Eosinophil granule proteins activate human heart mast cells

Eosinophilia in humans is often associated with heart disease and cardiac localization of eosinophil granule proteins, and several results suggest that granule proteins mediate endomyocardial damage. Here we investigated the in vitro effects of the four principal eosinophil granule proteins (eosinophil cationic protein (ECP), major basic protein (MBP), eosinophil-derived neurotoxin, and eosinophil peroxidase (EPO)) on the activation of effector cells of inflammation (mast cells) isolated from human heart tissue (HHMC). ECP and, to a lesser extent, MBP (0.3-3 microM), but not eosinophil-derived neurotoxin and eosinophil peroxidase stimulated the release of preformed (histamine and tryptase) and the de novo synthesis of vasoactive and proinflammatory mediators (PGD2) from HHMC. Activation of HHMC by ECP and MBP was Ca2+- and temperature-dependent and was abolished by preincubation (15 min, 37 degrees C) with 2-deoxy-D-glucose (10 mM) and antimycin A (1 microM). There was a significant correlation between the maximal percentage of histamine release induced by ECP and anti-IgE from HHMC (rs = 0.73; p < 0.005), by MBP and anti-IgE (rs = 0.79; p < 0.001), and by ECP and MBP (rs = 0.65; p < 0.005). A positive correlation was also found between histamine and tryptase secretion (rs = 0.71; p < 0.001) and between histamine and PGD2 release induced by ECP from HHMC (rs = 0.85; p < 0.001). This is the first demonstration that some eosinophil cationic proteins, namely ECP and MBP, found at the site of heart damage in patients with eosinophilia, act as complete secretagogues on HHMC. This observation indicates another mechanism by which infiltrating eosinophils and their metabolic products cause inflammatory reactions and thus endomyocardial lesions in patients with eosinophilia.     

Intrathoracic vagal nerve schwannoma preoperative diagnosis is correct because of clinical and characteristic CT findings--a case report and review of the literature

BACKGROUND: Blister 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 the present investigation these eosinophil granule proteins were studied in the conjunctiva of patients with ocular cicatricial pemphigoid (OCP). METHODS: Conjunctival biopsy specimens obtained from patients with subacute (n = 8) or chronic conjunctival disease (n = 13) were analysed histologically and immunohistochemically using antibodies directed against EG1 (stored and secreted ECP), EG2 (secreted ECP), MBP, CD45 (common leucocyte antigen), CD3 (pan T cell marker), and HLA-DR (class II antigen). RESULTS: Subepithelial mononuclear cells, mast cells, and neutrophils were detected in all specimens. The number of mononuclear cells, neutrophils, CD45+ cells, CD3+ cells, and the HLA-DR expression were significantly higher in the subacute than in the chronic disease group. Some eosinophils were found in specimens from five of eight patients with subacute OCP, but in none of the patients with chronic disease. The eosinophil granule proteins (ECP and MBP) were found in the epithelium and substantia propria in patients with subacute conjunctivitis. CONCLUSIONS: Subepithelial cell infiltration in the conjunctiva greatly differs between subacute and chronic ocular cicatricial pemphigoid specimens. The findings suggest that eosinophil granule proteins may participate in tissue damage in acute phase of inflammation in OCP.     

The eosinophil ribonucleases

The eosinophil ribonucleases, eosinophilderived neurotoxin (EDN/RNase 2) and eosinophil cationic protein (ECP/RNase 3) are two closely related proteins with intriguing functional and evolutionary properties. While both EDN and ECP maintain the structural and catalytic residues typical of the RNase A superfamily, the role of ribonuclease activity in the physiologic function of these proteins remains unclear. The biochemistry and physiology of EDN, ECP and the recently discovered ribonuclease k6 (RNase 6) will be reviewed in this chapter.     

Positive Darwinian selection after gene duplication in primate ribonuclease genes

Evolutionary mechanisms of origins of new gene function have been a subject of long-standing debate. Here we report a convincing case in which positive Darwinian selection operated at the molecular level during the evolution of novel function by gene duplication. The genes for eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) in primates belong to the ribonuclease gene family, and the ECP gene, whose product has an anti-pathogen function not displayed by EDN, was generated by duplication of the EDN gene about 31 million years ago. Using inferred nucleotide sequences of ancestral organisms, we showed that the rate of nonsynonymous nucleotide substitution was significantly higher than that of synonymous substitution for the ECP gene. This strongly suggests that positive Darwinian selection operated in the early stage of evolution of the ECP gene. It was also found that the number of arginine residues increased substantially in a short period of evolutionary time after gene duplication, and these amino acid changes probably produced the novel anti-pathogen function of ECP.     

