrence is not an artifact of the means of tissue handling, and that cytolysis of eosinophils may occur without prior extensive degranulation. We suggest that eosinophil cytolysis is a major activation mechanism, which occurs along with, but is distinct from, other types of degranulation. (J Allergy Clin Immunol 1998;102:286-94.)

Immunoelectron microscopic evidence for release of eosinophil granule matrix protein onto microfilariae of Onchocerca volvulus in the skin after exposure to amocarzine

The involvement of eosinophils in the host reaction to microfilariae (mf) of Onchocerca volvulus was studied by immunohistochemistry and immunoelectron microscopy. Skin biopsies were obtained from patients after transepidermal administration of the microfilaricide amocarzine. At 20-28 h after the application of amocarzine, mf were degenerated or dead and a marked eosinophil-parasite adherence (EPA) reaction was seen, with intense staining for intra- and extracellular eosinophil granule proteins such as eosinophil cationic protein (ECP) surrounding the mf. Immunoelectron microscopically the eosinophil granule matrix in intact and necrotic eosinophils was specifically labeled, whereas granules whose matrix had dissolved showed no specific gold particle binding. As specific labeling was seen on lowly electron-dense material adjacent to matrix-depleted granules, the material was regarded as released eosinophil granule matrix material. Intact and necrotic eosinophils, matrix-containing as well as matrix-depleted eosinophil granules, and released eosinophil granule matrix material were observed on the surface of damaged mf and between collagen fibers. The coincidence of mf degeneration, EPA reaction, and release of eosinophil granule matrix material on damaged mf and collagen fibers indicated a role of eosinophils and eosinophil granule matrix protein in the host reaction to mf after amocarzine application.

Exaggerated arthropod-bite lesions in patients with chronic lymphocytic leukemia: A clinical, histopathologic, and immunopathologic study of eight patients

Background: Unusual papulovesicular lesions resembling arthropod bites have been described in patients with chronic lymphocytic leukemia (CLL). Objective: Our purpose was to describe and characterize further the clinical, histopathologic, and immunopathologic features of these lesions. Methods: Eight patients were identified retrospectively who had CLL and characteristic skin lesions. Clinical and histologic features were recorded. Skin biopsy specimens were analyzed immunohistochemically for eosinophil granule major basic protein, eosinophil-derived neurotoxin, neutrophil elastase, and mast cell tryptase. Results: The clinical features, including the lesional distribution, suggested arthropod bites, although most patients could not recall having been bitten. Mixed T- and B-cell lymphoid cell infiltrates were present within lesions, along with prominent eosinophil infiltration and eosinophil granule protein deposition. Conclusion: Exuberant papulovesicular lesions develop in patients with CLL apparently as an exaggerated response to arthropod bites. Prominent eosinophil infiltration and degranulation within these lesions likely contribute to the severity of symptoms. (J Am Acad Dermatol 1998;39:27-35.)

Eosinophil major basic protein increases membrane permeability in mammalian urinary bladder epithelium.

The eosinophil granule protein major basic protein (MBP) is toxic to a wide variety of cell types, by a poorly understood mechanism. To determine whether the action of MBP involves an alteration in membrane permeability, we tested purified MBP on rabbit urinary bladder epithelium using transepithelial voltage-clamp techniques. Addition of nanomolar concentrations of MBP to the mucosal solution caused an increase in apical membrane conductance only when the voltage across the apical membrane was cell interior negative. The magnitude of the MBP-induced conductance was a function of MBP concentration, and the rate of the initial increase in conductance was a function of the transepithelial voltage. The MBP-induced conductance was nonselective for K+ and Cl-. Mucosal Ca2+ reversed the induced conductance, whereas mucosal Mg2+ partially blocked the induced conductance and slowed the rate of the increase in conductance. The induced conductance was partially reversed by changing the voltage gradient across the apical membrane to cell interior positive. Prolonged exposure resulted in an irreversible loss of the barrier function of the urinary bladder epithelium. These results suggest that an increase in cell membrane ion permeability is an initial step in MBP-induced loss of barrier function.

