Phenotype standardization of angioedema in the head and neck region caused by agents acting on the angiotensin system.

Angioedema is a potentially life-threatening adverse reaction to angiotensin-converting enzyme inhibitors and angiotensin receptor blockers. To study the genetic etiology of this rare adverse event, international consortia and multicenter recruitment of patients are needed. To reduce patient heterogeneity, we have standardized the phenotype. In brief, it comprises swelling in the head and neck region that first occurs during treatment. It should not coincide with urticaria or have another likely cause such as hereditary angioedema.

The stimulus-dependent release of eosinophil cationic protein and eosinophil protein x increases in apoptotic eosinophils.

Apoptotic cells are regarded as inert bodies that turn off intracellular processes and functional abilities. To study the changes in the ability of eosinophils to release their granule proteins while undergoing apoptosis. Eosinophils were cultured for up to 72 h. Living cells were separated from the apoptotic cells and their release of eosinophil cationic protein (ECP) and eosinophil protein X (EPX) was measured in response to serum-opsonized sephadex particles and phorbol 12-myristate 12-acetate (PMA). Changes in cell structure were examined by electron microscopy, and surface receptor expression of beta1- and beta2-integrins was investigated by flow cytometry. Stimulus-dependent release of the granule proteins ECP and EPX was found to increase in apoptotic eosinophils, whereas surface expression of beta1- and beta2-integrins was downregulated. Ultrastructural examination revealed that the granules of apoptotic eosinophils were translocated to the periphery of the cell, just beneath the plasma membrane. Apoptotic eosinophils are able to release their toxic granule proteins, which is probably because of the rearrangement of the cytoskeleton and spontaneous translocation of granules to the membrane. Our results suggest that apoptotic eosinophils are potentially harmful cells that have retained their ability to react to certain extracellular stimuli. The findings point to unexpected consequences of eosinophil apoptosis.

Eosinophil cationic protein is stored in, but not produced by, peripheral blood neutrophils.

BACKGROUND: Eosinophil cationic protein (ECP) is an eosinophil-derived protein, which has been shown to be present in circulating neutrophils. OBJECTIVE: To establish whether ECP is produced or internalized by peripheral blood neutrophils. METHODS: This was done using microscopy, flow cytometry, fractionation of cells and RT-PCR techniques. RESULTS: No ECP mRNA was detected after extensive cell purification to eliminate all traces of contaminating eosinophils. Examination of immunostained neutrophils by light, confocal, electron microscopy together with cell fraction experiments, established that ECP is present intracellularly and is mostly associated to cell granules. Uptake studies by flow cytometry and by using both cold and radiolabelled ECP showed that it is internalized by neutrophils and stored in some proportion in their primary granules. Upon stimulation with serum-treated Sephadex particles, the internalized ECP was partially released from cells. CONCLUSION: ECP is not produced but can be internalized by circulating neutrophils, which take it from the environment and partially store it in their primary granules.

(99m)Tc-HMPAO (Ceretec) is stored in and released from the granules of eosinophil granulocytes.

(99m)Tc-HMPAO (Ceretec) labelling of leucocytes is used clinically for the detection of inflammatory processes in the body. This study investigated the mechanisms by which (99m)Tc-HMPAO is taken up by eosinophils and neutrophils. Blood cells were labelled with (99m)Tc-HMPAO and the cells separated by means of their densities in Percoll gradients. For other purposes, eosinophils and neutrophils were purified by means of the MACS system and, after labelling these pure cellular preparations, the cells were ultrasonicated and the organelles separated on sucrose density gradients by means of ultracentrifugation. Organelles were characterized by their morphology on electron microscopy. Granulocytes were stimulated to secrete their granule constituents by means of exposure to complement-coated particles. ECP (eosinophil cationic protein) and MPO (myeloperoxidase) were measured using specific immunoassays. The uptake of (99m)Tc-HMPAO was 15--25-fold higher in eosinophils than in other leucocytes. (99m)Tc-HMPAO was predominantly stored in the secretory granules of eosinophils and released from the eosinophil, upon activation, together with ECP. A second storage compartment was a very light density organelle of unknown nature. These results indicated that, among leucocytes, (99m)Tc-HMPAO is preferentially taken up by eosinophils and stored in the secretory granules, which has to be taken into consideration when evaluating images based on this technique. Our findings suggest that (99m)Tc-HMPAO (Ceretec) may be used as a tool to follow eosinophil turnover and activity in disease.

