Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Silly, T.
Optimization of eosinophil cell culture technologies
Humanmedizin; [ Diplomarbeit ] Graz Medical University; 2015. pp. 71
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- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Böhm Eva
- Sedej Miriam
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- Abstract:
- Eosinophil granulocytes only comprise a small fraction of circulating peripheral blood leukocytes. Accordingly, the study of eosinophils is limited by difficulties in achieving sufficient cell number and purity. Thus, other models providing large quantities of fully differentiated eosinophils and permitting longer and stable cultivation for the in-depth study of eosinophil function are needed. Hence, we tested two different eosinophil cell culture systems: the murine bone marrow derived eosinophils (bmEos) and the human EoL-1 cell line. To explore the functional competence of these two cell lines, their responsiveness to eotaxin and PGD2, two potent eosinophil chemoattractants, was assessed. Unselected bone marrow was isolated from BALB/c mice. Progenitor cells were enriched in FLT3-L and SCF-containing medium for the first four days and thereafter differentiated into fully competent eosinophils in IL-5-medium. Different brands and quality grades of fetal calf serum (FCS) as well as different serum and IL-5 concentrations were tested. Using eotaxin and PGD2 as stimulating agonists, we furthermore compared the responsiveness of human whole blood eosinophils and bmEos regarding their expression of the surface molecule CD11b, as well as their chemotactic responsiveness. EoL-1 cells were cultured in a medium containing varying concentrations of FCS and IL-5 and differentiation was initiated using sodium butyrate. The expression patterns of the PGD2 receptors CRTH2 and DP were investigated by flow cytometry and functional assays such as Ca(2+)-influx and chemotaxis were performed. We observed that the development of bmEos strongly depends on the composition and the quality grade of the serum. We only found one serum that yielded large quantities of fully differentiated eosinophil granulocytes within 12 days of culture. Stimulation with eotaxin led to a significant upregulation of CD11b surface expression on bmEos, as well as on human whole blood eosinophils. In contrast, stimulation with PGD2 only affected human whole blood eosinophils, but was without effect in bmEos. Similar results were obtained in the chemotaxis assay. Mouse bmEos as well as human eosinophils migrated towards eotaxin, whereas PGD2 only induced chemotaxis in human eosinophils. A possible explanation for this finding might be the fact that IL-5 leads to internalization of CRTH2. Thus, we suggested that CRTH2 is internalized or inactivated due to the cultivation in IL-5 medium, since CRTH2 protein expression in bmEos was confirmed by western blotting. Furthermore, we found CRTH2 being expressed on the cell surface of EoL-1 cells while levels of DP expression were inconsistent. The cells responded with chemotaxis to stimulation with PGD2. In contrast, we found no PGD2-induced Ca(2+) flux in EoL-1 cells.