Related Experiment Videos
Characterization of an early dendritic cell precursor derived from murine lineage-negative hematopoietic progenitor
Sharon H Jackson1, Candido Alicea, Jennie W Owens
1The Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892-1886, USA. sjackson@niaid.nih.gov
Objective:
We define characteristics of a dendritic cell (DC) precursor generated from murine lineage-negative (Lin(-)) Sca1(+) hematopoietic progenitor cells (HPC).
Materials And Methods:
Lin(-)Sca1(+) HPC cultured 9 days in 100 ng/mL stem cell factor (SCF), 20 ng/mL interleukin-3 (IL-3), 50 ng/mL monocyte colony-stimulating factor (M-CSF), 5 ng/mL granulocyte-monocyte colony-stimulating factor (GM-CSF), and 25 ng/mL FLT3-ligand (FLT3-L) proliferate 387-fold and differentiate into DC precursors. Switch to > or =100 ng/mL GM-CSF + 1500 U/mL IL-4 or 500 U/mL tumor necrosis factor-alpha (TNF-alpha) for 3 days induces development into immature DC that are responsive to bacterial lipopolysaccharide (LPS)-induced maturation.
Results:
Lin(-)Sca1(+) HPC in the first 9 days of culture differentiate into DC precursors expressing surface CD11b(bright), CD11c(mod), CD86(low-mod), major histocompatibility class II antigen (MHC) II(low), DEC 205(low), but are surface CD40(-) and contain high levels of intracellular MHC II. Unlike immature DC described by others, these DC precursors are refractory to maturation with LPS and minimally stimulate allogeneic T lymphocytes in mixed leukocyte reactions (MLR). Switch to high-dose GM-CSF alone with IL-4 or TNF-alpha differentiates these DC precursors into immature DC. LPS treatment of the immature DC results in mature DC that express surface CD40(high) and CD86(high), secrete IL-1beta and IL-12, and strongly stimulate MLR.
Conclusions:
These studies define a distinct DC precursor derived from murine HPC that precedes development of immature and mature DC.