Abstract
Abstract Eosinophil cationic protein (ECP), is one of the many granular proteins released by eosinophils contains 160 amino acids with a molecular weight of 18,440 Da. ECP is cytotoxic to many invading foreign substances, including parasites and bacteria. The clinical importance of ECP is focused on its role in allergic reaction for the deposit of ECP in the epithelial lining of airway. Patients with severe asthma usually showed high ECP concentration in the affected area. This cytotoxic effect is thought to originate from ribonuclease activity associated with ECP. However, the results showed that ECP has far more complicated biological functions than what has been found. In the liposome experiment, ECP lysed liposomes made of phosphatidylcholine (PC16:0) easily. The cell membrane also appeared to be a target of ECP. We thus further investigated the hemolytic activity of ECP on human red blood cells. This report employed visible spectroscopy, fluorescence spectroscopy, light microscopy and fluorescence microscopy to investigate the lytic function of ECP. Our results indicated that (1) Fresh healthy human and porcine erythrocytes were destroyed by ECP at concentration as low as 10 ng/l and the rate of lysis depends on the concentration of ECP and source of RBC. (2) Morphological analysis of ECP-treated lung fibroblast cell by phase contrast microscopy demonstrated that this protein induced cells shrink, detachment of cells from cell culture substratum and cell death. (3) Observed under fluorescence microscope, fluorescence dye (calcein) leakage from RBC and lung fibroblast cell implied that ECP increase permeability of these membranes. This result also exhibited under fluorescence spectroscopy observation. (4) Inclusion hydrocortisone and methylprednisolone in the RBC incubation media greatly retarded the rate of hemolysis. A medium containing 40 mM hydrocortisone inhibited the hemolysis during the period of experiment (>15 min). Compared with hydrocortisone, methylpredisolone seemed to be more potent in inhibiting the hemolysis. On the contrary, cromolyn did not exhibit inhibition at 40 mM. (5) Hydrocortisone and methylprednisolone could insert into the outer leaflet of human red blood cells to caused crenation of the membrane as observed under microscope. (6) DPPC liposome fusion induced by ECP was observed under phase and fluorescence microscope.