Effect of Interleukin-9 on Cell Proliferation and Histamine Release of Cord Blood-derived Human Mast Cells / 소아알레르기및호흡기학회지

PURPOSE: Interleukin-9(IL-9), one of Th2-type cytokines, might be important in the pathophysiology of allergic diseases. We investigated the effect of IL-9 on human mast cells by assessing cell proliferation and histamine release. METHODS: Human umbilical cord blood cells were cultured in the presence of stem cell factor(SCF, 100 ng/mL) and IL-6(50 ng/mL) in liquid medium for 8 weeks. Then these cells were divided into 3 aliquots. Each aliquot was cultured for 4 more weeks in different conditions : SCF alone(100 ng/mL), IL-9 alone(50 ng/mL) and SCF+IL-9. Cell numbers were counted using hemocytometer. For evaluation of apoptosis, DNA fragmentation was determined by propidium iodide(PI) staining and flow cytometric analysis. Histamine concentration was measured by ELISA after stimulation with human IgE and anti-human IgE. RESULTS: Cell numbers increased significantly when they were cultured in the presence of SCF and IL-9 compared with SCF alone(P<0.05). Proliferation of mast cells was mediated by decreased apoptosis. Histamine release in activated mast cells was not different regardless of incubation with IL-9. CONCLUSION: IL-9 might be involved in allergic inflammation via proliferation of mast cells in target tissue.

Characterization of IL-3, IL-5, GM-CSF-induced Eosinophils from Human CD34+ Cord Blood Cells / 소아알레르기및호흡기학회지

PURPOSE: Eosinophils are cells of the granulocyte lineage that participate in host defence against parasitic disease and mediate allergic inflammation. In this study by using the combination of cytokines IL-3, IL-5 and GM-CSF, we explore the characterization of cultured eosinophils from CD34+ CBCs. METHODS: Mononuclear cells were isolated heparinized umbilical cord blood by Ficoll-paque (1.077 g/ml) density gradient centrifugation method. The CD34-bearing hematopoietic progenitor cells were collected by elution after their adhesion to a magnetic cell sepatation (MACS) column. The CD34+ cells were incubated in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and were cultured in the presence of IL-3, IL-5, and GM-CSF in a 6-well plate bottomed tissue culture plate at 37 degrees C for 7-28 days in humidified 5% CO2 and 95% air atmosphere. For the identification of cultured eosinophils Wright's and Giemsa staining, RT-PCR, Southern blotting and FACS analysis are used. RESULTS: We analyzed the cultured eosinophils Wright's and Giemsa staining, the total cell number of cells increased 50-fold by days 28 of culture. Also, using the sensitive RT-PCR technique, we monitered the appearance of mRNA transcrips of EPO. Identity of each RT-PCR product was confirms by southern blotting with independent gene-specific oligonucleotide probes and we found increasing of hybridization signals for EPO at 7th culture days. In addtion, we identified eosinophils in cultured CD34+ CBCs by flow cytometry. As s results, we succeeded in developing of pure eosinophils efficiently from CD34+ of CBCs in the presence of IL-3, IL-5 and GM-CSF. CONCLUSION: The in vitro growth of CD34+ CBCs may provide a useful system to study growth factor and stage-dependent adhesion molecule expression, as well as function on developing eosinophils.