Nancy J. Bigley (Advisor), Dawn P. Wooley (Committee Member), Marjorie Markopoulos (Committee Member)
Master of Science (MS)
Mouse models of eosinophil-associated diseases have been used to study the mechanisms of disease pathogenesis. In this study, mouse-derived bone marrow cells were used in long-term (6 and 9 months) cell cultures of differentiated eosinophils and macrophages. IL-5 was used to differentiate the stem cells to eosinophils and GM-CSF was used to propagate macrophages from the bone marrow stem cells. The maximum time period for observing the eosinophil cultures was 252 days which is censurably longer than the 18 days culture period observed by others. The results were assessed by describing the microscopic cell morphology by Wright staining, modified Giemsa staining and protein expression by immunofluorescent staining. The GMCSF-stimulated bone marrow cultures produced classically appearing monocyte/macrophages throughout the study and were used to compare the development of the eosinophils over the long-term period of observation. Differentiation of the BM cells was carried out using with growth factors (SCF, FLT3L) and cytokines (IL-5, GM-CSF) over the 252 days period. The most suitable culture plate for long-term of cell growth were thee 60 mm petri dishes. At 252 days, the eosinophils exhibited as bi-lobed nuclear shapes, comparable with human eosinophils. Dendrite-like ramifications were observed on the surfaces of these eosinophils. Long term culture of eosinophils in the presence of IL-5 contributed to formation of eosinophil extracellular traps (EETs) areas. Within the EETs the cells surface of eosinophils developed holes; the nucleus of such cells lost the “ring-like” or “lobular” morphology and appeared in de-condensed fashion. Within the EETs, the plasma membranes of eosinophils developed the protrusions containing cytoplasmic granules. The EETs and protrusions had not been observed previously in the mouse eosinophil models in vitro but were described in human eosinophils. Consequently, the long-term culture of mouse bone-marrow derived eosinophil cultures may be useful in identifying conditions leading to pathology of hypo-eosinophilia human diseases such as asthma and bullous pemphigoid (BP).
Department or Program
Microbiology and Immunology
Year Degree Awarded
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