Eosinophil markers in seasonal allergic rhinitis. Intranasal fluticasone propionate inhibits local and systemic increases during the pollen season

BACKGROUND: The purpose was to study activation markers of the eosinophil granulocytes in seasonal allergic rhinitis, and the impact of topical steroid therapy thereupon. METHODS: Sixty-three rhinitis patients with monoallergy to grass were examined before and at peak pollen season. Blood eosinophil count, eosinophil cationic protein (ECP), and eosinophil peroxidase (EPO) in serum and nasal lavage fluid were measured. During the season, patients were randomized to treatment with intranasal fluticasone propionate 0.1 mg o.d. (n=26), 0.2 mg o.d. (n=25), or placebo (n=12). Six healthy persons served as controls. RESULTS: During the season, all parameters, except nasal lavage ECP, increased in the placebo group (P<0.001-P<0.05). Significant differences were seen between the steroid groups and the placebo group for all parameters (P<0.001-P<0.05). Higher eosinophil count (P<0.05), serum EPO (P<0.02), and nasal lavage EPO (P<0.05) were found in patients before season than in controls. The following winter, 44 patients returned for repeated measurement. Lower levels of nasal lavage EPO were observed for patients than levels at the beginning of the season (P<0.0001). CONCLUSIONS: Intranasal fluticasone propionate reduced inflammation of the nasal mucosa, demonstrated locally by nasal lavage ECP and EPO, and systemically by blood eosinophils, serum ECP, and serum EPO. EPO seemed more sensitive than ECP as indicator of allergic inflammation. EPO demonstrated some perennial eosinophil activity in hay fever patients, increasing locally during spring.     

Elevated serum levels of the eosinophil granule major basic protein in patients with eosinophilia

A radioimmunoassay was established for the human eosinophil granule major basic protein (MBP). The mean level of MBP in sera from 105 normal control patients was 454 ng/ml, whereas in a sample of 188 patients with various forms of diseases, including the hypereosinophilic syndrome, levels as high as 14,000 ng/ml were measured. Serum levels of MBP did not correlate with eosinophil counts in normal subjects, but a positive correlation was seen in patients with eosinophilia; the patients with eosinophil counts greater than 350/mm3 generally showed increased levels of MBP. Many patients with skin disease and normal eosinophil counts had elevated levels of serum MBP. Monomer MBP has a molecular weight of 9,300, but in sera of patients with eosinophilia, the MBP activity was of high molecular weight, greater than 50,000. Analyses of serum by Sephadex G-200 and by electrofocusing suggest that MBP is not simply polymerized, but rather is bound to a larger carrier molecule. Monomeric MBP can be isolated from serum by reduction of serum with dithiothreitol, alkylation with iodoacetamide, and acidification to pH 2 followed by fractionation on Sephadex G-50 at pH 2. Under these conditions, up to 80% of the MBP emerges in monomeric form. The results indicate that eosinophil granule proteins circulate in blood covalently bound to serum proteins, and that elevated concentrations of serum MBP are present in some diseases associated with eosinophilia.     

