Systematic evaluation of the conditions required for the generation of immature rat bone marrow-derived dendritic cells and their phenotypic and functional characterization

Abstract

This study systematically evaluated the conditions required for generating immature rat bone marrow-derived dendritic cells (BMDCs) and characterized their phenotype. The culture of Wistar rat bone marrow cells for 7 days in an optimal cytokine environment (granulocyte macrophage-colony stimulating factor (GM-CSF), 10 ng/ml; IL-4, 5 ng/ml) resulted in adherent and non-adherent cell populations, but only the adherent population predominantly expressed the rat DC marker OX62. Adherent OX62 + cells were immature, in that they expressed lower levels of CD86 and MHC class II and were more phagocytic than their non-adherent OX62 + counterparts. Adherent BMDCs constitutively produced low levels of IL-12 and nitric oxide (NO), levels of both of which were markedly increased following lipopolysaccharide (LPS) activation. Activation also increased the proportion of OX62 + cells expressing CD40, CD54 and CD86 and their intensity of expression, however, unlike murine BMDCs, it had no effect on CD80 and MHC class II expression. Although the proliferation of allogeneic Lewis splenocytes in response to immature resting and LPS-activated (mature) Wistar BMDCs was of a similar magnitude, levels of IL-12 after 5 days were significantly higher in cultures containing LPS-activated BMDCs and the IFN-γ/IL-4 cytokine ratio differed markedly (2.35 vs. 6.66, respectively). This study systematically defines conditions for generating immature rat BMDC populations and demonstrates qualitative differences in the phenotype of immune responses induced by resting and LPS-activated BMDC populations.