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Eosinophil Cationic Protein (ECP) Is Processed during Secretion¹

Department of Medical Sciences, Clinical Chemistry, Uppsala University, Uppsala, Sweden

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.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This study was supported by the Swedish Society of Medicine, the Swedish Heart and Lung Foundation, the Faculty of Medicine of Uppsala University (Sweden), the Swedish Medical Research Council, and the Swedish Association against Asthma and Allergy.

² Address correspondence and reprint requests to Dr. Charlotte Woschnagg, Department of Medical Sciences, Clinical Chemistry, Uppsala University, 75185 Uppsala, Sweden. E-mail: charlotte.woschnagg{at}medsci.uu.se

³ Abbreviations used in this paper: ECP, eosinophil cationic protein; SELDI-TOF MS, surface-enhanced laser desorption/ionization-time of flight mass spectrometry; RT, room temperature; CTAB, cetyltrimethylammonium bromide.

⁴ The online version of this article contains supplemental material.