A H2O-producing NADH oxidase from the protozoan parasite Giardia duodenalis

Interleukin-2 (IL-2) is an essential growth factor for T cells. Previous studies have shown that human peripheral eosinophils respond to IL-2 in chemotaxis and express the IL-2 receptor (CD25). In addition, eosinophils have been shown to transcribe messenger RNA for IL-2. The aim of the present study was to determine whether eosinophils translate mRNA for IL-2 and to determine the site of intracellular localization. By immunocytochemistry, an average of 9% of cells showed cytoplasmic staining for IL-2 in freshly isolated unstimulated blood eosinophils obtained from asthmatic subjects who were not receiving oral corticosteroid treatment (n = 5). Freshly isolated, disrupted, highly purified eosinophils (> 99%, by CD16- immunomagnetic selection) contained an average of 6 pg/10(6) cells of IL-2 measured by a specific enzyme linked immunosorbent assay (ELISA) (n = 7). Purified eosinophil incubated with serum-coated Sephadex beads showed an increase in the amount of intracellularly-retained IL-2 (26.2 +/- 7.2 pg/10(6) cells) with some evidence for release of this cytokine but only in three out of six eosinophil preparations (range 1.3-5.8 pg/10(6) cells). The intracellular localization of IL-2 was determined by fractionation of the cells on a linear (0-45%) Nycodenz gradient in sucrose buffer followed by detection of IL-2 in the fractions using an IL-2-specific ELISA and dot blotting. The majority of the IL-2 detected co-eluted with known eosinophil granule markers (i.e. major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil peroxidase (EPO) and beta-hexosaminidase) but small quantities were also detected in the cytosolic (lactate dehydrogenase-(LDH) associated) and membrane (CD9+) fractions. Immunogold labelling of intact eosinophils using an anti-IL-2 monoclonal antibody confirmed IL-2 immunoreactivity in association with the eosinophil crystalline granule cores. These data are consistent with the hypothesis that eosinophils synthesize, release and store IL-2 largely within cystalloid granules. This stored IL-2 may serve as a reservoir for rapid release of IL-2 in inflammatory reactions associated with eosinophilia.     

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.     

Isolation and partial characterization of a major basic protein from rat eosinophil granules

The rat eosinophil granule possesses a major basic protein (MBP) similar to that previously isolated from human and guinea pig eosinophils. This conclusion is based on demonstration that the major protein of the rat eosinophil granule is a low molecular weight, highly basic material with an amino acid composition similar to that of the human and guinea pig MBP.     

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.     

Toxicity of an antitumor ribonuclease to Purkinje neurons

Purkinje cell toxicity is one of the characteristic features of the Gordon phenomenon, a syndrome manifested by ataxia, muscular rigidity, paralysis, and tremor that may lead to death (Gordon, 1933). Two members of the RNase superfamily found in humans, EDN (eosinophil-derived neurotoxin) and ECP (eosinophil cationic protein), cause the Gordon phenomenon when injected intraventricularly into guinea pigs or rabbits. We have found that another member of the RNase superfamily, an antitumor protein called onconase, isolated from Rana pipiens oocytes and early embryos, will also cause the Gordon phenomenon when injected into the cerebrospinal fluid of guinea pigs at a dose similar to that of EDN (LD50, 3-4 micrograms). Neurologic abnormalities of onconase-treated animals were indistinguishable from those of EDN-treated animals, and histology showed dramatic Purkinje cell loss in the brains of onconase-treated animals. The neurotoxic activity of onconase correlates with ribonuclease activity. Onconase modified by iodoacetic acid to eliminate 70% and 98% of the ribonuclease activity of the native enzyme displays a similar decrease in ability to cause the Gordon phenomenon. In contrast, the homologous bovine pancreatic RNase A injected intraventricularly at a dose 5000 times greater than the LD50 dose of EDN or onconase is not toxic and does not cause the Gordon phenomenon. A comparison of the RNase activities of EDN, onconase, and bovine pancreatic RNase A using three pancreatic RNA substrates demonstrates that onconase is orders of magnitude less active enzymatically than EDN and RNase A. Thus, another member of the RNase superfamily in addition to EDN and ECP can cause the Gordon phenomenon.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of estrogen treatment in female patients with dementia of the Alzheimer type

Increased numbers of eosinophilic granulocytes and eosinophil degranulation have been observed in various inflammatory conditions. Biopsies from the fallopian tubes of 28 patients were examined for the content of eosinophilic granulocytes. Peritoneal fluids (PF) from 89 patients were analyzed for the concentrations of eosinophilic granulocytes and the granule protein eosinophil cationic protein (ECP). The ECP levels in the PF were substantially elevated during genital inflammation (P < 0.001). Furthermore, one week after laparotomy for adhesiolysis there were both increased PF-ECP levels (P < 0.01) and raised concentrations of eosinophilic granulocytes (P < 0.001) when compared to the reference group. Staining of the biopsies with monoclonal antibodies revealed an increased total number as well as a number of activated eosinophilic granulocytes in specimens from adhesions and sactosalpinx compared to specimens from normal fimbriae. The findings indicate that eosinophilic granulocytes may have a possible role in the fibrotic process of pelvic adhesion disease.     

