ABSTRACT
OBJECTIVE: In Hodgkin lymphoma (HL), tumor eosinophilia indicates poor prognosis, probably caused by eosinophil-induced stimulation of tumor cells. Our aim was to investigate the effects of eosinophil cationic protein (ECP) on HL tumor cells in vitro. MATERIALS AND METHODS: A fluorometric microculture cytotoxicity assay was used to measure the survival index of cells from the HL cell lines: HDLM-2 (T-cell origin, nodular sclerosis histology), KMH2 (B-cell origin, mixed cellularity), and L428 (B-cell origin, nodular sclerosis) after incubation with ECP97arg variants with different glycosylations and with ECP97thr. Flow cytometry monitored the effects of ECP on markers of cell death. RESULTS: For KMH2 and L428, ECP was cytotoxic with a dose-response relationship similar to a previously investigated small-cell lung cancer cell line. HDLM-2 was more sensitive to ECP at low concentrations, but reached a plateau (survival index of 70%) at 0.018 µM. The IC(50) for KMH2 and L428 were 0.2 and 0.15 µM, respectively. The IC(50) was never reached for HDLM-2. All tested ECP variants displayed similar activity in HDLM-2, in contrast to KMH2 and L428, which were more sensitive to less glycosylated ECP. Positive DNA staining (propidium iodide) of HDLM-2 cells treated with ECP indicated cell death by necrosis. CONCLUSIONS: ECP is cytotoxic for HL tumor cells even at low concentrations, but heterogeneity between cell lines exists and not all tumor cells are eradicated. Two cell lines of B-cell origin, KMH2 and L428, were sensitive to ECP in a dose-response manner, but for HDLM-2, which is of T-cell origin, the cytotoxicity reached a plateau.
Subject(s)
Eosinophil Cationic Protein/isolation & purification , Eosinophil Cationic Protein/pharmacology , Eosinophils/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Dose-Response Relationship, Drug , Eosinophil Cationic Protein/chemistry , Flow Cytometry , Glycosylation , Hodgkin Disease/metabolism , Hodgkin Disease/pathology , Humans , Inhibitory Concentration 50 , Ki-1 Antigen/metabolism , Molecular Weight , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Eosinophil granules contain several toxic cationic proteins that contribute to the pathophysiology of allergic diseases. These include eosinophil peroxidase, two ribonucleases, and two forms of the major basic protein (MBP). Extraction of eosinophil granules by exposure to acid solution and fractionation on Sephadex G-50 characteristically yields a distinctive profile of three discrete peaks, and these proteins are usually recovered in good quantities, except for the eosinophil major basic protein homolog (MBP2). We investigated the effect of multiple granule extractions by dilute HCl on the recovery of granule proteins. Isolated granules were repetitively extracted, up to 31 times, in 0.01 M HCl, and the extracts fractionated on Sephadex G-50. Whereas initial extracts yielded the characteristic three-peak fractionation pattern, later extracts yielded four discrete peaks. Characterization of the novel fourth peak showed that it contained MBP2. These results indicate that repetitive extraction of eosinophil granules yields an increased amount of all granule proteins, and that MBP2 can now be recovered in good quantities and in a relatively pure form.
