Immunogenicity of dendritic cells pulsed with cartilage derived soluble alloantigens

Investigation of allo- and autoreactivity of cartilage components is useful not only for the potential allogeneneic transplantation but also for the treatment of tissue specific degenerative autoimmune diseases. We generated dendritic cells (DCs) to study articular cartilage immunogenicity, by using human peripheral blood mononuclear cells. Dendritic cells are extremely efficient antigen-presenting cells that initiate and modulate T-cell dependant immune responses. After isolation by density centrifugation, GM-CSF and IL-4 were added to adherent mononuclear cells. After 7 days of cultivation the cells in cell culture were: HLA-DR+, CD86+, CD14-, CD80-, CD83, CD19-, CD56-, CD3-. Dendritic cells were then pulsed with a supernatant taken from primary monolayer cell cultures of human allogeneic chondrocytes. The stimulating capacity of antigen-pulsed DCs was measured in a classical proliferation assay using tritium labelled thymidine as a marker of mitosis. Proliferative responses were significantly strong but became much stronger when DCs were concomitantly incubated with soluble antigens and human recombinant TNF-alpha and used subsequently as stimulators. Although TNF-alpha positively influenced the stimulating capacity of pulsed DCs, we were unable to detect major differences in expression of specific DCs' phenotype markers assessed by flow cytometry in the absence or presence of this cytokine.