Eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) were isolated from lysates of human eosinophil granules by gel filtration and ion exchange chromatography on heparin-Sepharose. Radioimmunoassay, using monoclonal antibodies, of fractions from the heparinSepharose chromatography showed one peak of EDN activity and two peaks of ECP activity (termed ECP-1 and ECP-2).EDN, ECP-1, and ECP-2 each exhibited heterogeneity in charge and molecular weight when analyzed by two-dimensional nonequilibrium pH gradient electrophoresis and NaDodSO4/PAGE. Digestion of EDN with endoglycosidase F (endo F) decreased its molecular weight and charge heterogeneity. Thus, EDN likely contains a single complex oligosaccharide. Endo F digestion of ECP-1 and ECP-2 decreased the molecular weight of both polypeptides, indicating that both likely contain at least one complex oligosaccharide. Amino acid sequence analyses showed that ECP-1 and ECP-2 are identical from residue 1 through residue 59 and that the sequences of EDN and ECP are highly homologous (37 of 55 residues identical). Both EDN and ECP NH2-termnu*al sequences showed significant homology to RNase, especially in regions of the RNase molecule involved in ligand binding. EDN, ECP-1, and ECP-2 had neurotoxic activity, causing the Gordon phenomenon at doses down to 0.15 ,ug when injected into the cisterna magna; the proteins were comparable in their activities. These results indicate that EDN and ECP are related proteins and suggest that they derived from genes associated with the RNase family.The human eosinophil granule contains several cationic proteins including the major basic protein (1-3), the eosinophil cationic protein (ECP) (4,5), the eosinophilderived neurotoxin (EDN) (6, 7), and the eosinophil peroxidase (EPO) (8). Major basic protein and ECP are potent helminthotoxins (9-11), and major basic protein is toxic to mammalian cells (9, 12). When injected intrathecally into rabbits or guinea pigs, both EDN and ECP, but not major basic protein or EPO, produce the Gordon phenomenon, a neurologic syndrome characterized in the rabbit by stiffness, ataxia, muscle weakness, and muscle wasting (6,7,13 MATERIALS AND METHODSEosinophil Granules. From nine patients with marked peripheral blood eosinophilia, eosinophils were collected by cytapheresis with hydroxyethyl-starch (15). The mean percentage of eosinophils in the concentrate was 79% (range, 64-94%); between 2 x 1010 and 2 x 1011 eosinophils were collected from a single patient. For purification of the eosinophil granules from the large numbers of cells obtained, we used procedures described previously (1-3, 7, 16). The enriched granule fractions were transferred to 2-ml freezing vials (Nunc) and stored in liquid nitrogen for up to 3 years.EDN and ECP Isolation. Frozen granule pellets were thawed, adjusted to pH 3 with 0.1 M HCl, and centrifuged at 40,000 x g for 20 min. Supernatants from the granule extracts were applied to a 1.2 x 48 cm Sephadex G-50 column equilibrated with 0.025 M NaOAc/0.15 M Na...

Eosinophils contain four principal cationic proteins, major basic protein (MBP), eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), and eosinophil peroxidase (EPO). To determine the quantities of these proteins in granulocytes and whether they are specific to eosinophils, their concentrations in lysates of human granulocytes were measured using specific radioimmunoassays. The effect of different methods for eosinophil lysis on the recovery of the proteins was also studied. Maximal recovery occurred at pH 2 for MBP and pH 5.6 for the other granule proteins. The proteins cosedimented with eosinophils and their concentrations (mean +/- SEM) in ng/10(6) eosinophils (and in nM/10(6) eosinophils) were: MBP, 8,982 +/- 611 (641.6); EDN, 3,283 +/- 116 (178.4); ECP, 5,269 +/- 283 (250.9); and EPO, 12,174 +/- 859 (171.5). Basophils from a normal person contained (in ng/10(6) cells) MBP, 2,374; EDN, 214; ECP, 77; and EPO, 17. Highly purified neutrophils contained (in ng/10(6) cells) MBP, 3 +/- 0.5; EDN, 72 +/- 9; and ECP, 50 +/- 12. Therefore we conclude that these proteins are mainly expressed in eosinophils, but that certain ones are present in basophils and neutrophils.