SNARE proteins are critical for regulated exocytosis of ECP from human eosinophils.

The SNARE hypothesis, describing a protein assembly-disassembly pathway, was recently proposed for the sequential steps of synaptic vesicle docking, activation, and fusion. To determine if SNARE proteins are involved in regulated exocytosis in eosinophils, the presence and functional role of SNAREs was examined in human blood eosinophils. Immunoblotting, subcellular fractionation, and immunocytochemistry documented that vesicle-associated membrane protein-2 (VAMP-2), a vesicle-SNARE, was expressed in human eosinophils. Syntaxin 4 and SNAP-25 were also detected. Sequencing of cloned RT-PCR products amplified from a domain conserved among VAMP isoforms revealed identity only to VAMP-2 but not to VAMP-1 or cellubrevin. Functional experiments revealed that tetanus toxin pretreatment, which cleaved VAMP-2 in eosinophils, significantly inhibited both IgE receptor- and phorbol ester-mediated exocytosis of eosinophil cationic protein (ECP) from streptolysin-O-permeabilized eosinophils. Thus, these results strongly suggest a critical role of SNAREs in regulated exocytosis in eosinophils.

SNARE proteins are critical for regulated exocytosis of ECP from human eosinophils.

The SNARE hypothesis, describing a protein assembly-disassembly pathway, was recently proposed for the sequential steps of synaptic vesicle docking, activation and fusion. To determine if SNARE proteins are involved in regulated exocytosis in eosinophils, the presence and functional role of SNAREs was examined in human blood eosinophils. Immunoblotting, subcellular fractionation, and immunocytochemistry documented that vesicle-associated membrane protein-2 (VAMP-2), a vesicle-SNARE, was expressed in human eosinophils. Syntaxin 4 and SNAP-25 were also detected. Sequencing of cloned RT-PCR products amplified from a domain conserved among VAMP isoforms revealed identity only to VAMP-2 but not to VAMP-1 or cellubrevin. Functional experiments revealed that tetanus toxin pretreatment, which cleaved VAMP-2 in eosinophils, significantly inhibited both IgE receptor- and phorbol ester-mediated exocytosis of eosinophil cationic protein (ECP) from streptolysin-O-permeabilized eosinophils. Thus, these results strongly suggest a critical role of SNAREs in regulated exocytosis in eosinophils.

Piecemeal degranulation of peripheral blood eosinophils: a study of allergic subjects during and out of the pollen season.

The variability of serum and plasma levels of eosinophil granule proteins in different clinical conditions, interpreted as the result of different patterns of cytokine priming, suggests a selective mobilization of granule proteins. Inasmuch as piecemeal degranulation (PM) is the mechanism proposed for the differential release of eosinophil granule proteins, we decided to investigate whether blood eosinophils from allergic subjects show characteristics of PM during natural allergen challenge. Eosinophils from three birch-sensitive subjects were studied before and during the pollen season. Electron microscopy analysis showed that during the season, eosinophils presented morphologic features of PM. By immunogold labeling, eosinophil cationic protein (ECP) was detected not only in normal specific granules but also in the cytoplasm, in the vicinity of partially lucent specific granules. These results were confirmed by subcellular fractionation, where the amount of ECP associated with compartments containing small vesicles increased 2-fold during the pollen season. A study of the distribution of ECP, eosinophil peroxidase, and hexosaminidase in eosinophils of different densities showed that the profile of each of these proteins differed depending on cell density. All of these proteins decreased in the specific granule of hypodense cells and increased in other cell compartments. We conclude that allergen exposure causes PM of the peripheral blood eosinophils of allergic subjects, and that the density of these cells reflects the degree of degranulation. Our results provide novel information for the understanding of the selective mobilization of granule proteins into the circulation.