Immunohistochemical Study of E–cadherin and ZO–1 in Allergic Nasal Epithelium of the Guinea Pig

Nasal epithelial damage during allergic inflammation was studied by observing the distribution of cell adhesion molecule E–cadherin and tight junction (zonula occludens) cell–cell contact associated protein ZO–1. The guinea pig model of nasal allergy, sensitized with intraperitoneally administered ovalbumin (OA) and subsequently challenged with OA intranasally, was used. In control epithelium, E–cadherin immunoreactivity was detected continuously along neighboring epithelial cell borders. ZO–1 spot–like immunoreactivity was detected in the apicolateral portion of epithelial cells corresponding to the tight junction (TJ) position, but no changes in immunoreactivity were found between control and challenged epithelia. In the challenged epithelium of sensitized animals, marked infiltration of eosinophils and structural changes, such as widening of the intercellular spaces and detachment of adjacent epithelial cells, were observed concurrently. In addition, spots negative for E–cadherin immunoreactivity were noted in the epithelium, associated with the extracellular deposition of eosinophil granule proteins. Immunoelectron microscopy revealed a decrease or disappearance of E–cadherin immunoreactivity, which took place not only in regions where intercellular spaces were wide and adjacent epithelial cells were detached, but also at the point of contact between infiltrating eosinophils and epithelial cells. Approximately 87% of eosinophils observed in the challenged epithelium were associated with such loss of E–cadherin immunoreactivity. These results suggest that the intimate epithelial cell contact mediated by E–cadherin is loosened as a consequence of eosinophil infiltration, which may trigger the initial step of subsequent epithelial destruction in allergic states.

Familial eosinophilic cellulitis, dysmorphic habitus, and mental retardation

Background: Eosinophilic cellulitis is a polymorphous, chronic disease characterized by eosinophil infiltration and granulomatous inflammation. Objective: Our purpose was to describe the clinical, histologic, and immunohistologic findings in three family members who have had eosinophilic cellulitis since childhood associated with mental retardation and abnormal body habitus. Methods: Family members were evaluated. Multiple skin biopsy specimens were obtained and examined after hematoxylin-and-eosin staining, by immunofluorescence and by electron microscopy. Blood specimens were analyzed by immunoassays for eosinophil granule proteins and eosinophil active cytokines. Results: Three short-statured, mentally retarded family members with abnormal body habitus in at least two generations had recurrent eosinophilic cellulitis. Peripheral blood and bone marrow eosinophilia was present. Plasma eosinophil granule major basic protein and eosinophil-derived neurotoxin levels were elevated with normal plasma eosinophil cationic protein levels. Eosinophil survival in culture was increased by patients’ plasma and was blocked with monoclonal interleukin-5 antibody. The level of plasma interleukin-5 was elevated. Lesional skin biopsy specimens showed massive staining for three eosinophil granule proteins. Electron microscopy showed eosinophil disruption. Conclusion: Eosinophilic cellulitis, mental retardation, and abnormal body habitus were likely inherited as a dominant syndrome in this family in which eosinophil involvement was striking. (J Am Acad Dermatol 1998;38:919-28.)

Respiratory Syncytial Virus-Infected Pulmonary Epithelial Cells Induce Eosinophil Degranulation by a CD18-Mediated Mechanism

Respiratory syncytial virus (RSV)-induced bronchiolitis in infants is characterized by wheezing, respiratory distress, and the histologic findings of necrosis and sloughing of airway epithelium. High concentrations of eosinophil cationic protein (ECP), a cytotoxic protein contained in the granules of eosinophils, have been found in the airways of RSV-infected infants. The mechanisms of eosinophil degranulation in vivo remain largely unknown. Since RSV-infected respiratory epithelial cells are a rich source of cytokines with eosinophil-activating properties, our studies were designed to mimic in vitro the interaction between RSV, pulmonary epithelial cells (A549), and eosinophils in the airway mucosa. We report in this work that, in the absence of epithelial cells, neither RSV, in the form of purified virions, nor UV-irradiated culture supernatant of RSV-infected epithelial cells (RSV-CM) induced eosinophil degranulation. On the other hand, eosinophils released significant amount of ECP when cultured with RSV-infected A549 cells. Uninfected A549 cells, which failed to induce eosinophil degranulation, were equally effective in triggering ECP release if they were cultured with eosinophils in the presence of RSV-CM. Although RSV-CM induced the up-regulation of the beta2 integrin CD11b on eosinophils and the expression of ICAM-1 on A549 cells, release of ECP was inhibited significantly by anti-CD18 mAb, but not by anti-ICAM-1 mAb. These results suggest a novel mechanism by which respiratory viruses may trigger the detrimental release of eosinophil granule proteins in the airway mucosa.

