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The eosinophil granulocyte is an inflammatory cell involved in allergic diseases such as asthma and rhinitis. Eosinophil cationic protein (ECP) is a basic and potentially cytotoxic granule protein that is released from the eosinophil upon activation. The aim was to study secretion of molecular variants of ECP from blood eosinophils with the hypothesis that the stored noncytotoxic ECP is altered into cytotoxic species upon release from the cell. Eosinophil granulocytes were purified to >95% from venous blood from birch pollen allergic subjects, with symptoms of rhinitis, and from healthy control subjects during the birch pollen season. The cells were stimulated with IL-5, GM-CSF, or serum-opsonized Sephadex particles. Concentration of ECP in cells or supernatants was measured by means of a fluoroenzyme immunoassay, and ECP heterogeneity was studied using an affinity capture assay with the surface-enhanced laser desorption/ionization-time of flight mass spectrometry technique. Extracts of unstimulated eosinophils contained 10 major ECP variants, with molecular masses ranging from 16.1 to 17.7 kDa. Stimulation with particles mainly induced the secretion of two molecular variants at 16.1 and 16.3 kDa, while cytokine stimulation gave rise to a different secretion profile. ECP variants in the pellet extracts remained unaffected by cell activation. The modifications of secreted ECP were partly explained by differences in N-linked glycosylations. Secretion of ECP from eosinophils involves protein modification. The molecular masses of released ECP have acquired the masses of the cytotoxic species.

Proteins from human eosinophils were separated bidimensionally and identified by mass spectrometry (336 spots/bands, 98 different proteins). Of these, 24.7% belonged to the cytoskeleton/migration group. Highly basic proteins (11.3%) were concentrated in the granule-containing cell fraction. We detected novel hyperacidic forms of cofilin-1, profilin-1 and adenylyl cyclase-associated protein, and hyperbasic forms of eosinophil-derived neurotoxin/eosinophil protein X and major basic protein homologue. We also found evidence of the triglycosylation of the heavy chain of eosinophil peroxidase. In addition, through comparative 2D image analysis, spot quantification and MS, it was found that hsc70, actin-capping protein and hyperacidic forms of eosinophil peroxidase heavy chain are overexpressed in cells from birch pollen allergic subjects, at the peak of a season. The link between these findings and an increased cellular antigen-presenting capacity and motility are discussed.

Eosinophil cationic protein (ECP), a secretory protein of the eosinophil granulocyte, is a basic and highly heterogeneous protein. This heterogeneity is dependent on polymorphisms in the ECP gene and post-translational modifications, and it affects the functional properties of the protein in terms of cytotoxicity. The aim of this study was to further investigate the molecular heterogeneity, hence, an affinity capture assay based on an antigen-antibody interaction with the surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS) technique was developed. Of three monoclonal antibodies tested, that is, EG2, 614, and 652, the 614 mab was chosen for the experiments. ECP heterogeneity of single individuals was studied in extracts of purified blood eosinophils, and the presence of approximately 5 major molecular species was demonstrated in each subject. ECP from subjects with different ECP 434(G>C) genotypes (arg97thr) showed mass differences corresponding to the amino acid shift from arginine to threonine. ECP purified from pooled leukocytes of large numbers of healthy blood donors demonstrated an extensive mass heterogeneity with approximately 10 major molecular species. By the use of a variety of glucosidases it was shown that this heterogeneity was mainly due to N-linked oligosaccharides on which sialic acid, galactose, and acetylglucosamine was positioned. We conclude that the SELDI-TOF MS technique using specific monoclonal antibodies is a convenient and versatile tool; by means of this technique, we could detect both genetic and post-translational causes of the molecular heterogeneity of the eosinophil cationic protein.

In neutrophils, coupling of chemoattractants to their cell surface receptor at low temperature (