Administration of G-CSF to healthy subjects: the effects on eosinophil counts and mobilization of eosinophil granule proteins.

Any influence of G-CSF on eosinophils is mostly negative, although reports which have studied this relationship are few with varied results. The aim of this study was to investigate the influence of G-CSF administration to healthy subjects on eosinophils in peripheral blood. Blood eosinophil counts, serum levels of eosinophil cationic protein (ECP), eosinophil peroxidase (EPO) and eosinophil protein X (EPX), as well as cell morphology were studied. 14 healthy volunteers received 7.5 microg (n = 8) or 10 microg/kg body weight (n = 6) G-CSF daily for six consecutive days. ECP and EPX were assessed by specific RIAs and EPO by a specific FEIA. Cell morphology was examined by electron microscopy. During G-CSF administration, eosinophil counts increased from 0.22 +/- 0.04 x 10(9)/l to 0.61 +/- 0.098 x 10(9)/l (P = 0.001), serum ECP from 12.39 +/- 2.45 microg/l to 61.82 +/- 7.38 microg/l (P = 0.0014), serum EPX from 28.05 +/- 4.54 microg/l to 87.86 +/- 9.84 microg/l (P = 0.002) and serum EPO from 8.89 +/- 2.2 microg/l to 19.98 +/- 5.1 microg/l (P = 0.003). All variables returned gradually to initial values after discontinuation of G-CSF. Distinct changes in the morphology of secondary granules were observed 24 h after G-CSF administration. The granules became irregular and their matrix less electron dense. We conclude that administration of G-CSF to healthy humans increases the number of circulating eosinophils and affects the mobilization of eosinophil granule proteins.

The differential release of eosinophil granule proteins. Studies on patients with acute bacterial and viral infections.

BACKGROUND: Earlier in vitro studies have suggested that the eosinophil may release its granule proteins selectively depending on the stimulus to which the cell is exposed. OBJECTIVE: The object of the present study was to study the question of selective release in vivo by means of serum measurements of the two eosinophil granule proteins eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO) in acute infections. METHODS: Fourty-six subjects with acute infections were studied before treatment, 20 with bacterial infections and 26 with viral infections. Serum ECP, EPO and MPO were measured by specific RIA. RESULTS: In acute bacterial infections ECP, but not EPO, was significantly raised in serum (P < 0.0001) compared with non-infected healthy subjects. In acute bacterial infections ECP was significantly correlated to the levels of the neutrophil marker myeloperoxidase (MPO) (rs = 0.96, P < 0.0001) but not to EPO. In acute viral infections neither ECP nor EPO were on average raised. However, almost 20% the patients had elevated levels of bot proteins. In the viral infections the serum-levels of ECP and EPO were correlated (rs = 0.63, P < 0.001), but no correlation was found with MPO. CONCLUSION: It is concluded that eosinophils are activated during acute bacterial infections and that this activation results in the preferential mobilisation of ECP. The simultaneous assay of the two eosinophil proteins, ECP and EPO, may give new insight into the role of the eosinophil in disease.

[Neutral endopeptidase (NEP)--modulator of neurogenic inflammation]. / Obojetna endopeptydaza (NEP)--modulator zapalenia neurogennego.

Stimulation of the sensory nerves causes the release of neuropeptides such as substance P, CGRP, neurokinin A and B. NEP cleaves and inactivates a proportion of the peptide thus limiting the neurogenic inflammation. The role of NEP have been discussed.