Eosinophilia and gastrointestinal symptoms after ingestion of shiitake mushrooms

Background: Shiitake mushrooms are a dietary staple in Asia and are increasingly popular worldwide. A cholesterol-lowering study with shiitake showed that 17 of 49 participants withdrew because of rash or abdominal discomfort, and two had marked eosinophilia. One of these latter participants was subsequently challenged for 14 days with shiitake powder and again had eosinophilia. Objective: We investigated whether ingestion of shiitake mushroom powder induces eosinophilia or symptoms. Methods: We studied 10 normal persons. Each participant ingested 4 gm shiitake powder (open label) daily for 10 weeks (trial 1), and the protocol was repeated in these same subjects after 3 to 6 months (trial 2). Blood counts and serum samples were obtained biweekly (trial 1) or weekly along with stool specimens (trial 2). Eosinophil major basic protein and IL-5, IgE, and IgG antishiitake antibodies were measured in sera. Eosinophil-derived neurotoxin was measured in stool extracts. We defined responders as subjects having peak eosinophil counts four or more times their average baseline counts. Results: Each trial had four responders, and trial 2 had one new and three repeat responders. Eosinophilia ranged from 400 to 3900/mm 3. Responders had increased blood eosinophils, serum major basic protein, stool eosinophil-derived neurotoxin, and factors that enhanced eosinophil viability. Antishiitake IgE was not detected, and antishiitake IgG increased in two responders. Gastrointestinal symptoms coincided with eosinophilia in two subjects. Symptoms and eosinophilia resolved after discontinuing shiitake ingestion. Conclusions: Daily ingestion of shiitake mushroom powder in five of 10 healthy persons provoked blood eosinophilia, increased eosinophil granule proteins in serum and stool, and increased gastrointestinal symptoms. Shiitake ingestion suggests a model to study the eosinophil's role in the blood and gastrointestinal tract. Finally, our report raises concerns of possible adverse systemic reactions to this increasingly popular food.(J Allergy Clin Immunol 1998;101:613-20.)

Molecules in focus Eosinophil cationic protein (ECP)

ECP (eosinophil cationic protein) was first purified from human myleoid cells in 1971 and identified as an eosinophil granule protein in 1975. ECP is a heterogeneous protein with molecular weights of the variants from 16–24 kDa. ECP is extremely basic with a pI of pH 10.8. The gene for ECP is found on chromosome 14 adjacent to other proteins of the ribonuclease family, with which ECP shares some sequence homologies. ECP has a variety of biological activities interacting with other immune cells and plasma proteins such as coagulation factors and proteins of the complement system. The cytotoxic activity, however, is the most conspicuous. The different isoforms of ECP seem to have different biological properties with respect to cytotoxicity and the effects on fibroblasts. ECP can be measured in biological fluids, by means of sensitive immunoassays, as an indication of eosinophil turnover and activity in vivo.

Eosinophil granule proteins expressed in ocular cicatricial pemphigoid

BACKGROUNDBlister formation and tissue damage in bullous pemphigoid have been attributed to the release of eosinophil granule proteins—namely, to eosinophil derived cationic protein (ECP) and major basic protein (MBP). In...

Serum eosinophil cationic protein, eosinophil protein X and eosinophil peroxidase in relation to pulmonary function in cystic fibrosis

Recently, increased serum levels of eosinophil cationic protein (ECP) in cystic fibrosis (CF) have been reported which were closely related to the levels in sputum. In the present study we investigated other eosinophil proteins such as eosinophil peroxidase (EPO) and eosinophil protein X (EPX) in sera of patients with CF and their relation to pulmonary function. Serum samples from 42 patients with CF and from 25 healthy control subjects were measured for ECP, EPO and EPX. Lung function tests were performed by using whole body plethysmographic technique, and the results were correlated with the levels of eosinophil granule proteins. Serum ECP (median: 20.9 microg/L), EPO (median: 30.3 microg/L) and EPX (median: 37.9 microg/L) levels were significantly increased in CF compared with healthy controls (3.5 microg/L, P < 0.0001, 5.6 microg/L, P < 0.0001 and 14.6 microg/L, P < 0.0001, respectively) whereas eosinophil counts were not different. There was a strong correlation between the levels of eosinophil proteins and variables of pulmonary function, like between ECP and forced vital capacity (r = -0.764, P < 0.0001). In addition, ECP concentrations were significantly related to the levels of EPO and EPX, albeit, in some patients with low ECP levels, increased EPO and EPX concentrations were observed. These results indicate that in patients with CF, ECP, EPO and EPX concentrations also were increased with a significant relationship between these three eosinophil proteins. Since eosinophil activity in patients with CF is strongly correlated with pulmonary function, the assessment of eosinophil granule proteins might be useful for clinical monitoring in CF.