Serum levels of eosinophil cationic protein and eosinophil protein x in pollen atopic patients with stable asthma and its relation with bronchial hyperresponsiveness

Eosinophils are important effector cells in allergic inflammation described in allergic rhinitis (AR) and allergic bronchial asthma (BA). During the pollen season serum levels of eosinophil cationic protein (ECP) and eosinophil X protein/eosinophil-derived neurotoxin (EPX/EDN) are increased in BA. The aim of the present study was to evaluate the serum levels of ECP and EPC in pollen atopic patients with AR and BA during the winter. 92 patients were studied. They were divided into three groups: I 29 patients with AR, II 51 patients with BA and III 12 healthy subjects. Allergic rhinitis and bronchial asthma were diagnosed by routine clinical tests: clinical history, skin tests, total IgE and specific IgE. In addition ECP and EPX were determined in serum. All patients were asymptomatic, stable and without medical treatment. Methacholine challenge test (MCT) was performed in all patients. MCT were positive in 4 patients of group I and 45 patients of group II. ECP levels (ug/l) were: 21 (I), 24 (II) and 7 (III). EPX levels (ug/l) were 35 (I), 45 (II) and 21 (III). Statistical differences (p < 0.01) were observed both in ECP and EPX levels in patients with MCT positive in relation to patients with MCT negative, and in allergic patients (I and II) in comparison with the healthy subjects (III) (p < 0.01). ECP and EPX serum levels are increased in patients with a positive MCT in the winter, out of the pollen season, when patients are asymptomatic, stable and without treatment. This fact suggests that eosinophils play an important role in the pathogenesis of bronchial asthma.     

Effect of a bed covering system in children with asthma and house dust mite hypersensitivity

Allergen avoidance is regarded as an important approach to management of atopic asthma. The effect of Intervent bed covering systems on house dust mite (HDM) allergen exposure, asthma symptoms and markers of inflammation was investigated in 31 HDM sensitive asthmatic children. Dust concentrations of Dermatophagoides pteronyssinus allergen 1 (Der p 1) were monitored before and after covering the mattress, duvet and pillow with active and placebo covers for 3 months, in a single-blind, cross-over trial. Twice daily peak expiratory flow rate (PEFR), daily symptom scores and treatment schedule were recorded. Bronchial hyperresponsiveness was monitored by histamine challenge (provocative concentration of histamine causing a 20% fall in forced expiratory volume in one second (PC20)), and inflammation by measuring eosinophil cationic protein (ECP), eosinophil protein X (EPX), eosinophil peroxidase (EPO), and soluble interleukin-2 receptor (sIL-2R) in serum. There was a significant reduction in Der p 1 when the mattress, duvet and pillow were covered with the active bedding. There was no significant improvement in symptoms of asthma, PEFR, bronchodilator usage of PC20. Also, ECP, EPX, sIL-2R concentrations did not change for either treatment. EPO concentrations were significantly lower in the active compared to the placebo period. The active bed covers reduced retrievable Dermatophagoides pteronyssinus allergen 1 (Der p 1) from the bedding, with short term clinical benefit.     

Further evidence for allergic pathophysiology in allergic fungal sinusitis

Controversy continues over whether allergic fungal sinusitis represents a true allergy, an infection, or a point somewhere along a spectrum between allergy and infection. The present study describes two experiments that add weight to the argument that allergic fungal sinusitis (AFS) is truly an immunologically mediated hypersensitivity and not a form of infection. In the first experiment, eight patients with Bipolaris culture-positive AFS were prospectively evaluated with Bipolaris antigen skin testing and with inhibition radioallergosorbent (RAST) and enzyme-linked immunosorbent assay (ELISA) for Bipolaris-specific IgE and IgG antibodies. The Bipolaris AFS cases were compared with 10 control patients with no history of AFS. All eight AFS cases demonstrated positive skin testing to Bipolaris and in addition, all tested positive by RAST and ELISA for IgE and IgG Bipolaris antibodies, respectively. In the control group one patient had a positive skin test, ELISA, and RAST and one additional patient had a positive ELISA only. Good correlation was noted between skin test, RAST, and ELISA results. In the second experiment, sinus mucosa from 14 AFS patients and 10 control patients with other forms of surgical sinus disease was analyzed by immunohistocytochemistry for the eosinophilic inflammatory mediators major basic protein (MBP) and eosinophil derived neurotoxin (EDN) and the neutrophil mediator neutrophil elastase. All AFS cases demonstrated evidence of eosinophilic mediator release, and MBP and EDN predominated over neutrophil elastase. In the control group eosinophil and neutrophil mediator release in sinus mucosa was equal. The two experiments support the concept that AFS is an antigen-triggered, IgE- and IgG-mediated hypersensitivity response with a late-phase inflammatory reaction involving release of eosinophilic mediators.     

