An improved method to generate equine dendritic cells from peripheral blood mononuclear cells: divergent maturation...

Equine dendritic cells (eqDC) can be generated from peripheral blood monocytes by propagation in GM-CSF and IL-4. Despite similarities with the generation of human DC, we found significant improvements for eqDC generation and functional influences on eqDC maturation. The fractionation of peripheral blood mononuclear cells (PBMC) by two subsequent gradients at densities of 1.090 and 1.077 as well as an adherence step in AIM V((R)) medium on dishes coated with extracellular matrix components (Primaria) improved the purity and yield of DC. After 3 days, eqDC cultures with GM-CSF alone developed into three subsets of (i) MHC II(neg) cells, (ii) MHC II(low) immature, endocytic cells and (iii) MHC II(high) spontaneously mature, non-endocytic DC. The immature DC fraction of the GM-CSF cultures matured, as detected by MHC II up-regulation, upon LPS exposure overnight. DC cultures in GM-CSF plus IL-4 resulted in higher cell yields, a loss of the immature MHC II(low) population but increased mature MHC II(high) DC, suggesting maturation. However, the MHC II(high) DC fraction was still endocytically active and did not lose their endocytic function after LPS treatment. They marginally up-regulated MHC II expression but this did not result in an enhanced stimulation of an allogeneic mixed lymphocyte reaction. However, LPS treatment clearly induced mRNA for IL-12p35 and p40, which was not observed by addition of IL-4 alone. Together our data indicate that IL-4 and LPS induce two different maturation programs. IL-4 induces a semi-maturation where the cells are still endocytic, which can be further matured to secrete cytokines in a second step by LPS.