Differentiation and culture of dendritic cells from equine peripheral blood mononuclear cells

Abstract

Dendritic cells(DCs) derived from myeloid cells are the most potent antigen-presenting cells. In the mouse, it is possible to differentiate a number of DCs from bone marrow. However, the horse has trouble in sacrificing with economic problems. To our knowledge, the culture of equine DCs has not been performed yet in our country. Furthermore, the cytokine set for culture of equine DCs has not been established in this area. Thus, we tried some sets of cytokines and milogen for this purpose. Mononuclear cells were isolated from blood by Ficoll histopaque, versene solution and an adhesion process and then cultured for 4 days. Specific morphology and viability of DC were determined by visual observation using an optical microscope. Human Granulocyte Macrophage-Colony Stimulating Factor(GM-CSF) and equine interleukin(lL)-4 were certainly effective for culturing equine DCs. Antigen-stimulatory ability of equine DCs was confirmed by Mixed Leukocyte Reaction using <sup>3</sup>H-thymidine incorporation assay. To verify antigen-uptake capability of DC, we usedfluorescein isothiocyanate-dextran and analyzed by using FACSCalibur<sup>Ⓡ</sup> flow cytometer(BD Biosciences). It was evident from our results that human GM-CSF could be used as an alternative cytokine for equine GM-CSF due to its genetic homogeneity. Taken together, this study demonstrated that human GM-CSF and equine IL-4 were effective to diflerentiate and proliferate DCs from peripheral blood mononuclear cells and enhanced its viability. The information from this study may provide the study of horse immunity with new insights and basic technology.