Thallium poisoning presenting with abdominal colic, paresthesia, and irritability

We have determined the complete nucleotide sequence for the cDNA encoding rat eosinophil major basic protein (MBP) using the rapid amplification of cDNA ends (RACE) procedure. The deduced amino acid sequence revealed that the rat prepro-MBP has three functional domains, namely the signal peptide, the acidic peptide that contains numerous acidic amino acids, and the mature MBP, as in human and guinea pig MBP.     

Hemodialysis-induced increase in serum lactoferrin and serum eosinophil cationic protein as signs of local neutrophil and eosinophil degranulation

Transient reduction in circulating polymorphonuclear granulocytes and eosinophils were observed early in hemodialysis. About a threefold increase in serum-lactoferrin occurred 2 h from the start of hemodialysis. Increments of the serum levels of eosinophil cationic protein (ECP) were observed as early as 1 h after initiation of hemodialysis, reaching maximum levels (about a fourfold increase from initial levels) 1 h later. When fresh blood was circulated through a dialyzer without having a patient in the circuit considerable increases of lactoferrin and ECP were also found. The intracellular contents of lactoferrin and ECP in granulocytes isolated from peripheral blood were unaffected throughout the dialysis period. Sera obtained at different times during dialysis induced no release of granular proteins from isolated granulocytes in vitro. The raised serum concentrations of lactoferrin and ECP during dialysis suggest that a local degranulation of neutrophils and eosinophils may take place probably in the dialyzer.     

Induced sputum eosinophil cationic protein (ECP) measurement in asthma and chronic obstructive airway disease (COAD)

BACKGROUND: Induced sputum is a useful way to monitor airway inflammation in asthma, but cell counts are time-consuming and labour intensive. OBJECTIVE: The aim of this study was to evaluate a novel processing method using eosinophil cationic protein (ECP) as a biochemical marker of sputum eosinophil number and activation in subjects with asthma and other airway diseases. METHODS: Sputum was dispersed with dithiothreitol and centrifuged to yield cell free supernatant and a cell pellet. The pellet was treated with a cellular lysis buffer to release cell-associated ECP. ECP was measured in sputum supernatant and in the lysed cell pellet and was compared with sputum eosinophil counts in 31 adults with asthma, chronic obstructive airway disease (COAD), bronchiectasis and healthy controls. The ratio of supernatant to pellet ECP was evaluated as an index of eosinophil degranulation. The effect of sputum processing reagents and storage time on ECP measurement was also evaluated. RESULTS: ECP measured in the cell pellet lysate correlated closely with sputum absolute eosinophil counts across a range of subject groups (r = 0.72, P = 0.004). Sputum eosinophil counts were less well correlated with supernatant ECP levels (r = 0.54, P < 0.05). Incubation with dithiothreitol or lysis buffer did not influence ECP measurement and sputum ECP levels were stable over a 6-9 month period. Sputum supernatant and pellet lysate ECP concentrations were increased in stable asthma, asthma exacerbations and COAD/bronchiectasis (P < 0.05). The ratio of supernatant to pellet ECP was used as an index of eosinophil degranulation and found to be elevated in asthma exacerbations, COAD and bronchiectasis, but not in stable asthma. CONCLUSION: The measurement of ECP in the sputum cell pellet provides a reliable and efficient estimate of sputum eosinophil counts which can potentially be used in clinical trials and epidemiological surveys. The ECP ratio may be a useful marker of eosinophil activation, and was increased in asthma exacerbation and COAD. The increased ECP in COAD reflects a non-selective accumulation of eosinophils in this condition.