Eosinophils are multifunctional leukocytes implicated in the pathogenesis of asthma and in immunity to certain organisms. Associations between exposure to an environmental fungus, such as Alternaria, and asthma have been recognized clinically. Protease-activated receptors (PARs) are G protein-coupled receptors that are cleaved and activated by serine proteases, but their roles in innate immunity remain unknown. We previously found that human eosinophils respond vigorously to Alternaria organisms and to the secretory product(s) of Alternaria with eosinophils releasing their proinflammatory mediators. In this study, we investigated the roles of protease(s) produced by Alternaria and of PARs expressed on eosinophils in their immune responses against fungal organisms. We found that Alternaria alternata produces aspartate protease(s) and that human peripheral blood eosinophils degranulate in response to the cell-free extract of A. alternata. Eosinophils showed an increased intracellular calcium concentration in response to Alternaria that was desensitized by peptide and protease ligands for PAR-2 and inhibited by a PAR-2 antagonistic peptide. Alternaria-derived aspartate protease(s) cleaved PAR-2 to expose neo-ligands; these neo-ligands activated eosinophil degranulation in the absence of proteases. Finally, treatment of Alternaria extract with aspartate protease inhibitors, which are conventionally used for HIV-1 and other microbes, attenuated the eosinophils’ responses to Alternaria. Thus, fungal aspartate protease and eosinophil PAR-2 appear critical for the eosinophils’ innate immune response to certain fungi, suggesting a novel mechanism for pathologic inflammation in asthma and for host-pathogen interaction.

Eosinophils are important effector cells in defense against helminth infection and in allergic diseases. To identify novel eosinophil proteins, large scale sequencing of a cDNA library prepared from interleukin-5-stimulated umbilical cord precursor cells was performed, and the major genes expressed by maturing eosinophils were determined. This resulted in the identification of a cDNA with 64% identity to human prepro-major basic protein (hprepro-MBP). This cDNA was designated hprepro-MBP homolog (hprepro-MBPH). Interestingly, the calculated pI values for hMBPH and hMBP differed by >100-fold, with pI values of 8.7 and 11.4, respectively. Given this pronounced basicity difference, the homolog transcript's abundance (1.1%), and MBP's critical role in eosinophil biological activity, we further characterized the homolog. Reverse transcription-polymerase chain reaction detected transcription of hprepro-MBPH in bone marrow only, and this result was confirmed by analysis of a large cDNA data base (electronic Northern). hMBPH was isolated from human eosinophil granule lysates, and its identity was verified by amino acid sequencing and by mass spectrometry. Analyses of the biological activities showed that hMBPH had effects similar to hMBP in cell killing and neutrophil (superoxide anion production and interleukin-8 release) and basophil (histamine and leukotriene C 4 release) stimulation assays, but usually with reduced potency. Overall, this novel homolog's unique physical properties indicated that the high net positive charge of hMBP is important but not essential for biological activity.

Nitration of tyrosine residues has been observed during various acute and chronic inflammatory diseases. However, the mechanism of tyrosine nitration and the nature of the proteins that become tyrosine nitrated during inflammation remain unclear. Here we show that eosinophils but not other cell types including neutrophils contain nitrotyrosine-positive proteins in specific granules. Furthermore, we demonstrate that the human eosinophil toxins, eosinophil peroxidase (EPO), major basic protein, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), and the respective murine toxins, are post-translationally modified by nitration at tyrosine residues during cell maturation. High resolution affinity-mass spectrometry identified specific single nitration sites at Tyr 349 in EPO and Tyr 33 in both ECP and EDN. ECP and EDN crystal structures revealed and EPO structure modeling suggested that the nitrated tyrosine residues in the toxins are surface exposed. Studies in EPO ؊/؊ , gp91 phox؊/؊ , and NOS ؊/؊ mice revealed that tyrosine nitration of these toxins is mediated by EPO in the presence of hydrogen peroxide and minute amounts of NOx. Tyrosine nitration of eosinophil granule toxins occurs during maturation of eosinophils, independent of inflammation. These results provide evidence that post-translational tyrosine nitration is unique to eosinophils.Human eosinophils are bone marrow-derived, non-dividing granulocytes of the innate immune system, which store the highly cationic proteins eosinophil peroxidase (EPO), 3 major basic protein (MBP), eosinophil-derived neurotoxin (EDN), and eosinophil cationic protein (ECP) in secondary granules (1, 2). In rodents, eosinophil proteins with similar structure and activity have been identified (3-6). In response to allergen provocation or parasitic infection, expanded eosinophil populations from the bone marrow are selectively recruited to affected tissues (1). The release of cationic toxins from activated eosinophils by degranulation is regarded as a dominant effector function of these cells, mediating lysis of helminths and protozoae (7-10). The positive net charge conveys tight toxin binding to negatively charged cell surfaces where EPO causes oxidation of membrane components in the presence of hydrogen peroxide (H 2 O 2 ) (8). MBP increases membrane permeability and perturbation following insertion of apolar residues into the membrane (11), whereas ECP creates membrane channels (12). Both EDN and ECP exert ribonuclease activity (13). Due to their unspecific binding to membranes and their cytolytic activities, eosinophil granule proteins also cause host tissue damage during parasite infections and inflammatory disorders such as allergic asthma (14 -16). Despite advances in elucidating the mechanisms of action for the eosinophil granule proteins (1, 2), the cationic nature of these proteins (pI values Ͼ 10) would predict electrostatic repulsion and exclude interaction/cooperation of single granule proteins and among different granule proteins. Because sequen...